Stoichoimetric graphene fluoride monolayers are obtained in a single step by the liquid-phase exfoliation of graphite fluoride with sulfolane. Comparative quantum-mechanical calculations reveal that graphene fluoride is the most thermodynamically stable of five studied hypothetical graphene derivatives; graphane, graphene fluoride, bromide, chloride, and iodide. The graphene fluoride is transformed into graphene via graphene iodide, a spontaneously decomposing intermediate. The calculated bandgaps of graphene halides vary from zero for graphene bromide to 3.1 eV for graphene fluoride. It is possible to design the electronic properties of such two-dimensional crystals. Comparative theoretical calculations on a series of hypothetical graphene derivatives  suggest that the thermodynamic stability of the fluorinated layers is significantly greater than that of the other derivatives, making their neat isolation from the pristine layered solid by solvent extraction a realistic goal. Theoretical calculations also demonstrate that electronic properties of graphene derivatives, such as the bandgap, can be tuned by varying their substituents. The differences in thermodynamic stability between the graphene halides can be exploited: CF can be converted via halide exchange to graphene iodide (CI), which is unstable and undergoes spontaneous decomposition to afford carbon nanostructures (graphene and nanodiamond).1F1, Aldrich) was suspended in 50 mL sulfolane  and the mixture was sonicated in a 135 W sonicator bath for 1 h at 50 °C. The resulting suspension was left to rest for two weeks at 80 °C in order to allow any insoluble particles to settle out. Then, the dilute, colloidally dispersed supernatant was carefully collected for microscopic analysis. Note that sulfolane is normally a solid under ambient conditions (mp: 28 °C). Therefore, in order to maintain its fluidity throughout our experimental process, it was necessary to keep the working temperature >40 °C.In a typical procedure, 250 mg of purchased graphite fluoride (CKI (2 g) was completely dissolved in 50 mL of colloidal-sulfolane dispersion and the solution was heated under stirring at 240 °C for 2 h. The resulting precipitate was centrifuged and washed several times with water (4 × 50 mL) and ethanol (2 × 50 mL), each wash being followed by further centrifugation.α radiation) equipped with an X´Celerator detector. Samples were spread on zero-background Si slides and scanned in the 2θ range of 10–100° in steps of 0.017° for 720 s per step. IR spectra were acquired using a Fourier-transform infrared (FTIR) spectrometer (Bruker Equinox 55/S) and KBr pellets. Raman spectra were obtained using an inVia Reflex microRaman spectrometer (Renishaw) with a 100× objective lens and a crystal laser excitation of 514.5 nm operating at 0.1 mW.TEM and HRTEM were carried out using JEOL JEM2010 and 3010 microscopes operating at 200 and 300 kV, respectively. The instruments were each equipped with a LaB6 cathode, an energy dispersive X-ray (EDX) detector. A very dilute drop of the colloidal dispersion was placed on a carbon-coated copper grid and allowed to dry by evaporation at ambient temperature. AFM images were obtained using an NTEGRA Aura (NT-MDT) microscope. A sample of the diluted dispersion was placed on synthetic mica as an atomically smooth support and evaporated at room temperature. The measurements were performed in air at room temperature in noncontact mode, with Si tips of the 1650–00 type at resonance frequencies ranging from 180 to 240 kHz. X-ray powder diffraction (XRD) experiments were performed with a PANalytical X´Pert PRO instrument   and on related halogenated systems . The first model (henceforth denoted model A) consists of perhydrocoronene and its halogenated analogues, i.e., C, Br see . The secPBC) see . All calBecause of the unusually high polarity of sulfolane, which exceeds that of dimethylformamide (DMF) or dimethyl sulfoxide (DMSO), it is capable of dissolving a wide range of compounds, including hydrocarbons. TEM images reveal that the sheets obtained by single-step exfoliation of graphite fluoride with sulfolane are extraordinarily transparent, with lateral dimensions ranging between 200 nm and 2 μm see . Some of2  is calculated from the total energies of the reactants and products according to Ab initio theoretical calculations were used to elucidate the thermodynamical stability and structural features of graphene derivatives. The C–C bond lengths predicted on the basis of the two chosen models are similar for both CH and for CF, but they are ≈0.03 Å greater than those reported in the literature  and maximal direct gaps (Egmax). CH, CF, and CCl are direct bandgap materials, while CBr is an indirect bandgap material. The bottom of the conduction band and the top of the valence band are located at the Γ point in the first Brillouin zone for CH, CF, and CCl  and G (1585 cm−1) bands of carbon materials. This spectrum is dramatically different from that observed for the pristine fluorinated graphite.3 carbons. Hence, elimination of the halide substituents and the subsequent condensation of adjacent layers might provide a pathway for the growth of a diamondlike phase. Clearly, the ease with which this elimination can occur will be dependent on the halide, e.g., iodine might be expected to be more labile than fluorine. Thus, the graphene fluoride sheets can be simply transformed to metastable graphene iodide, which quickly disproportionates to carbon and iodine −1 disappears and a new peak corresponding to the C–I unit (745 cm−1) is observed (see To confirm this reaction mechanism, we reacted CF with KI using DMF as the solvent; under these conditions, the reaction occurs at just 150 °C. The IR spectrum of the reaction mixture was monitored; over the course of the reaction, the peak corresponding to the C–F stretch at 1220 cmThis study provides the first experimental and theoretical evidences for the existence of thermodynamically stable, stoichiometric graphene derivative: graphene fluoride. The bandgap width of this monolayered graphene derivative have been compared with those calculated for graphane and other graphene halides. The halide-exchange process can be exploited for the transformation of graphene fluoride to graphene, with nanodiamonds being formed as a minor by-product.Note added at proof: During the review process other papers dealing with various “graphene fluoride” systems were published.


