The decreases in IRS-1/2 protein were paralleled by increases in GRB10 Ser501/503 and S6K1 Thr389 phosphorylation, two mTORC1 targets that can destabilize IRS proteins. PKB Ser473 phosphorylation was higher from 3–6 days, and this was associated with increased TSC2 Thr939 phosphorylation. The phosphorylation of TSC2 Thr1345 (an AMPK site) was also elevated, whereas phosphorylation at the other PKB site, Thr1462, was unchanged at 6 days. In agreement with the phosphorylation of Thr1345, SA led to activation of AMPKα1 during the initial growth phase, lasting the first 9 days before returning to baseline by day 12. The UPR markers CHOP and BiP were elevated over the first 12 days following ablation, whereas IRE1α levels decreased. These data suggest that during supraphysiological muscle loading at least three potential molecular brakes engage to downregulate mTORC1.The goal of the current work was to profile positive  and negative  pathways that might regulate overload-induced mTORC1 (mTOR complex 1) activation with the hypothesis that a number of negative regulators of mTORC1 will be engaged during a supraphysiological model of hypertrophy. To achieve this, mTORC1-IRS-1/2 signaling, BiP/CHOP/IRE1α, and AMPK activation were determined in rat plantaris muscle following synergist ablation (SA). SA resulted in significant increases in muscle mass of ∼4% per day throughout the 21 days of the experiment. The expression of the insulin-like growth factors (IGF) were high throughout the 21st day of overload. However, IGF signaling was limited, since IRS-1 and -2 were undetectable in the overloaded muscle from  During periods of growth, the rate of protein synthesis is greater than degradation -I and insulin  and nutr1345  is activated by chronic overload . Since A1345  or rapto345 , and potential catabolic signals (ER stress and metabolic stress/AMPK activation) during overload-induced skeletal muscle hypertrophy and determine whether alterations in any of these factors are associated with altered mTORC1 activity during supraphysiological hypertrophy induced by synergist ablation. We hypothesized that a number of molecular brakes will be engaged to restrain mTORC1 activity.All procedures on Female Wistar rats  were approved by the University of Dundee Research Ethics Committee and performed under UK Home Office project licence no. 60/3441. All surgical and collection procedures took place under inhaled anesthetic using a 2.5% concentration of isoflurane throughout. Animals were first sedated in an anesthetic induction box before placing the animal into the surgical field. Animals were allowed to recover for the appropriate time period post-stimulation and were terminated after muscle collection under anesthesia.n = 4), 6 days (n = 4), 9 days (n = 4), 12 days (n = 4), and 21 days (n = 4) of muscle hypertrophy induced by unilateral ablation of the gastrocnemius (GTN) and soleus (SOL) muscles. Animals were anesthetized, and the area above the incision was shaved and sterilized. The GTN and SOL muscles were isolated and severed at the Achilles tendon, and the distal two-thirds of the GTN and the whole SOL were removed, leaving the plantaris (PLN) intact. The overlying skin and fascia were sutured separately, and the animal was then moved to a temperature-controlled environment to recover. All animals returned to normal activity within 1 h. In the recovery period prior to collection, animals were monitored on a daily basis for signs of pain or postoperative infection. However, none of the animals showed any sign of undue discomfort or distress. On the day of collection, animals were anesthetized, and hypertrophied and contralateral control muscles were rapidly removed, snap-frozen in liquid nitrogen, and stored at −80°C until processing. Following muscle collection, animals were terminated by cervical dislocation.Twenty rats were used for this study, and they were randomly assigned to five treatment groups, 3 days (4-Na2(PO4)2, and 0.1% DTT]. The homogenate was briefly vortexed and centrifuged at 4°C for 10 mins at 10,000 g to remove insoluble material. Protein concentrations were determined using the DC protein assay .Muscles were rapidly removed, rinsed of blood, snap-frozen in liquid nitrogen, and stored at −80°C. For processing, muscle was powdered on dry ice using a mortar and pestle and polytron homogenized in 10-fold mass excess of ice-cold sucrose lysis buffer . AMP, [γ-32P]ATP (200 cpm/pmol), and the peptide substrate AMARAASAAALARRR were added to the immunoprecipitate at a final concentration of 200 μm. The assays were carried out for 15 min at 30°C and terminated by applying 30 μl of the reaction mixture to P81 papers . ATP incorporation into the peptide substrate was inhibited by placing the filter paper in 1% orthophosphoric acid. Phosphate incorporation into the peptide was measured as previously described  Triton X-100] per 20 mg of tissue and further homogenized using a hand-held polytron. The samples were left to rotate at 4°C for 30 min, and then cell debris was pelleted by centrifugation at 17,600 2VO4). The immunocomplexes were resuspended in 30–50 μlof 1× Laemmli sample buffer (LSB). The samples were boiled and spun briefly to pellet the beads, and 15 μl was loaded per well for SDS-PAGE separation, and transferred as described above.Rabbit TSC2 antibody (2 μg) was incubated with 1 mg of PLN lysate overnight at 4°C. The following day, TSC2 was immobilized on protein G-Sepharose for 1 h. Immunocomplexes were washed two times in sucrose lysis buffer and two times in TNE . Total RNA was then subjected to electrophoresis to check mRNA quantity and quality.taq-ready mix and 96-well plates (Fisher). PCR reactions were performed according to the manufacturer's instructions with the following primers: IGF-I , IGF-II , MGF , IRS-1 , and GAPDH  used at a concentration of 10 pM. PCR conditions were as follows: initial denaturation at 95°C for 15 min followed by 40 cycles of denaturation at 94°C for 15 s, annealing at 56°C for 30 s, and extension at 72°C for 30 s. A melt curve was then performed to assess the specificity of the primers and the presence of primer dimers. PCR efficiencies were determined for each primer set. The ratio of expression of the gene of interest normalized against the standard was computed according to the equation proposed by Pfaffl et al.  was used to synthesize first-strand cDNA with AMV reverse transcriptase and oligo(DT) primers  using the manufacturer's recommendations. cDNA was diluted 1:15 before use in qPCR. qPCR was performed using an Eppendorf mastercycler ep real-time thermocycler  along with a SYBR Green jump start l et al. .n = 3 or 4 per group. For the analysis of Western blot data where different sets of animals were used across a time course but samples were paired within a time point, the data were modeled using linear mixed effects with treatment, time, or the interaction between treatment and time as a fixed effect and animal as a random effect. We used the approach of Kenward and Roger . Interestingly, the time course of peak S6K1/GRB10 phosphorylation paralleled IRS-1/2 suppression and recovery. In addition to the phosphorylation changes there was a nonsignificant trend for t-S6K1 levels to be elevated at day 3 and a nonsignificant trend for t-GRB10 to be elevated at every time point post-ablation.mTORC1 has been proposed to negatively regulate IRS-1/2 content via two mechanisms; first via S6K1 and second via GRB10. We therefore measured S6K1 Thre period B. S6K1 pd; day 6 , A and B308 and Ser473  but returned to baseline by day 12  S6K1 activation does not drive the loss of IRS-1/2 protein; 2) the small increase in S6K1 phosphorylation and activity in this context may have a significantly greater effect on IRS-1/2 protein, possibly due to a difference in cellular localization; or 3) the longer duration of S6K1 activation is the key factor driving the feedback inhibition. Interestingly Harrington et al. . This is consistent with recent work from the Hornberger laboratory demonstrating that, although phosphorylation of Raptor at alternative serine/threonine sites is an important process in mechanical activation of mTORC1, the phosphorylation status of raptor at the AMPK site Ser792 was detectable only when Raptor was overexpressed  to K. Baar.The work was supported in part by a project grant from the No conflicts of interest, financial or otherwise, are declared by the author(s).Author contributions: D.L.H., A. Philp, and K.B. conception and design of research; D.L.H., A. Philp, M.G.M., A. Patton, and M.C.T. performed experiments; D.L.H., A. Philp, M.G.M., A. Patton, M.C.T., I.J.G., and K.B. analyzed data; D.L.H., A. Philp, I.J.G., S.C.B., and K.B. interpreted results of experiments; D.L.H., A. Philp, and K.B. prepared figures; D.L.H., A. Philp, and K.B. drafted manuscript; D.L.H., A. Philp, M.G.M., I.J.G., S.C.B., and K.B. edited and revised manuscript; D.L.H., A. Philp, M.G.M., A. Patton, M.C.T., I.J.G., S.C.B., and K.B. approved final version of manuscript.


In vitro, RVC20 and RVC58 were able to neutralize all 35 rabies virus (RABV) and 25 non‐RABV lyssaviruses. They showed higher potency and breath compared to antibodies under clinical development  and commercially available human RIG. In vivo, the RVC20–RVC58 cocktail protected Syrian hamsters from a lethal RABV challenge and did not affect the endogenous hamster post‐vaccination antibody response.Currently available rabies post‐exposure prophylaxis (PEP) for use in humans includes equine or human rabies immunoglobulins (RIG). The replacement of RIG with an equally or more potent and safer product is strongly encouraged due to the high costs and limited availability of existing RIG. In this study, we identified two broadly neutralizing human monoclonal antibodies that represent a valid and affordable alternative to RIG in rabies PEP. Memory B cells from four selected vaccinated donors were immortalized and monoclonal antibodies were tested for neutralizing activity and epitope specificity. Two antibodies, identified as RVC20 and RVC58 , were selected for their potency and broad‐spectrum reactivity.  Rhabdoviridae, genus Lyssavirus, and causes acute encephalitis in mammals. Lyssaviruses are enveloped single‐stranded (‐) RNA viruses which have helical symmetry and display on the outer surface of the virion envelope the G protein, which is the target antigen of virus‐neutralizing antibodies.Rabies virus (RABV) belongs to the family et al, et al, et al, et al, et al, et al, et al, et al, RABV is the first of fourteen lyssavirus species identified to date  is recommended to prevent the advancement of infection and thus the clinical disease; however, it must be administered as early as possible. According to the World Health Organization (WHO)  were tested for their ability to neutralize a panel of 12 pseudotyped lyssaviruses representing RABV and non‐RABV lyssaviruses isolates of phylogroup I, II, and III viruses . The neutralization profile of the other lyssavirus species varied considerably in all donors tested, but in a few cases, all species were neutralized. It was interesting to note that HRIG showed only modest activity against non‐RABV phylogroup I species and no cross‐reactivity with phylogroup II and III viruses.In order to isolate broadly neutralizing antibodies against not only RABV isolates but also non‐RABV lyssaviruses, sera from 90 RABV vaccinees were screened for the presence of high titers of antibodies that bind to the RABV CVS‐11 isolate) G protein by ELISA  ranging from 0.01 to 317 ng/ml  and CpG, as previously described , all antibodies did not bind to RABV G protein under reducing conditions, thus suggesting that the epitopes recognized, in most cases, are conformational .et al, et al, Competition studies were then performed to determine the spatial proximity of each of the conformational epitopes recognized by the selected neutralizing monoclonal antibodies. The two reference antibodies CR57 and CR4098 were previously shown to recognize G protein antigenic sites I and III, respectively  and with antibodies that bind to sites different from site III, or I, suggesting that the RVC58 antibody recognizes a yet undefined epitope that only partially overlaps with the one recognized by CR4098. The binding of RVB181, RVC56, RVB185, RVC21, RVB161, and RVC111 was blocked by antigenic site III antibodies, but reciprocally, these antibodies did not block binding of several other antigenic site III antibodies, such as CR4098, RVC4, and RVC69. In interpreting competition results, it should be taken into account that when two epitopes overlap, or even when the areas covered by the arms of the two antibodies overlap, competition should be almost complete and mutually cross‐competitive. Thus, only marked mutual cross‐competition should be taken as unequivocal evidence of overlapping epitopes, since weak or one‐way inhibition may simply reflect a decreased affinity due to steric or allosteric effects. Thus, the latter results suggest that these antibodies form a third cluster that recognizes a distinct, hereafter dubbed III.2, antigenic site. Three additional sites were further defined and named A, B, and C. Site A is defined by the unique antibody RVB686, whose binding compromises the binding of the majority of the labeled antibodies in the panel, but reciprocally the binding of the labeled RBV686 is not blocked by any antibody in the panel. These results might suggest that RVB686 binding induces an allosteric effect on the G protein that compromises the binding of most other antibodies. Site B is defined by antibody RVC44, whose binding is not blocked by any other antibody in the panel. Similarly, site C is defined by antibodies RVB143 and RVC68, which also recognize a unique and distinct site as compared to all the other antibodies.® (HRIG) were included for testing against a large panel of lyssaviruses using pseudotyped (N = 22) and infectious viruses (N = 16) covering RABV, LBV, MOKV, DUVV, EBLV‐1, EBLV‐2, ABLV, IRKV, KHUV, ARAV, SHIBV, BBLV, IKOV, and WCBV species .Twelve of the 21 antibodies were selected for testing based on their neutralizing potency and recognition of distinct sites on the RABV G protein. In addition, CR57, CR4098, RAB1, and Berirabcies Fig A and B , although with IC90 values 10‐ to 100‐fold higher than compared to RVC20 and RVC58  neutralize all phylogroups I and II isolates tested  was able to neutralize 68% of all non‐RABV lyssaviruses tested and, remarkably, all the phylogroup I non‐RABV lyssaviruses tested Fig C. In comn = 35, 26 viruses and 9 pseudotyped viruses), which are representative of all circulating lineages  , neutralizing six and three RABV isolates, respectively, with IC50 > 1,000 ng/ml, a concentration which is likely not to be effective in PEP. This analysis was extended to an additional 7 RABV isolates for which we tested the ability of the antibodies to bind to G protein‐transfected cells by flow cytometry  Fig . All 35 ytometry . All theet al, et al, In order to better define the epitope specificity of the 12 selected human monoclonal antibodies, they were tested against chimeric and mutant RABV and LBV pseudotyped viruses. In particular, the amino acid changes K226E, K226N, G229E, and N336D found in the previously described CR57 and CR4098 viral escape mutants . While LBV was not neutralized by the antigenic site III and III.2 antibodies, the chimeric LBV pseudotyped virus L3C carrying the CVS‐11 antigenic site III was neutralized efficiently by CR4098, RVA144, RVB185, and RVC21. These results indicate that antibodies RVA144, RVB185, and RVC21 recognize an epitope which is similar to that recognized by CR4098. In the case of the RVC38 antibody, the swapping of antigenic site III from LBV into CVS‐11 (C3L) did not affect its neutralizing activity but, similar to the other antigenic site III antibodies described above, the L3C pseudotyped virus is neutralized by RVC38. This finding suggests that the recognition of antigenic site III residues by the RVC38 antibody is sufficient to neutralize the chimeric LBV but also that its epitope in RABV CVS‐11 is formed by additional residues surrounding antigenic site III. Finally, in the case of RVA122, RVC58, and RVC111 antibodies, the swapping of antigenic site III residues into LBV resulted in partial or complete loss of neutralizing activity, thus suggesting that their epitopes comprise additional residues to those of antigenic site III.Finally, all antibodies, including CR4098, but with the exception of RAB1 (data not shown), RVC111 and RVC68, were able to neutralize the CR4098 CVS‐11 escape mutants N336D, thus indicating that this mutation does not block the binding of these antibodies to their cognate epitopes in the context of the CVS‐11G protein. These results indicate that some antibodies, such as RVA144, RVB185, and RVC21, recognize an epitope in antigenic site III that is similar to that recognized by CR4098, while others, such as RVC58, RVB492, RVC38, and RVC111 recognize distinct epitopes in the antigenic site III region. In addition, all the antibodies in our panel directed against antigenic site III, RVC58 in particular, showed a greater breadth of reactivity against non‐RABV lyssaviruses as compared to CR4098 . As described above for antigenic site I, we therefore analyzed the degree of conservation of antigenic site III amino acid residues in the panel of 2566 RABV isolates. We found that positions 330, 331, 334, 335, and 337 are highly conserved (> 99.61%), while residues 332, 333, 336, and 338 are polymorphic Figs B. RVC58 in vivo, we tested the two antibodies in a Syrian hamster (Mesocricetus auratus) model  were challenged intramuscularly with a lethal dose of RABV CVS‐11 and subsequently received post‐exposure prophylaxis containing either HRIG (PEP) or an equimolar cocktail of RVC20 and RVC58 antibodies (exPEP) at different concentrations (0.045 or 0.0045 mg/kg).To investigate whether the antibodies RVC58 and RVC20 display neutralizing activity against a lethal RABV infection Eleven out of 12 animals (92%) that were not treated after infection succumbed between day 6 and 8 Fig A. The stin vivo experiment was performed in hamsters. Briefly, vaccine immunogenicity was assessed through serological testing, in the presence of HRIG or RVC20 and RVC58 cocktail administration. The results demonstrated that both HRIG (20 mg/kg) and RVC58 + RVC20 (0.045 mg/kg) did not reduce the endogenous hamster IgG‐binding antibody response to the RABV G protein , we have identified several others of interest, whose specificity and properties will require further investigations. Of note, one of these antibodies, namely RVC68, showed an extraordinary breadth of reactivity across phylogroups I and II lyssaviruses and recognized a linear epitope yet to be determined.Since they were first generated in 1975 . Indeed, only about 1 million doses of RIG  are produced and sold every year and about 60% of the people with category III exposures do not receive RIGs  that would allow long‐term storage as well as more convenient supply to rural areas. According to WHO recommendations, the best approach to replace RIG with monoclonal antibodies is based on the development of a cocktail of antibodies able to reduce the risk of PEP failure. The best characteristics for a cocktail of RABV‐neutralizing antibodies are as follows: (i) high