The alpha-isoform of calcium/calmodulin-dependent kinase II (αCaMKII) is a major synaptic kinase that undergoes autophosphorylation after NMDA receptor activation, switching the kinase into a calcium-independent activity state. This αCaMKII autophosphorylation is essential for NMDA receptor-dependent long-term potentiation (LTP), induced by a single tetanus, in hippocampal area CA1 and in neocortex. Furthermore, the αCaMKII autophosphorylation is essential for contextual long-term memory (LTM) formation after a single training trial but not after a massed training session. Here, we show that in the absence of αCaMKII autophosphorylation contextual fear conditioning is hippocampus dependent and that multi-tetanus-dependent late-LTP cannot be induced in hippocampal area CA1. Furthermore, we show that in the absence of αCaMKII autophosphorylation contextual LTM persists for 30 days, the latest time point tested. Additionally, contextual, but not cued, LTM formation in the absence of αCaMKII autophosphorylation appears to be impaired in 18 month-old mice. Taken together, our findings suggest that αCaMKII autophosphorylation-independent plasticity in the hippocampus is sufficient for contextual LTM formation and that αCaMKII autophosphorylation may be important for delaying age-related impairments in hippocampal memory formation. Furthermore, they propose that NMDA receptor-dependent LTP in hippocampal area CA1 is essential for contextual LTM formation after a single trial but not after massed training. Finally, our results challenge the proposal that NMDA receptor-dependent LTP in neocortex is required for remote contextual LTM. T286A) mice . Theref) Figure . Twenty-T286A mutant mice to investigate several mechanistic questions of contextual LTM formation. Firstly, we have shown that like contextual LTM formation, contextual STM formation is impaired in the absence of αCaMKII autophosphorylation after 3, but not 5, conditioning trials. In principle, this result is consistent with the idea that STM may be a prerequisite for LTM formation. However, in earlier studies we found that αCaMKII autophosphorylation is required for the induction of immediate-early gene (IEG) expression by contextual fear conditioning [[Here, we have used the αCaMKIItioning [; Radwanstioning , is indtioning [. In thisT286A mutants. This is the opposite to contextual LTM formation in CREBαΔ-/- mutants that is enabled by spaced, but not massed, training [T286A mutants shows that spaced training is not always more beneficial than massed training for LTM formation. It suggests that massed training may be more beneficial for LTM formation when synaptic signaling is impaired, whereas spaced training may be more beneficial for LTM formation when transcription in the nucleus is affected.We found that massed, but not spaced, training enables contextual LTM formation in αCaMKIItraining . It is nT286A mutants is hippocampus dependent and that multi-tetanus-dependent L-LTP cannot be induced in hippocampal area CA1 in these mice. Together, with previous hippocampal slice recordings [We have shown that contextual fear conditioning in the αCaMKIIcordings  this strcordings . Howevercordings . Consistcordings . Thus, wT286A mutants, the finding of normal remote contextual LTM in these animals suggests that NMDA receptor-dependent LTP in neocortex is not required for the reorganization of contextual LTM. This suggestion is in contrast with an earlier suggestion from a study with heterozygous αCaMKII null mutants that have impaired NMDA receptor-dependent LTP in neocortex [T286A mutants have normal neurogenesis in adult dentate gyrus [T286A mutants. For example, αCaMKII has a structural function that is independent from the T286 autophosphorylation [T286A mutants, have impaired remote contextual LTM.We also found that αCaMKII autophosphorylation is not required for remote contextual LTM after massed training. As NMDA receptor-dependent LTP in the neocortex is blocked in the αCaMKIIeocortex . Howevereocortex , which heocortex . Consistte gyrus  and normT286A mutants are impaired in contextual, but not cued, LTM formation. However, more systematic aging studies are required to further establish whether aged αCaMKIIT286A mutants have impaired contextual LTM formation. Nonetheless, our preliminary findings suggest that αCaMKII autophosphorylation-dependent plasticity in the hippocampus is important to delay age-dependent memory impairments. This idea may be of relevance for understanding Alzheimer's disease that has been suggested to be associated with