potency, (ii) recognition of distinct non‐overlapping antigenic sites, and (iii) high breadth of reactivity for complete coverage of field RABV isolates. Another important feature is that the activity of the antibodies forming the cocktail should not rely on a synergistic effect. Indeed, although synergy may result in potent neutralization of a virus harboring all epitopes of the individual cocktail antibodies, this potency could be significantly reduced for a virus in which any of these epitopes is not available. Importantly, the RVC20 and RVC58 antibodies did not show any synergistic neutralizing activity in vitro .in vivo fitness and this might particularly be the case for viral escape from multiple, broadly reactive antibodies. An additional important feature of RVC20 and RVC58, similar to HRIG, is their ability to neutralize all the RABV tested within a narrow and similar range of antibody concentrations, in contrast to CR57, CR4098, and RAB1. In previous studies, it was found that the antigenic site I CR57 antibody was not able to neutralize 2 out of 26 viruses tested and that from the analysis of a database of 229 isolates ~1% contained mutations that would most likely abrogate binding of CR57 . Considering the marked reduction in antibody production costs  from vaccinees for their ability to bind to two distinct antigenic sites (sites I and III) on the RABV G protein. In addition to this, they were able to potently neutralize each RABV isolate in our panel, representing all lineages, and all phylogroup I non‐RABV isolates. Indeed, the identification of rare, broadly reactive antibodies such as those selected in this study can increase the barrier to the occurrence of resistance since they can cope with a higher degree of variability in their cognate epitopes. In this regard, it is important to note that viral escape mutants might have an impaired  Kelley, , this amIn conclusion, the combination of the RVC20 and RVC58 antibodies represents a treatment with an unprecedented breadth and potency for the development of a low‐risk and affordable product to replace RIGs in rabies PEP.+ memory B cells were isolated from cryopreserved PBMC using CD22 microbeads (Miltenyi Biotec), followed by depletion of cells carrying IgM, IgD, and IgA by cell sorting, and immortalized with EBV and CpG in multiple replicate wells as previously described  was incubated with doubling dilutions of sera or antibodies for 1 h at 37% (5% CO2) before the addition of 3,000 BHK‐21 cells. These were incubated for a further 48 h, after which supernatant was removed and 15 μl SteadyLite reagent (Perkin Elmer) was added. Luciferase activity was detected 5 min later by reading the plates on a Synergy microplate luminometer (BioTek) . ID50 values for the sera correspond to the dilution at which RLU were reduced 50%. Antigenic site swapping between the CVS‐11 (accession no. EU352767) and LBV.NIG56‐RV1 (accession no. EF547431) G genes was undertaken using overlapping PCR  were used for production of the lentiviral pseudotypes. Neutralization assays were undertaken on BHK‐21 cells clone 13 (ATCC CCL‐10). In a 384‐well plate, pseudotyped virus that resulted in an output of 50–100 × 10A standard ELISA was used to determine binding of serum antibodies or monoclonal antibodies to RABV G protein (CVS‐11). Briefly, ELISA plates were coated with RABV G protein at 5 μg/ml, blocked with 10% FCS in PBS, incubated with sera or human antibodies, and washed. Bound antibodies were detected by incubation with AP‐conjugated goat anti‐human IgG (Southern Biotech). Plates were then washed, substrate  was added and plates were read at 405 nm. The relative affinities of sera binding or monoclonal antibody binding were determined by measuring the dilution of sera (ED50) or the concentration of antibody (EC50) required to achieve 50% maximal binding at saturation.et al, ™ Western Blotting Detection Reagent  and the LAS4000 CCD camera system.Purified RABV G protein  and further adapted on BSR cells (a clone of BHK‐21). Two protocols slightly modified from fluorescent antibody virus neutralization (mFAVN) and from rapid fluorescent foci inhibition (mRFFIT) test . Briefly, one hundred microliters of sample suspension were used for the extraction, and RNA was eluted in a final volume of 60 μl and stored at −80°C. One‐step RT–PCR amplification was performed using the Qiagen OneStep RT–PCR kit  according to the manufacturer's instructions. Primers used for the amplification of complete G gene sequences are available upon request. PCR products were analyzed for purity and size by electrophoresis in 2% agarose gel after staining with GelRedTM Nucleic Acid Gel Stain . Amplicons were subsequently purified with ExoSAP‐IT  and sequenced in both directions using the Big Dye Terminator v3.1 cycle sequencing kit . The products of the sequencing reactions were cleaned up using the Performa DTR Ultra 96‐well kit  and analyzed on a 16‐capillary ABI PRISM 3130xl Genetic Analyzer .Sequencing of complete G gene of the original specimens as well as of the cell‐adapted lyssaviruses used as challenge viruses in the et al, The occurrence of different amino acid identities at antigenic site I and antigenic site III was analyzed by downloading all RABV glycoprotein sequences present in National Center for Biotechnology Information (NCBI) Entrez Protein database ]. The protocol was authorized by the Italian Ministry of Health (Decrees 128/2011‐B and 115/2014‐PR) before experiments were initiated and approved by the Committee on the Ethics of Animal Experiments of the IZSVe.Mesocricetus auratus) of 6–7 weeks of age (average weight 105 g) (Charles River Laboratories). Animals were housed in individually HEPA‐filtered ventilated cages, three individuals per cage, at a temperature of 22 ± 1°C, on a 12L:12D light cycle, with free access to water and food. Pressed cotton pads, mouse houses, and litter bags were used as environmental enrichment, and the standard rodent feed was weekly integrated with autoclaved sunflower seeds. In order to minimize the effect of subjective bias during allocation, animals were randomly assigned to treatment or control groups. No blinding of investigator was implemented. No samples nor animals were excluded from the analyses.All experiments were performed on female SPF Syrian hamsters (6.76 MICLD50/ml) dilution of the CVS‐11 strain. Animals were given biologics or PBS (negative control) by the end of day 0. For each treated group (n = 12), a commercial vaccine  was administered intramuscularly (i.m.) in the left hind limb at days 0, 3, 7, 14, and 28 post‐infection (p.i.). Hyperimmune treatment consisted of HRIG  or candidate antibodies (0.045 mg/kg and 0.0045 mg/kg) of an equimolar mixture of RVC20 and RVC58 antibodies administered i.m. on day 0 in the right hind limb in a final volume of 50 μl. Challenged animals were observed twice a day and promptly euthanized at the onset of one of clinical signs of rabies . Central nervous system (CNS) tissues, namely brain, cerebellum, brainstem and spinal cord, were collected to confirm rabies virus infection by means of a standard technique, fluorescent antibody test (FAT)  with 0.05 ml of a 1:100 (10n = 12 per group) and vaccinated with a commercial purified chicken embryo cell vaccine . Vaccine was administered on day 0, 3, 7, 14, and 28 i.m. in the left hind limb. Three groups were concomitantly administered on day 0 with HRIG  or an equimolar mixture of RVC20 and RVC58 at 0.045 or 40 mg/kg (referred as HD) that were injected i.m. in the right hind limb and in the right and left hind limbs for HD antibody cocktail administration. Sera were collected from all animals (n = 48) on day 44 post‐vaccination (p.v.) and tested by FAVN  was calculated using Fisher's exact conditional test for two proportions  and power 1‐β = 0.80 (α = 0.05). A Wilcoxon matched‐pair signed‐rank test was used to analyze differences in mean values between groups. Mann–Whitney and Mantel–Cox tests were also used as described in legends. P‐values of < 0.05 were considered statistically significant.The number of individuals in each experimental group  protocols, human deaths due to rabies are still a burden in endemic areas, with a roughly estimated 60,000 deaths each year. More than 95% of cases occur in Asia and Africa, 50% of which affect children. Current protocols of PEP include the first‐aid treatment of the wound and the administration of a rabies vaccine, which is coupled with rabies immunoglobulin (RIG) administration in specific high‐risk cases. More than 15 million people a year are treated after an episode of suspect or confirmed rabies exposure. However, the limited production and the high costs of RIGs make them scarcely available on the market, especially in developing countries where they are mostly needed.We immortalized memory B cells from four vaccinated donors and then tested monoclonal antibodies for their rabies neutralizing activity and epitope specificity. We selected two antibodies, identified as RVC20 and RVC58 as highly potent and broadly neutralizing a panel of 60 lyssaviruses (including rabies and non‐rabies viruses), widely representatives of the global viral diversity. The potency and breadth shown by the two selected antibodies were higher than those detected in commercially available human RIG and in previously discovered antibodies under clinical development. Our antibody cocktail (RVC20 + RVC58), experimentally administered as alternative to commercially available RIG according to current PEP protocols, was able to protect Syrian hamsters from a lethal rabies virus challenge and did not impact on vaccine immunogenicity in hamsters.The cocktail of antibodies proposed in our study represents a treatment with an unprecedented breadth and potency to be eventually adopted as a valid alternative to RIG in the frame of human rabies PEP. Importantly, considering the high potency of our cocktail, the amount needed in PEP would be compatible with a considerably lower price if compared to that of currently available RIG.AppendixClick here for additional data file.Expanded View Figures PDFClick here for additional data file.Review Process FileClick here for additional data file.Source Data for Figure 4Click here for additional data file.Source Data for Figure 6Click here for additional data file.Source Data for Figure 7Click here for additional data file.


Seiridium cardinale is the major threat to Cupressus sempervirens worldwide. We investigated the production of terpenes by canker-resistant and susceptible cypresses inoculated with S. cardinale, the effect of these terpenes on fungal growth, and the defensive biotransformation of the terpenes conducted by the fungus. All infected trees produced de novo terpenes and strongly induced terpenic responses, but the responses were stronger in the canker-resistant than the susceptible trees. In vitro tests for the inhibition of fungal growth indicated that the terpene concentrations of resistant trees were more inhibitory than those of susceptible trees. The highly induced and de novo terpenes exhibited substantial inhibition (more than a fungicide reference) and had a high concentration-dependent inhibition, whereas the most abundant terpenes had a low concentration-dependent inhibition. S. cardinale biotransformed three terpenes and was capable of detoxifying them even outside the fungal mycelium, in its immediate surrounding environment. Our results thus indicated that terpenes were key defences efficiently used by C. sempervirens, but also that S. cardinale is ready for the battle.The canker-causing fungus  To our knowledge, no other terpenes of C. sempervirens have been tested, and the terpene biotransformation capacity of this fungus has never been investigated. To fill these gaps in our understanding of the arms race between the tree and the fungus, we studied the terpenic composition and response of C. sempervirens trees selected for resistance against canker (Agrimed) and trees not selected for resistance (NR) to S. cardinale infection using gas chromatographic/mass spectrometric (GC-MS) analyses of control, wounded, and infected phloem tissues. We then used in vitro growth inhibition tests using both in planta and arbitrary concentrations to determine the antifungal activity of 15 terpenes in healthy and cankered C. sempervirens. We also studied the biotransformative and detoxificant capabilities of S. cardinale inside  and outside  the mycelium with GC-MS analyses of in vitro inhibition test plugs.de novo terpenes exclusively found in the infected states of both groups (oxygenated MT2 only in infected Agrimed). Other compounds, such as ocimene, thymyl methyl eter, and MT4 were only found in the wounded and infected states. The concentrations of these terpenes were usually low (post hoc test P < 0.05 or P < 0.10)  , Table 1 < 0.10) . The pro < 0.10) . AgrimedS. cardinale of the C. sempervirens terpenes varied substantially when tested in vitro, ranging from complete growth inhibition (e.g. (+)-α-terpineol and (−)-terpinen-4-ol) to growth stimulation (e.g. (+)-α-pinene and limonene)  -α-pinene, (−)-bornyl acetate and limonene. Infected NR concentrations were more inhibitory than the control concentrations for (+)-sabinene, terpinolene, (+)-cedrol and (+)-manool, and infected Agrimed concentrations were more inhibitory than infected NR concentrations for (+)-sabinene, (+)-δ-3-carene, and (−)-terpinen-4-ol. The mean of all inhibitions of infected Agrimed (24.1%) was significantly higher than that of infected NR (18.4%) and the control (15.0%) .The antifungal activity against imonene) , Table 2imonene) . Several < 0.05) . The in −1 malt agar extract (MEA). (+)-Totarol was more inhibitory than azoxystrobin, a reference fungicide. At 0.50 mg g−1 MEA, some MT hydrocarbons began to show moderate rates of inhibition (ca. 25%), the oxygenated MTs substantially increased their inhibition, whereas STs and DTs maintained similar inhibitions to growth. The most concentrated test, 1.0 mg g−1 MEA, exhibited the strongest inhibitions, led by oxygenated MTs, half of which inhibited growth completely and overcame the inhibition caused by the fungicide, followed by DTs, STs, and MTs. We calculated the concentration-dependence of inhibition for each terpene within that concentration range . The most concentration-dependent compounds were the de novo terpenes (68.2%), followed by induced terpenes (22.1%), and the major terpenes (9.0%)  -cedrol, and the DTs had substantial effects on fungal growth in the fixed concentration tests  at 0.25 on range  by subtr < 0.01) .S. cardinale grew on MEA plates containing (+)-camphor, (−)-bornyl acetate, or (+)-cedrol (the transformation test), but only the terpene substrate was found on MEA plates containing these three terpenes but without the fungus (the terpene test) -cedrol produced six new compounds that could not be identified. The tests also produced quantitative differences among these three terpenes . The H and HF samples of the transformation test also presented several differences, with H usually having higher concentrations of biotransformation products than HF (P < 0.05).Several biotransformations in both H (hyphae) and HF  plugs were detected in the biotransformation tests where ne test) . The Petterpenes , and in  than HF . A test  than HF  showed hde novo oxygenated MT and more inductions in concentrations and proportions. Agrimed also had several higher concentrations and proportions of various terpenes than NR in the infected treatments -sabinene, (+)-δ-3-carene, and (−)-terpinen-4-ol), so these results suggest that, by day 30, MTs could be the class of terpenes responsible for conferring the higher canker resistance to Agrimed  in concentrations about 1–2 mg g−1 (fresh weight). As said above, S. cardinale began to infect cypress a few decades ago, and we hypothesize that a lack of co-evolution could explain our results.The majority of terpenes showed a concentration-dependent inhibition of fungal growth12 Agrimed . The lowin planta inhibition tests have two important limitations: i) mean concentrations in phloems are applied, which does not represent the real variability of concentrations, and ii) the application of the same concentrations in the MEA as those found in phloems may not be quantitatively appropriate. Our results suggested that X mg g−1 MEA were more inhibitory than X mg g−1 phloem. The fixed concentration tests allowed a comparison of the inhibitory powers of the terpenes and can help to predict inhibition in canker-infected cypress stems or more advanced states of infection (e.g. day 90), which should exhibit higher concentrationsDespite being a useful tool for studying inhibition more realistically, de novo terpenes (+)-sabinene hydrate3812S. cardinale. The de novo compounds produced by an infected conifer can have very strong inhibitory effects on the infecting pathogen27in planta concentrations but would likely have been stronger inhibitors at the ca. fifteen-fold higher concentrations reported in our previous studyS.cardinale -α-pinene, (+)-δ-3-carene, (+)-cedrol, and (+)-totarol), differed little between 0.25 and 1.0 mg g−1 MEA  S. cardinale excretes terpene substrates along with some biotransformed products away from fungal cells, which would act as a detoxification mechanism to lower the cellular terpene levelsii) S. cardinale, suggested to release exoenzymes that play a role in systemic pathogenesisS. cardinale, because the fungus would encounter partially detoxified defences, and thus a less aggressive environment to colonise.The biotransformation of (+)-camphor to bornane-2,5-dione was observed for the first time in C. sempervirens reacts to the early stages of infection (day 30) by increasing the concentration of MTs but may be preparing itself for more advanced stages by beginning to generate several phytoalexins and increasing the concentrations of the most inhibitory compounds currently known for this pathogen. Cypress devoted more resources to increasing the concentrations of minor than of major terpenes, corroborating the observations of our previous studyS. cardinale may be able to tolerate some of the most inhibitory terpenes of C. sempervirens, detoxifying them by biotransformation and changing its immediate environment for its behalf. Further studies should determine the identity of the biotransformed compounds, and investigate the biotransformation and detoxification mechanisms of this fungus. It would also be very interesting to see if other pathosystems react similarly and corroborate the tree defence mechanisms suggested here. In more practical terms, the strong actions of the most inhibitory terpenes warrant further efforts to test their viability as natural fungicides against S. cardinale.The differences in constitutive and induced terpene responses to infection between NR and Agrimed, along with the inhibitory power of these compounds, suggest that part of the Agrimed resistance to cypress bark canker may be due to its stronger and more inducible terpenic profile. Inhibition tests suggest that Cupressus sempervirens L. trees grown in pots were divided into two groups: 18 were not selected for resistance to cypress bark canker (NR) and 18 were the Agrimed n°1S. cardinale (Wagener) Sutton & Gibson standard isolate ATCC 38654 was used for the artificial inoculations and the inhibition and biotransformation tests. The fungus was grown on malt agar extract (MEA) at 25 °C in the dark for 15 days.Thirty-six five-year-old grafted Three treatments were applied to both tree groups in August 2013: control , wounded , and infected (inoculation wound + fungus). The phloems of three randomly chosen main branches of the trees were inoculated following standard proceduresThe phloem sub-samples of each replicate were bulked and ground with a pestle in 50-ml Teflon tubes containing liquid nitrogen to avoid evaporation and facilitate the grinding. One ml of pentane containing dodecane  was added to the ground tissues, and the solution was stored overnight at −20 °C. Three-hundred μl of the supernatant were analysed by GC-MS. The Teflon tubes were dried to constant weights, weighed in a precision balance, cleaned thoroughly, dried, and reweighed to tare the tubes. One blank was analysed for every six samples.−1 to 150 °C and maintained for 5 min, thereafter at 30 °C min−1 to 250 °C and maintained for 5 min, and finally at 30 °C min−1 to 280 °C and maintained for 5 min. Total run time was 29 min, the helium flow was set to 1 ml min−1, and the split was 1:10. The terpenes were identified by comparing the mass spectra with known standards and published spectra . Calibration curves for terpene quantification were prepared with dodecane and commercial standards of four MTs , four STs  and two DTs (totarol and phytol). All terpenes were purchased from Fluka Chemie AG, Buchs, Switzerland, and had purities superior to 95%. Four different concentrations were used to perform terpene calibration curves, with correlations between signal and concentration always highly significant (r2 ≥ 0.99). The main terpenes had similar sensitivities (differences < 5%).Two microlitres of the phloem extract were injected into a capillary column  of a GC  with an MS detector . Initial temperature was maintained at 35 °C for two minutes, increased at 15 °C min®, Syngenta), commonly used against cypress bark cankerThe terpenes tested for antifungal activity were selected by their performance in our previous studyin vitro tests were performed in 6-cm Petri dishes containing 5 g of 2% MEA. The test solutions were prepared by mixing the terpenes with 60 μl of acetone, whereas the acetone controls contained only acetone. The solutions were gently shaken, pipetted, and then spread over the MEA surface with a spatula. A 5-mm disk of a S. cardinale colony was then placed in the centre of the Petri dishes, which were immediately tightly sealed with Parafilm® and incubated at 25 °C for 6 d in the dark. All tests were replicated four to five times. Three in planta concentrations corresponding to those found in the C. sempervirens GC-MS (in mg g−1 phloem tissue) analyses were tested for each terpene in vitro (in mg g−1 MEA). The control (mean concentration of both groups of trees), infected NR, and infected Agrimed  were calculated by:The  Agrimed  concentrDa is the average mycelial diameter of each sample test and Db is the average mycelial diameter of the acetone control.where −1 MEA inhibition tests  were sampled immediately after the measurement of fungal growth at day 6 to study fungal biotransformations. Three supplementary tests, agar , fungus , and terpene , were also sampled 6 d after test application. No terpenes were detected in the agar and fungus tests. Three plugs were taken from the transformation test, one from the margin of the colony , one of MEA 5 mm from the mycelial border , and one identical to HF to incubate for two weeks for verifying the absence of hyphae . Plugs were extracted with a 5-mm cork borer and were placed in refrigerated Eppendorf tubes and immediately stored at −80 °C. Sample analyses and identification were performed as described above but with individual calibration curves for each terpene and a split of 1:3 to maximise compound detection. The GC-MS analyses of the MEA found no MTsMEA plugs (5 mm diameter) from the 1.0 mg gS. cardinale on the fungus mycelial growth was assessed in vitro. Two groups (detoxification and control) of 12 Petri dishes containing MEA and 1 mg g−1 of (+)-camphor, or (−)-bornyl acetate or (+)-cedrol  solved in pentane (4 replicates for each terpene) were prepared following the procedure described before. In each Petri dish of the first group (detoxification treatment), a 5-mm plug of S. cardinale mycelium was added as described above. No S. cardinale plug was added to the second group of Petri dishes (control treatment). The Petri dishes were stored at the previously described conditions for 6 days.The inhibitory activity of the terpenes biotransformed by S. cardinale colony was trimmed from each Petri of the detoxification treatment. In the control Petri dishes, we trimmed the equivalent ring of agar from an equivalent position. The trimmed rings of MEA from each Petri dish were separately soaked with 3 ml of pentane in hermetic vials and kept overnight at −20 °C in constant shaking (150 rpm) in order to extract all the terpenes and the products biotransformed by the fungus. Next morning, we concentrated the resulting extract with a flux of gaseous nitrogen until we reached a final volume of 100 μl. This final solution was pipetted and spread in the surface of two new groups of MEA Petri dishes (4 replicates each): Biotransformed substrate  and Non-biotransformed substrate (extract of MEA with non-biotrasformed terpenes). Petri dishes were incubated as previously described conditions and fungal growth was measured after 3 and 6 days.Then, a 10 mm width ring of MEA+terpene (hyphae free) surrounding the post hoc tests (P < 0.05). Outliers were removed using absolute deviation around the medianThe data were analysed by one-way ANOVAs, and treatments and tests were compared with Tukey’s How to cite this article: Achotegui-Castells, A. et al. Terpene arms race in the Seiridium cardinale - Cupressus sempervirens pathosystem. Sci. Rep.6, 18954; doi: 10.1038/srep18954 (2016).