impaired αCaMKII autophosphorylation [Finally, we provide some evidence that 18 month-old αCaMKIIrylation -31.One of the main conclusions from our studies is that contextual fear conditioning in the absence of αCaMKII autophosphorylation is hippocampus-dependent. Thus, hippocampal plasticities that do not require αCaMKII autophosphorylation appear to be sufficient for contextual LTM formation. Further, NMDA receptor-dependent LTP in hippocampal area CA1 that requires αCaMKII autophosphorylation seems to have a specific role in contextual LTM formation: it is needed for one-trial LTM formation but not for LTM formation after a session of massed training trials. Additionally, our findings suggest that NMDA receptor dependent LTP in neocortex is not required for reorganization of contextual LTM as previously proposed , as remoad libitum. Male and female homozygous αCaMKIIT286A mutants and control wild-type (WT) littermates were obtained in the 129B6F4-6 background by intercrossing heterozygous mutants. Genotyping was carried out by PCR analysis, as described previously [The subjects were housed in groups of two to five and maintained on a 12 h light/dark cycle with food and water For all of the experiments background contextual fear conditioning was used. Background contextual fear conditioning involves the hippocampus more strongly than conditioning without a tone presentation . BackgroOn the conditioning day, the mice were brought from the housing room into a holding room where they were allowed to acclimatise for 30 min before training. During training, the mice were placed individually in the chamber and after a 120 s introductory period a tone  was presented for 30 s, the last 2 s of which coincided with a foot shock (0.7 mA). Depending on the intensity of the training protocol the mice received a further 2 or 4 tone-shock pairings at 60 s intervals, and for spaced training they received two training trials with a single tone-shock pairing and an inter-trial interval of 1 h. After training mice were returned to their home cage.In order to test for short-term contextual memory mice were re-exposed to the conditioning chamber for a period of 5 min 2 h after training. Long-term contextual fear memory was assessed by re-exposing the mice to the conditioning chamber for 5 min 24 h after training. Long-term cued fear memory was assessed by placing each mouse in a novel chamber 48 h after training. Following 180 s without a tone (Pre-CS) the tone was presented for 180 s (CS) to assess cued fear memory. In order to test for the stability of the contextual and cued fear memories the mice were re-tested 30 and 31 days respectively after training.The behaviour of the mice was recorded and freezing behaviour  was scored for 2 s in every 5 s.T286A mutants and their WT littermates 24 h after training. The mice were anaesthetized with a mixture of isoflourane  and O2 and then mounted in a Kopf stereotaxic frame. The scalp was shaved and surgically cleaned and then a midline incision was made that exposed the skull. The skull overlying the target area was removed and bilateral injections of ibotenic acid (Sigma) dissolved in phosphate buffer saline (PBS) at a concentration of 10 mg/ml  and were perfused with physiological saline and 4% paraformaldehyde in PBS. The brains were removed and stored in 4% paraformaldehyde until they were coronally sectioned at 40 μm and stained with cresyl violet.3, 1.24 mM NaH2PO4, 2.5 mM CaCl2, 1.3 mM MgSO4, 10 mM D-glucose, bubbled with a mixture of 95% O2 and 5% CO2. Mice were anesthetized and decapitated. The hippocampus was dissected and cut in 450 μm-thick slices with a tissue chopper. The slices were transferred into the recording chamber and kept in interface at 28°C for 1.5 h. The perfusion rate of ACSF was 1 ml/min. Bipolar twisted nickel-chrome electrodes (50 μm each) were used to stimulate Schaffer's collaterals.Hippocampal slices were perfused with artificial cerebrospinal fluid (ACSF) with the following composition: 124 mM NaCl, 5 mM KCl, 26 mM NaHCOExtracellular field excitatory postsynaptic potentials (fEPSP) were recorded in the stratum radiatum of the CA1 region with low resistance (2-5 megaOhm) glass microelectrodes filled with ACSF. Test stimuli were biphasic (0.08 ms for each pulse) constant-voltage pulses delivered every minute with an intensity adjusted to evoke an approximate 40% maximal response. The slope of the fEPSP was measured on the average of four consecutive responses. For each slice, an input-output curve was established from the responses obtained