In the present study the effect of hydrolysis with non-commercial Cucurbita ficifolia serine protease on a reduction of the IgE and IgG binding capacity of whey protein concentrate and αs-casein was investigated. The intensity of the protein degradation was analyzed by the degree of hydrolysis, the free amino groups content and RP-HPLC. The ability to bind the antibodies by native proteins and their hydrolysates was determined using a competitive ELISA test. Deep hydrolysis contributed to a significant reduction of immunoreactive epitopes present in WPC. In the case of IgE and IgG present in the serum pool of children with CMA, the lowest binding capacity was detected in the 24 h WPC hydrolysate, where the inhibition of the reaction with native WPC was ≤23 and ≤60 %, respectively. The analysis of the IgG reactivity in the antiserum of the immunized goat showed that the lowest antibody binding capacity was exhibited also by 24 h WPC hydrolysate at a concentration of 1000 μg/ml where the inhibition of the reaction with nWPC was ≤47 %. One-hour hydrolysis of α-casein was sufficient to significant reduction of the protein antigenicity, while the longer time (5 h) of hydrolysis probably lead to the appearance of new epitopes reactive with polyclonal. Food allergies, i.e. adverse reactions to food are an abnormal immune response to a specific food component. The reaction are known to cause many health problems  and α-lactalbumin (15–20 %) are in the majority. Other minor proteins are bovine serum albumin, immunoglobulins and lactoferrin. Due to their outstanding nutritional traits and properties whey proteins are used as food ingredients in functional food products, such as infant formula, yogurt, meat and bakery products.Studies conducted on large population of infants with CMA have shown, that the major milk allergens are BLG and α-S1-casein Wal . BLG—whiReduction or elimination of the major milk allergens by application of effective methods and technologies is essential to patients allergic to milk. However, it is very difficult to entirely remove the protein components from diet, and it is even harder to perform in case of infants who cannot be breast-fed   manufactured from sweet whey and spray dried was provided by Davisco Foods International, Inc. Alpha-s-casein was obtained according to the method of Thompson and Kiddy  or by the addition of 10 % trichloroacetic acid (TCA) (1:1 V/V).The degree of hydrolysis The course of the hydrolysis was monitored by the determination of soluble peptide concentration in 5 % TCA in relation to total protein. The concentration of the trichloroacetic acid-soluble product in the supernatant was measured spectrophotometrically at λ 280 nm  was determined using trinitrobenzene sulfonic acid  according to the method described by Kuchro et al.  Peptide profiles were determined by RP-HPLC with an Agilent 1100 Series system. The peptide preparations were solubilized in the even volume of phase A (0.1 % TFA in H2O) before loading on the chromatographic HPLC column (Zorbax Eclipse XDB-C18 Agilent column (50 × 4.6 mm). Separation was performed at a flow rate of 1 ml/min at 30 °C. Peptide fractions, varying in hydrophobicity, equal from the column in linear gradient of phase B (0.1 % TFA in acetonitrile). Absorbance measurement was made at λ = 230 nm .Determination of protein content Protein content was determined by colorimetric method of Lowry et al.  and WPC-80 hydrolysates was determined using a competitive ELISA test according to the modified method described by Pescuma et al. .450nm ≥0.5) in the reaction with WPC. A pool of 5 sera, with high IgG reactivity (OD450nm ≥0.5) with nWPC, taken from children not exhibiting any clinical symptoms of CMA was used as a control.A pool of sera from 20 children (3–5 years old) with persistent cow’s milk allergy, as defined by a positive result in specific serum IgE was used in the study. Sera were selected from 37 analyzed samples and were characterized by the highest absorbance levels (ODMaxi-Sorp 96-well plates (Nunc) were coated with 100 of 1 μg/ml nWPC solution in 0,1 M carbonate buffer (pH 9.5) per well. The plates were incubated for 2 h at 37 °C and then overnight at 4 °C. In the next step the plates were washed with PBST buffer (PBS pH = 7.4 with 0.05 % Tween 20) and blocked with a 2 % solution of Tween (300 μl per well) for 2 h at 37 °C. Before use, sera were preincubated  with solutions of nWPC proteins or hydrolysates after 1, 3, 5, 24 of hydrolysis .A pool of sera with various protein solutions was mixed at a ratio of 1:1 and incubated for 2 h at room temperature on rotary platform in 48-well plates. After incubation the mixture was loaded in triplicates on plates previously coated with nWPC solution and blocked and incubated for approximately 18 h at 4 °C on a rotary platform. As the secondary antibodies goat anti-human IgE conjugated to HRPO , or rabbit anti-human IgG conjugated to HRPO  were used in amount of 100 μl of conjugate per well. Afterwards the mixture was incubated for 1.5 h at room temperature with gentle rotation. The reaction was developed for 30 min using the TMB Super Sensitive substrate . Readings were performed at 450 nm using a Quantum reader (Biotek).The percentage of antigen-binding inhibition was calculated using the following equation:o−Absx)/Abso] × 100, where Abso was the mean absorbance value of no-antigen triplicates (serum was incubated with PBST at a ratio of 1:1); Absx was the mean absorbance value of triplicates obtained with different antigen concentrations.Inhibition rate (%) = [ were administered every 14 days. Twelve days after the last injection, blood was collected from the cervical vein and the obtained serum was stored at −20 °C until use .The binding capacity of IgE and IgG by native αs-casein (nα-c) and αs-casein hydrolysates was determined using a competitive ELISA.450nm ≥ 0.5) in the reaction with nα-c. A pool of 5 sera, with high IgG reactivity (OD450nm ≥ 0.5) with nα-c was taken from children not exhibiting any clinical symptoms of CMA was used as a control.A pool of sera from 20 children (3–5 years old) with persistent cow’s milk allergy, as defined by a positive result in a specific serum IgE was used in the study. Sera were selected from 37 analyzed samples and were characterized by the highest absorbance levels , followed by a Duncan multiple range test to determine the significant difference between sample at The elimination of all cow’s milk products, without appropriate substitutions, can lead to malnutrition and/or specific nutrient deficiencies at a time when infants and children are growing. Hence there is a need for reduction or elimination these major milk allergens by effective methods and technologies. Proteolysis may be considered an efficient way of removing allergenic epitopes in proteins and increasing their digestibility  particular fractions were almost undetectable with the SDS-PAGE method. 24 h hydrolysis resulted in DH exceeding 21 %. Also Vázquez-Lara et al. (The course of the enzymatic hydrolysis of WPC-80 and αs-casein was monitored by determination of the degree of hydrolysis (DH) (%) Table . The deta et al. , who stua et al.  demonstrAnalysis of the IgE reactivity in the sera of children with cow’s milk allergy Fig. a.1 showeThis observation may suggest that the obtained hydrolysates have the ability to bind similar amounts of antibodies, and thus the degree of degradation did not reduce the amount of reactive epitopes. It should be noted however, that this observation may be due to the competition for binding sites (epitopes) between antibodies of different classes, i.e.: IgE, IgG and IgM present in the analyzed samples.The competition for epitopes may interfere with the reactivity of IgE antibodies, particularly as the concentration of these antibodies in the serum is much lower than the concentration of IgG. At higher hydrolysates concentrations (>50 μg/ml), where the number of potential sites for antibody binding was higher, the lowest IgE-binding capacity was detected in the 24 h WPC hydrolysate, where the inhibition of the reaction with nWPC was ≤23 %. Deep hydrolysis contributed to a reduction of higher amounts of reactive epitopes present in WPC.Lactobacillus strain, at the concentration of 1 mg/ml, considerably reduced (32 %) its recognition by the IgE of allergic children as compared to heat-denatured BLG. Although the degradation of this protein was incomplete, the IgE-binding reaction was still observed.The research by Pescuma et al.  indicateIn the case of IgG present in the serum pool of children with CMA Fig. b.1 a higAnalysis of the reactivity of IgG present in the sera of healthy children Fig. c.1 showeIt has been shown that exposure to cow milk protein during the first 3 months of life results in high levels of IgG subclass antibodies, detected even at 8 years of age  longer hydrolysis did not decrease the binding capacity for anti-WPC antibodies by the resulting peptides. For all hydrolysates it was found that the inhibition of antibody reactivity for nWPC at a similar level (≤30 %). A difference was found for higher concentrations of hydrolysates (≥5 μg/ml), where the progress of hydrolysis reduced the antigenicity of the whey proteins. The lowest antibody binding capacity was exhibited by hydrolysates 5 h and 24 h, at a concentration of 1000 μg/ml the inhibition of the reaction with nWPC was ≤63.4 and ≤47 %, respectively. This experiment confirmed that the hydrolysis procedure caused a partial reduction in reactive epitopes, which lead to a reduction of antigenicity of the WPC proteins.Analysis of the reactivity of IgE antibodies present in the serum pool of children with an allergy to cow’s milk Fig. a.2 showeThe time of hydrolysis caused a decrease in IgE-binding capacity of the resulting peptides compared to control, but the most preferred effect was observed after the 1 and 24 h hydrolyses, but not after the 5-hour hydrolysis . One-hour hydrolysis of α-casein was sufficient to significant reduction of the protein antigenicity, while the longer time (5 h) of hydrolysis probably lead to the appearance of new epitopes reactive with polyclonal anti- αs-casein IgE which were then degraded by further hydrolysis (24 h).To date, the presence of nα-c specific IgG and IgE has been demonstrated in the serum of patients with CMA, as well as IgG antibodies in healthy individuals . These findings suggest that hydrolysates with such traits may have the potential to be administrated in the prophylactic treatment of infants with a high risk of allergy.The results obtained in the present study demonstrate that hydrolysis using a non-commercial serine protease isolated from


The Late Pliocene epoch is a potential analogue for future climate in a warming world. Here we reconstruct Plio-Pleistocene East Antarctic Ice Sheet (EAIS) variability using cosmogenic nuclide exposure ages and model simulations to better understand ice sheet behaviour under such warm conditions. New and previously published exposure ages indicate interior-thickening during the Pliocene. An ice sheet model with mid-Pliocene boundary conditions also results in interior thickening and suggests that both the Wilkes Subglacial and Aurora Basins largely melted, offsetting increased ice volume. Considering contributions from West Antarctica and Greenland, this is consistent with the most recent IPCC AR5 estimate, which indicates that the Pliocene sea level likely did not exceed +20 m on Milankovitch timescales. The inception of colder climate since ∼3 Myr has increased the sea ice cover and inhibited active moisture transport to Antarctica, resulting in reduced ice sheet thickness, at least in coastal areas. The behaviour of Antarctic ice sheets during warm climates of the past is poorly understood. Here, the authors combine cosmogenic nuclide exposure ages and numerical simulations in an effort to assess changes in East Antarctic ice sheet thickness since the Pliocene. Thick Cenozoic glaciomarine sequences are exposed at Mac Robertson Land (MRL) and the Transantarctic Mountains , suggesting that the ice margin was located several hundred kilometres inland of its present location during the Miocene or Pliocene21in situ cosmogenic nuclide exposures ages together with model simulations of Antarctic ice thickness and distribution. We first reconstruct Pliocene EAIS configuration from the cumulative exposure time of bedrock, as reflected by the concentration of in situ produced cosmogenic nuclides  in quartz from ice-free regions. The altitude of each sampling sites in turn provides a minimum estimate of ice sheet thickness at that location. We then compare this result with our model-based reconstruction to make inference on the ice sheet history of currently glaciated areas, such as the Wilkes Subglacial and Aurora Basins, from which geologic data are scarce. We finally use the ratio of two cosmogenic nuclides  to determine exposure history (simple or complex).To better constrain EAIS dynamics during the Pliocene, we present newly acquired and recently compiled 10Be and 26Al data presented here were obtained from material collected at the central and eastern portions of the Sør Rondane Mountains, Droning Maud Land (DML) by the National Institute for Polar Research during JARE 27, 28, 31 and 32 (New 1 and 32 . The Sør1 and 32 . There aPreviously published exposure ages are compiled from five ice-free areas of East Antarctica and grouped on the basis of distance from the ice sheet edge . Area 1 252728292533343810Be and six 26Al ages from the Sør Rondane Mountains. Two samples exhibit a complex exposure history (2510Be dating) obtained from a higher altitude raises the possibility that the ice sheet was ∼600 m above present on at least one occasion before 4 Myr. Exposure histories are predominately complex during the Late Pleistocene, with simple exposure histories predominating for samples with larger exposure ages. The subsequent thinning pattern varies between the upstream and downstream portions of the ice sheet. Upstream ice thickness began decreasing during the Pliocene, declining to ∼200 m above present by ∼2.5±0.5 Myr. Downstream ice thickness declined more rapidly, beginning at ∼1.0±0.1 Myr, and reached the current level at ∼0.4 Myr. These results are consistent with geomorphologic evidence from glacial tills indicating that the ice sheet was 400 m thicker than present before 4 Myr , we interpret these data to indicate that the EAIS at this location was thicker by at least 750 m than present, at least once before 3 Myr, before thinning by ∼350 m by 1 Myr  are overlapping such that we cannot discern any significant age differences. Nonetheless, we acknowledge that these are local sites distributed over a wide spatial area, and there is the possibility that some of these ages reflect local dynamics. Considered individually, however, each of the areas exhibits an overall decrease in thickness towards the present.As illustrated in the above compilation, there is a wide range in reconstructed thickness across each of the sites of Areas 1–3, which indicates between 150 and 800 m increase in thickness relative to present, at least once before ∼4 Myr. The variability in thickness is similar to the differing altitudes of each sampling location. For example, the maximum altitude of the Grove Mountains is ∼200 m above the present height of the EAIS, making this region unable to record any additional increase in thickness. Individual ages appear to range over 1–2 Myr period over the Pliocene, with an average ∼1-Myr age uncertainty. Except for the >4-Myr age, all error bars We utilize the Ice sheet model for Integrated Earth system Studies (IcIES)42For the Pliocene, the model produces an ∼−3-m SLE change in the WAIS volume. The ice sheet interior in East Antarctica is generally thicker, consistent with the individual exposure age sites .Prepared samples were then measured using accelerator mass spectrometry (AMS) at the University of Tokyo11; ref. ) and KNA11; ref. ). The ba0Be ref.  and 0.706Al ref. . The morBecause nuclide production rate scales with atmospheric thickness−1. The enhancement factor and the geothermal heat flux were assumed to be uniform and, respectively, retuned to 3 and 54.6 mW m−2 to simulate the volume of the present-day Antarctic ice sheet. In addition, the basal sliding parameterization was revised to allow submelt sliding following ref. −10 m8 yr−1 N−3), which is eight times larger than that used in ref. The numerical experiments were performed using the Ice sheet model for IcIES, as described in refs 2, 405 versus 280 p.p.m.  as well as differing biome distributions, land sea mask and ice sheet configuration/topography on the basis of the PRISM3 data set446845Two sets of experiments using IcIES were conducted, one for the ‘modern'  and the other for the ‘Pliocene' situation using different climatic conditions and initial conditions, assuming the Antarctica ice sheet responds to equilibria under different climatic conditions. The climatic anomaly difference was obtained and simplified from the MIROC Pliocene experiment44Y.Y., M.Y. and A.A.-O. designed the research; K.M. collected the rock samples; M.Y. and H.M. carried out AMS measurements; M.Y. compiled and recalculated exposure ages. All authors contributed to the interpretation of the data. A.A.-O. and F.S. performed model simulations. S.O., M.Y., Y.Y. and A.A.-O. wrote the paper together with input from all other authors.How to cite this article: Yamane, M. et al. Exposure age and ice-sheet model constraints on Pliocene East Antarctic ice sheet dynamics. Nat. Commun. 6:7016 doi: 10.1038/ncomms8016 (2015).Supplementary Figures 1-16, Supplementary Tables 1-4, Supplementary Notes 1-4 and Supplementary References


N- and C-termini and in the core domain. The phosphorylation sites are relatively less mutated than other residues in p53. To understand why and how p53 phosphorylation sites are rarely mutated in human cancer, using a bioinformatics approaches, we examined the phosphorylation site and its nearby flanking residues, focusing on the consensus phosphorylation motif pattern, amino-acid correlations within the phosphorylation motifs, the propensity of structural disorder of the phosphorylation motifs, and cancer mutations observed within the phosphorylation motifs. Many p53 phosphorylation sites are targets for several kinases. The phosphorylation sites match 17 consensus sequence motifs out of the 29 classified. In addition to proline, which is common in kinase specificity-determining sites, we found high propensity of acidic residues to be adjacent to phosphorylation sites. Analysis of human cancer mutations in the phosphorylation motifs revealed that motifs with adjacent acidic residues generally have fewer mutations, in contrast to phosphorylation sites near proline residues. p53 phosphorylation motifs are mostly disordered. However, human cancer mutations within phosphorylation motifs tend to decrease the disorder propensity. Our results suggest that combination of acidic residues Asp and Glu with phosphorylation sites provide charge redundancy which may safe guard against loss-of-function mutations, and that the natively disordered nature of p53 phosphorylation motifs may help reduce mutational damage. Our results further suggest that engineering acidic amino acids adjacent to potential phosphorylation sites could be a p53 gene therapy strategy.p53 protein has about thirty phosphorylation sites located at the  In adp53 phosphorylation ,7 at almGiven the functions controlled by p53 phosphorylation, it is expected that the specificity and dynamics of p53 phosphorylation are carefully regulated, and mutations would affect p53 phosphorylation pattern and function. Phosphorylated p53 mutants were found to accumulate in tumor tissues ,28. p53 C-terminal homo-oligomerization domain and becomes phosphorylated predominantly at Y99, and also at Y121 and Y240 . It. It46PDDF1B1/p62 .N-terminal transactivation domain (TA), a DNA-binding core domain, a C-terminal tetramerization and a regulatory domain. In addition, p63 and p73 also have a sterile alpha motif domain (SAM) at the end of the C-termini. Most of the N-terminal TA domain and C-terminal regulatory domain are highly flexible and have no well-defined structure. Therefore, p53 can also be classified as a typical intrinsically unstructured protein [The structures of p53/63/p73 contain an  protein ,100, whi protein ,101. The protein ,103.It was suggested that natively disordered proteins are more likely to be associated with cancer . p53 is p53 gene transfer in human glioma correlates with site-specific phosphorylation [Gene therapy to restore p53 function is among the clinical approaches currently under investigation . For exarylation ,104, sugrylation . It is krylation . While siYj=i+1, XiYj=i+2, and XiYj=i+3, which are equivalent to the dipeptide XY, tripeptide X × Y, and tetrepetide X ×× Y motifs, respectively. Then we calculate the propensity of amino acid pair association by normalizing the frequencies of the overall counts of individual amino acid in the p53, p63, or p73 families:XiYj is the propensity of amino acid pair association in XiYj=i+1, XiYj=i+2, and XiYj=i+3 positions, respectively. NXiYj is the total number amino acid pair counts in XiYj=i+1, XiYj=i+2, and XiYj=i+3 positions in all sequences in each family, respectively. The NX and NY are total numbers of amino acid type (X and Y) in all sequences in each p53, p63, or p73 families [XiYj is similar to that used by Vonderviszt and Simon [XiYj/(NXY × PX × PY) to measure the dipeptide propensity . PXiYj in equation 1 changes more smoothly with the variation of NX and NY in the calculation of the dipeptide propensities across evolution [For each sequence in a protein family, we count the two amino acids (X and Y) in the positions of Xfamilies . Thus, and Simon , who usevolution .XiYj by adding PXiYj and PYiXj for non-diagonal elements in the correlation matrix.We calculate the degenerate dipeptide pair correlation propensity DPThus, we do not distinguish between XY and YX and label such amino acid combinations X/Y, and the propensity X/Y is obtained from the combined XY and YX counts.Disorder propensities were calculated using the webserver FoldUnfold . The aveWe use ClustalX 2.0  to alignp53 gene therapy.The existence of almost thirty phosphorylation sites in p53 poses a considerable evolutionary pressure on p53 to selectively bind to specific kinase with the right phosphorylation motif to respond to correct phosphorylation signals. What are the sequence and structural features in p53 phosphorylation motifs? Why and how p53 phosphorylation sites are rarely mutated in human cancer? In our study, we focused on the phosphorylation sites and nearby flanking residues and computationally examined the consensus phosphorylation motif pattern, amino acid correlations within the phosphorylation motifs, the propensity of structural disorder of the phosphorylation motifs, and cancer-related mutations observed within the phosphorylation motifs. Among the many factors potentially contributing to the safeguarding mechanism against mutations in p53, we found two patterns that appear unique and correlated with p53 mutations. p53 proteins have high propensities for acidic amino acids adjacent to potential phosphorylation sites. The negative charge near a phosphorylation site might make a mutation of the phosphorylation site less deleterious, since the motif already has a negative charge and may still have certain electrostatic interactions with other binding partners. The above conclusion is consistent with experimental observations. p53 phosphorylation motifs are mostly disordered. Even though cancer-related mutations largely decrease the disorder propensity of the phosphorylation motifs, the disordered nature of phosphorylation motifs might still help to compensate for mutational effects on p53 phosphorylation. Our computational results suggest that engineered acidic amino acids adjacent to potential phosphorylation sites could be a potential strategy in