for various stimuli. LTP was induced by applying four trains . In both protocols, the potentiated response was recorded for 4 h.For each slice, the fEPSP slopes were normalized with respect to the mean slope of the fEPSPs recorded during the 30-minute period, preceding induction of LTP. To determine whether or not the normalized fEPSP of a group of slices submitted to the same experimental conditions was significantly potentiated (p < 0.05), the percentages of baseline measured after induction of LTP were compared by a two-way ANOVA and several student t tests at different times after LTP induction.Normally distributed data were analyzed by analysis of variance (ANOVA) followed by Student-Newman-Keuls post-hoc analysis. Not normally distributed data were analyzed by ANOVA on ranks.αCaMKII: alpha-isoform of the calcium/calmodulin-dependent kinase II; LTM: long-term memory; LTP: long-term potentiation; STM: short-term memory;The authors declare that they have no competing interests.T286A mutants. EEI performed the behavioral studies with the mice. AD and LR performed the electrophysiological recordings. EEI, KR, EG, LR and KPG designed the experiments and they wrote the paper. All authors have read and approved the final version of this manuscript.EEI, CN and WL bred and genotyped the αCaMKII


Human papilloma virus (HPV)-16 is the most prevalent high-risk mucosal genotype. Virus-like-particle (VLP)-based immunogens developed recently have proven to be successful as prophylactic HPV vaccines, but are still too expensive for developing countries. Although vaccinia viruses expressing the HPV-16 L1 protein (HPV-L1) have been studied, fowlpox-based recombinants represent efficient and safer vectors for immunocompromised hosts due to their ability to elicit a complete immune response and their natural host-range restriction to avian species.L1) was engineered and evaluated for the correct expression of HPV-L1 in vitro, using RT-PCR, immunoprecipitation, Western blotting, electron microscopy, immunofluorescence, and real-time PCR assays.A new fowlpox virus recombinant encoding HPV-L1 (FPL1 recombinant correctly expresses HPV-L1 in mammalian cells, which are non-permissive for the replication of this vector.The FPL1 recombinant represents an appropriate immunogen for expression of HPV-L1 in human cells. The final aim is to develop a safe, immunogenic, and less expensive prophylactic vaccine against HPV.This FP Cervical cancer caused by human papilloma virus (HPV) is the second leading cause of malignancies in women worldwide, and the first in developing countries. Persistent infection with one of the approximately fifteen high-risk oncogenic HPV types can cause the onset of cervical intraepithelial neoplasia and progression to invasive cervical cancer . ApproxiThe absence of specific antiviral drugs has stimulated the search for prophylactic vaccines against HPV-16 and HPV-18, which are the HPV genotypes that are most commonly associated with the disease. In particular, HPV-16 is by far the most prevalent high-risk mucosal genotype, and, as it is present in around 50% of all cervical cancers ,5, it haAlthough therapeutic vaccines are of high priority for the control of progression to neoplasia in subjects who are already infected, prophylactic vaccines are important to limit the diffusion of infection. They are therefore the best choice for intervention against HPVs, as they can neutralise the incoming virus and prevent disease progression.The preparation of a new vaccine has been hampered for a long time because of the restricted host range of the virus and its selective tropism for differentiated squamous epithelium, which makes its growth in cell culture difficult. Expression of the L1 major capsid protein of HPV-16 (HPV-L1) in eucaryotic cells results in the formation of empty virus capsids   and quadImmunisation with vaccinia virus recombinants expressing HPV genes or other viral antigens has been demonstrated to be a good candidate for stimulation of the immune system . HoweverThe avipox viruses, and the fowlpox virus (FP) in particular, represent alternative vectors due to their natural host-range restriction to avian species ,25, to tL1) was engineered and evaluated for its correct expression in vitro, with the final aim of developing a prophylactic vaccine. This recombinant should express foreign genes intracellularly and allow cross-presentation of the antigen in association with both MHC class I and II complexes, thus eliciting a complete humoral and cellular protective immunity.In the present study, a new FP recombinant encoding HPV-L1  cells, and MRC-5 human lung fibroblasts were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated calf serum , 100 U/ml penicillin and 100 μg/ml streptomycin (P/S). Specific-pathogen-free primary chick embryo fibroblasts (CEFs) were grown in DMEM with 5% heat-inactivated calf serum, 5% tryptose phosphate broth , and P/S.2HPO4, 6 M guanidine-HCl, pH 8). After clarification for 30 min at 17,000 × g at 4°C, the supernatant containing the HPV-L1 preparation was supplemented with 1% Triton X-100/20 mM imidazole, pH 8, in buffer A. This was then incubated with Ni-NTA agarose resin (Qiagen) for 30 min at room temperature. After washing once with 1% Triton X-100 in buffer A, twice with buffer A, and multiple times with buffer C  up to a final optical density of 0.013, the protein was eluted into different fractions with 1 M imidazole, pH 8. After their analysis by 15% PAGE, the fractions enriched in the recombinant HPV-L1 were pooled. The HPV-L1 was dialysed overnight at 4°C using a slide-A-lyser cassette , and soaked in dialysis buffer . The protein was quantified, stored at -80°C and used as a control in the Western blotting.The pQE30 expression plasmid  engineered to contain the L1 gene of HPV-16 -labelled HPV-L1 probe, and then subjected to multiple cycles of plaque purification. One clone was selected for correct and highest expression of the HPV-L1 gene by Western blotting. The FPL1 recombinant was amplified in CEFs and purified on a sucrose gradient, as described previously -methionine and [35S]-cysteine were added, using the same medium supplemented with 2% dialysed foetal bovine serum (Gibco). Sixteen hours p.i., the cells in 2 ml of medium were harvested by resuspending them in 1 ml lysis buffer  and 0.6 TIU aprotinin  per Petri dish, with scraping into microcentrifuge tubes . The lysate was clarified by centrifugation at 9,000 × g for 20 min at 4°C. Immunoprecipitation was performed either with 2 μl anti-HPV-L1 preadsorbed polyclonal mouse serum (C. Giorgi) or with a monoclonal antibody . Proteins were resolved using 12.5% SDS-PAGE, and fluorographed.The cells were infected with 5 PFU/ml in DME methionine-, cystine-, and L-glutamine-free medium , boiled for 5 min, and loaded onto polyacrylamide gels. After running through a 4% stacking gel for 1 h at 20 mA and through a 15% running gel for 1.5 h at 40 mA, the proteins were transferred onto 0.2 μm nitrocellulose membranes  for 1 h at 100 V in cold transfer buffer  using a transblot apparatus (Bio-Rad). Soon after the protein transfer to the nitrocellulose, the positions of protein markers  (5 μl/gel) were marked on the nitrocellulose. The nitrocellulose membranes were incubated for 30 min in 0.5% glutaraldehyde , washed in H2O, and incubated for 1 h with 5% skimmed milk  in Ca2+-free and Mg2+-free phosphate-buffered saline (PBS-). After three rinses in wash buffer (20% Tween in PBS-), the nitrocellulose membrane was incubated overnight at 4°C with a 1:5,000 dilution of the specific anti-HPV-L1 CamVir-1 antibody (BD Biosciences). The washing was followed by a 1-h incubation with a goat anti-mouse horseradish peroxidase antibody , and 2-h washes, before the HPV-L1 was revealed using the ECL system . The densitometric analysis of the Western blotting bands of the L1 protein was performed using the ImageJ software  Ct, sample = Ct,L1 - Ct, RPS7, [delta] Ct, sample is the Ct value for any sample normalised to the RPS7 endogenous housekeeping transcripts and [delta] [delta]Ct = [delta]Ct, sample - [delta]Ct, wt. In all of the samples, the 2-[delta] [delta]Ct refers to an N-fold increase of HPV-L1/GFP copy number relative to the control.RNA was extracted from Vero cells infected with 2 PFU/cell FPL1 recombinant was tested over time by RT-PCR. The mRNA isolated from infected Vero cells showed that the gene carried by the FPL1 recombinant amplified as a band of 530 bp . Complete VLPs  were seen in Vero cells at different times p.i., although in limited amounts and mainly enclosed in pseudo-vacuoles .Structural capsid proteins can self-assemble into VLPs spontaneously. Non-permissive Vero Figure  and MRC-s Figure . These ps Figure , whereasTo determine whether the limited amount of VLPs can be ascribed to the interaction of the HPV-L1 and GFP genes driven by the VVH6 and SP promoters, respectively, a quantitative comparison between the HPV-L1 and GFP transcripts was performed over 3 weeks by real-time PCR at different days p.i. Figure . The GFP®, Merck; Cervarix®, Glaxo SmithKline Biologicals) and approved for