Reviewing clinical trial site performance identifies strategies to control outcomes. Performance across 5 geographical regions  was investigated in a study that randomised 322 HIV-infected individuals.Regional performance was compared using descriptive analysis for time to site opening, recruitment, quality of data and laboratory samples. Follow-up consisted of 10 visits (96 weeks), electronic data collection (EDC) within 7 days of a visit and serious adverse events (SAEs) reported within 24 hours of site awareness.Median days to site opening was 250 (188 to 266), ranging from 177 (158 to 200)  to 265 (205 to 270) (Europe). Median days to ethics and regulatory approval was 182 (120 to 241) and 218 (182 to 341) days, respectively. Within regions, time to approval ranged from 187 (91 to 205) days  to 276 (175 to 384) days (Europe). Time to first randomisation ranged from 282 (250 to 313) days  to 426 (420 to 433) days (North America).Recruitment was lower than forecasted in Asia, Australia, Europe and North America at 89%, 77%, 91% and 43%, respectively. The converse was true in Latin America where despite ethics, regulatory and contractual delays, recruitment was 104% of predicted.Median days to EDC was 7 (3 to 16), ranging from 3 (1 to 16) (Asia) to 13 (8 to 14) days (North America). Median days for initial SAE submission to sponsor was 6 (2 to 20), ranging from 4 (2 to 18) (Latin America) to 24 (5 to 46) days . Sites took longer to submit final reports, overall median of 28 (7 to 91) days, ranging from 7 days  to 67 (23 to 103) days (Europe).Population availability and time to ethics and regulatory approvals influence recruitment; therefore accurate feasibility assessments are critical to site selection. Time to ethics and regulatory approval may not limit site inclusion if compensated by rapid recruitment. Identifying potential delays and methods for reduction can decrease time and costs for sponsors.NCT00335322. Date of registration: 8 June 2006Clinical Trials.Gov identifier:  Poor site selection in multicentre randomised clinical trials can result in delayed start-up, unmet target recruitment, poor data quality and/or research integrity, thereby contributing to cost inefficiencies in resource and time allocation. Therefore, tracking a site’s operational performance from a sponsor perspective can identify ways to improve processes for future studies and to enable performance comparison between sites.Identifying where processes can be improvedIdentifying where resource allocations can changeManaging the workload effectively across staffEstablishing performance benchmarks, andProviding data-driven rationale to management to request additional resources.Use of performance metrics is two-fold: to improve processes internally (for continuous improvement efforts and future research), and to strengthen relationships with sponsors. Benefits for internal operations include:Identifying areas of strong competitive advantage, andAbility to complete site feasibility questionnaires with accurate data .Benefits from the sponsor relationship perspective include:Leading indicators - information that can be acted on immediately to get the trial back on trackLagging indicators - information that can be used for future trials or for process improvement effortsCycle-time - measures the time taken to complete a taskTimeliness - measures whether a particular milestone was metEfficiency - measures the amount of resource required to complete a task or set of tasks versus that expected, andQuality - measures how well an output from a process meets the requirements of the customer of that process .Metrics can enable comparison of operational performance between sites -5 and thMetric #1: Cycle Time from Draft Budget Received from Sponsor to Budget Finalisation: the time (days) between the date that the first draft budget is received and the date that the sponsor sends approval of the budget. Long cycle times can signal the site to identify areas where the process is being delayed and ways to improve the process. Sites that have short cycle times for this metric can use the information to demonstrate their responsiveness to sponsors [sponsors Metric #2: Cycle Time from Institutional Review Board (IRB) Submission to IRB Approval: the time (days) between the dates of initial submission to the ethics committee and the protocol approval. If the site is experiencing delays in receiving ethics approval, process improvement should be investigated between the site and ethics committee. If the site approves new trials in a timely fashion, sponsors are more likely to provide repeat business. Ethics approval is one of the first milestones in the life cycle of a clinical trial and the variability between sites at this step is beneficial; therefore, a good track record for this metric is the site’s advantage when promoting abilities [bilities Metric #3: Cycle Time from Full Contract Execution to Open to Enrolment: the time (days) between the date of an executed contract and the date subjects may be enrolled. Recruitment is a significant challenge across the industry, and as with other metrics, long cycle times could indicate that the site should try to identify areas where the process is being delayed. The metric should be tracked over time to verify if any changes have had a positive impact. If the site’s performance for this metric is good, it should be used to leverage during budget negotiation. Sites have more time to enrol subjects if they are able to get protocols open to recruitment faster. Sites with a history of good performance for this metric will be selected first for future trials [e trials Metric #4: Volumetric - Number of Active Protocols: the number of protocols that are not in long-term follow-up with one of the following statuses: Open to Recruitment, Closed to Recruitment, or Suspended within a given month. This metric determines the site’s clinical research operations capacity and efficiency [ficiency Metric #5: Volumetric - Number of New Subject Recruitments: the number of subjects recruited during a given month. This metric measures the site’s ability to deliver on recruitment targets. Sites with a history of successfully meeting recruitment targets are selected first for new trials [w trials .Examples of site performance metrics include:Recruitment into multicentre, randomised clinical trials is often slower or more difficult than initially expected, with many trials failing to reach their planned sample size within the originally envisaged timeframe and funding ,12. ConsDuring site selection, sponsors request feasibility information on how a site performed on similar studies; that is, number of enrolled subjects versus what was contracted. According to the Tufts Centre for the Study of Drug Development, two thirds of sites do not meet patient enrolment requirements of a given study . This meTemporal - number of days to obtain review approvals to the first subject visitQuantity - number of subjects a site can enrolQuality - number of queries per case report form (CRF) .There is limited published international, multicentre, randomised clinical trial data in this area. One study outlined the use of benchmarking clinical trials conducted in Europe , evaluatThe study concluded that benchmarking provided an effective tool for improving data quality, reducing time to market, and declining development costs of international, multicentre clinical trials. Furthermore, a matrix of performance measures was established to evaluate site compliance for the South African National Defence Force-sponsored Project Phidisa . The priOperational data can be tracked within a clinical trial management system (CTMS) or on a spreadsheet; however the challenge lies in analysing and interpreting the data in a way that leads to beneficial change. Therefore, careful consideration is required when setting up a study to ensure all required operational metrics are defined, and can be captured appropriately.The aim of the current exploratory analysis is to describe regional performance in the Altair study using operational metrics relating to start-up, recruitment, data collection, and stored samples, to plan efficient implementation of future studies.The Altair study, sponsored by The Kirby Institute, University of New South Wales, Australia, has been described previously . BrieflyThirty-six sites in 15 countries  participated in this study. The sites represented 5 regions including Asia (n = 5 sites), Australia (n = 10), Europe (including Israel) (n = 7), Latin America (n = 11), and North America (n = 3). Ethical approval was received at all sites, although local regulatory authority approval was not required in Australia, North America and Israel. Moreover, public hospitals in Australia, Ireland and United Kingdom underwent governance evaluation of the protocol. Prior to study conduct, written informed consent was obtained from each participant. A list of ethics committees that approved the Altair study is provided in TDF/FTC was supplied as the fixed-dose combination (Truvada) to all sites after packaging and labelling of Gilead clinical trial supply by ALMAC Clinical Services, Craigavon, Northern Ireland. The drug was not registered or commercialised at study initiation in Chile, Hong Kong, Malaysia, Singapore, Taiwan and Thailand.There were 10 study visits during 96 weeks of follow-up. The Oracle Electronic Data Capture (EDC) platform  was used for data collection. Data entry was required within 7 days of the study visit and serious adverse events (SAEs) reported to the sponsor within 24 hours of site awareness.Randomised Altair study patients provided informed consent before permitting storage of plasma and buffy coat samples at 10 time points. At week 0 or baseline, a buffy coat was collected for DNA isolation with the aim of conducting future investigations related to HIV disease. Furthermore, storage of samples for future genomic testing occurred depending on patient consent. Samples were shipped to a central facility  once the final visit (week 96) was concluded. Paper inventories were requested at week 48 and prior to shipping at week 96. The number and quality of samples were interrogated upon receipt in Sydney and were compared by region.Time from protocol release to ethics and/or regulatory submission and approvalTime from protocol release to First Participant Randomisation (FPR) and Last Participant Randomisation (LPR)Start-up:Time from site opening to FPRTime from FPR to LPRActual versus estimated recruitmentRecruitment:Time from actual visit to EDC initiationTime from EDC initiation to completionNumber of missing values per participantNumber of queries opened per participantNumber of missed visits per regionNumber of SAEs reportedTime from SAE occurrence to initial reportTime from initial SAE report to final reportData collection:Number of plasma samples collected versus protocol-mandated samples to be collectedNumber of buffy coat samples collected versus protocol-mandated samples to be collectedQuality of laboratory samples collectedStorage samples:A descriptive analysis of the operational metrics listed below evaluates regional performance across Asia, Australia, Europe, Latin America, and North America:Median days from protocol release to site opening was 250 188 to 266), ranging from 177 (158 to 200)  to 265 (205 to 270) (Europe). Figure 88 to 266Figure Figure Table There were a total of 50 missed visits during the course of the study. The median number of missed values per participant was highest in Europe and lowest in Latin America. The remaining three regions had a similar numbers of missed values per participant.The median number of queries raised per participant was lowest in North America; Asia followed closely behind and the remaining regions reported similar values.There were no SAEs reported in North America Table . Asia, AOne unreported SAE was identified in Europe during a site monitoring visit conducted by the project team.The expected number of buffy coat and plasma samples for collection during the study was 30,800. Overall, 82% of protocol-mandated plasma samples were received at the central laboratory; ranging from 79% (Asia) to 99% (North America) , Latin America and North America. Document translation contributed to the submission delays in these regions ,19 and tStart-up fees for the site, pharmacy and laboratoryArchiving and close outLead site fees for additional administrationEthics and governance review fees for the main study and substudiesPharmacy fees for dispensing, destruction, call back, drug transfersInstitutional overheadA limitation of this analysis is insufficient data capturing detailed reasons for delayed ethics and governance submission and subsequent approvals. Delayed ethics submission could be due to late receipt of translated documents from the sponsor, prolonged review of site-specific documents by the sponsor, ability of the site to prepare a high-quality submission package, availability of department heads to obtain signatures on documents, and the study coordinator’s current workload . In addition to the difficulty posed when establishing site contracts that require translation, delayed governance submissions could also be due to prolonged budget negotiations between the site and the sponsor, and can indicate that the proposed budget is insufficient for a site to cover costs. Therefore, during feasibility and site selection, it would be useful to obtain a site’s standard setup costs including:Sites that are yet to develop a schedule of fees for clinical trial services should consider the above factors and conduct a time and motion study for participant visit-related procedures; for example, time taken to conduct informed consent, assess eligibility criteria, medical history and conduct physical examination by the principal investigator (PI).Recruitment was lower than pre-study estimations by sites in Asia, Australia, Europe, and North America due to a lower naïve patient population in the latter three regions and a competing naïve study in Australia. Despite multiple delays during start-up, recruitment in Latin America exceeded the estimated total within a 3-month period.This metric demonstrates the importance of accurate data when sites are completing feasibility questionnaires. In order to estimate the recruitment target and the intended recruitment period, sites should be asked to provide the total number of active protocols, number of active protocols per study coordinator (SC), number of new subject recruitment and screen failures during a given month, number of previous similar studies and respective recruitment, and number of competing studies.Knowing how much time SCs spend on various study-related tasks is extremely useful and beneficial from both site and sponsor perspectives. It demonstrates where there may be inefficiencies and room for process improvement, allows improved planning, and enables accurate budgeting for the time and effort required to conduct a study. Documenting work effort requires staff to log their time, whether in a spreadsheet, a CTMS, or other system. With complete and up-to-date logs filled out, sites can view a detailed report of staff time across activities and protocols. Therefore, a site should invest in calculating an average full-time equivalent allocation to ensure staff is neither overburdened nor under-utilised.Other factors that may impact recruitment at a site may include the process of identifying suitable participants  and methods of effective advertising.Following this analysis, the Kirby Institute implemented a more rigorous feasibility questionnaire to be completed during site selection for future studies.Only ten participants were randomised in North America although initial data entry following the visit was delayed by a week. In contrast, sites in Asia and Latin America that randomised over 100 participants began data entry within a week after the visit. The number of missing values per participant varied largely from 15 in Latin America to 53 in Europe; however, the number of queries opened per participant remained fairly consistent across regions. The rate of missed visits was very low across all regions.The majority of the reported SAEs (83%) were hospitalisations, hence extending the reporting timelines between the initial and final reports. There were no SAEs reported from the North American region and most (63%) were from Latin America and Asia, corresponding to the proportion of global recruitment. A total of 63% of the SAEs reported from Thailand were hospitalisations reflecting the current environment for receiving medical treatment covered by health insurance. Strict confidentiality and privacy laws surrounding access to medical records from local hospitals also influenced timelines for completing follow-up SAE reports from this region.Prolonged data entry, a high number of missing values and missed visits indicate that site staff may require additional protocol and data entry training. Moreover, the SC’s current workload may also be represented within these measures; that is if the workload is high, there is less time to focus on data quality.The Kirby Institute implemented weekly remote monitoring of data entry for all future studies to ensure improved data quality and integrity.Clinical trials of immunologic therapies provide opportunities to study the cellular and molecular effects of those therapies and may permit identification of biomarkers of response. When the trials are performed at multiple centres, transport and storage of clinical specimens become important variables that may affect blood and tissue specimens ,21. Thercollection of buffy coat samples and under-collection of plasma samples identified a need for additional laboratory training during the lifetime of the study; however, as paper inventories were utilised to document sample collection and shipments to the central laboratory occurred at study closure, the sponsor was not alerted to this problem in real-time. Furthermore, the extra buffy coat samples resulted in additional shipping costs borne by the sponsor, and the smaller quantity of plasma ensured fewer samples to conduct future investigations in HIV disease.Over-The outcome from this analysis proved a need to improve stored sample processes for future studies using several strategies such as improving communication with local laboratory staff, increasing the number and quality of laboratory monitoring visits and greater frequency of shipping to the central laboratory (even if incurring more expense). As a result of the Altair study sample shipments, The Kirby Institute studies now employ an electronic inventory to allow real-time monitoring of the type and number of samples collected by sites. This has allowed early identification and resolution of errors prior to shipment.The proposed strategy for comparing performance of service providers in clinical trials was intended to help interpret differences due to factors affecting start-up, recruitment, data and storage sample collection across regions. It was hoped to provide metrics to establish compliance for future studies in order to meet required timelines and improve cost-efficiency.Multicentre randomised clinical trials that have been conducted since the Altair study have focused attention on site selection strategies tailored specifically to address the target population required for the protocol, as well as attention to recruitment, data and stored sample collection. The site selection process itself is dependent on multiple factors in addition to the quantity of suitable and available participants at the site and PIs are encouraged to be realistic in their recruitment forecasts. In an attempt to reduce cost and time inefficiency, some studies implemented a cut-off on recruitment estimated by sites and others targeted sites that have a good track record in conducting large, randomised clinical trials with known PIs. The Kirby studies also commenced tracking of recruitment more closely, and maintained frequent communication with site PIs and SCs to identify ways to improve recruitment. Remote monitoring of data collected occurred weekly to ensure data entry was timely, missing values were not a recurring problem, and any missed visits were followed up with the site to confirm the reason. Stored samples are being tracked via an electronic inventory to ensure the correct number and type of samples are being collected.It was apparent with the Altair study that certain regions are more worthwhile than others in terms of cost efficiency and timeliness of recruitment. For example, sites in Asia and Latin America took longer than sites in Australia to commence enrolment, however participant recruitment and data quality in these regions compensated for the initial delays. Moreover, sites in Asia excelled in data quality and storage sample collection relative to other regions.