immunisation programmes in several countries [+ T-lymphocytes in association with MHC class II molecules, and require boost inoculations to maintain high antibody levels and immune cell memory. Recombinant live viral vaccines driving HPV transgene expression represent an attractive alternative as they might induce humoral and cellular immune responses and a long-lasting immunity without the need for numerous recalls.HPV-16 infection is the major risk factor for the development of cervical neoplasia and invasive cancer , and itsountries , are effountries ,14,47. HIn the present study, we have described the construction, transcription and protein expression of a new FP recombinant containing the humanised L1 gene of HPV-16, and we have demonstrated that: (1) HPV-L1 is correctly expressed in mammalian cells, as demonstrated by RT-PCR, immunoprecipitation, Western blotting, and real-time PCR; (2) in spite of the abortive replication of the FP virus vector in mammalian cells, the transcription of HPV-L1 is long lasting; (3) HPV-L1 is localised at the nuclear level, as seen by immunofluorescence; (4) L1 self-assembles both as T1 particles and as VLPs, and it can be found both in non-human and human primate cells.L1 recombinant construction. However, detection of HPV-L1 also in non-human primate Vero cells by Western blotting instead suggests that although present, only the non-native HPV-L1 structure can be detected in these cells. The absence of any specific band in FPL1-infected CEFs both by immunoprecipitation and Western blotting can be ascribed to the prevalent expression of the early and late genes of the vector and to its cytopathogenicity, which might prevent expression of the heterologous HPV-L1 transgene.The long-lasting expression of the HPV-L1 transcripts in mammalian cells confirms the possibility for a non-replicating recombinant vector to induce a good immune response . As the On the other hand, the HPV-L1 expression in CEFs determined by immunofluorescence performed 6 h p.i. demonstrates that HPV-L1 can also be detectable in replication-permissive cells, although only at early times of infection, when the vector is expressing non-structural enzymatic proteins with poor cytopathic effects. Indeed, HPV-L1 expression was sometimes also found in CEFs by immunoprecipitation, when the samples were loaded in excess (our unpublished data). This suggests early/low expression in these cells, which makes it rarely detectable.L1, which, as with other constructs [Similarly, HPV-L1 capsid proteins never appear to be correctly structured as VLPs in CEFs, although they were present in Vero and MRC-5 cells. While it is not surprising that VLPs are not detectable in cells where the recombinant replicates, as vector protein synthesis might be detrimental to HPV-L1 production and VLP assembly, the reason for the low VLP production in Vero and MRC-5 cells is not completely clear. This cannot be ascribed to the L1 gene coding sequence inserted into FPnstructs ,49,50 thL1 recombinant has the NLS, nuclear localization is expected, in spite of the vector transgene expression in the cytoplasm. T1 capsomers were found in the nucleus, which were still not assembled as complete VLPs. VLPs were seen in pseudo-vacuoles, although very rarely, as if they were in the cytoplasm as a result of the tangential section of a plasma membrane invagination. We suggest therefore that the VLPs in the pseudo-vacuoles might represent extracellular VLPs that were previously assembled in the nucleus and were then released by already lysed cells.As already described , L1 capsWe hypothesise that the GFP gene co-inserted with the HPV-L1 transgene into the vector to track the expression of the recombinant, the expression of which is driven by the strong SP synthetic promoter, might interfere with HPV-L1 expression. Although other studies have also used constructs containing GFP for recombinant selection ,52, the The similar decrease over time in both HPV-L1 and GFP mRNA transcripts shown by real-time PCR does not correlate with the low amounts of VLP production. However, data from our laboratory with recombinants that co-express transgenes and GFP suggest that SP promoter activity and/or GFP expression might interfere negatively, by limiting the synthesis of the L1 protein and VLP assembly. This might justify the good mRNA expression for both of these transgenes up to 10 days p.i. with very low VLP amounts.It is therefore likely that HPV-L1 protein expression, rather than mRNA, is responsible for the low VLP assembly, which can justify the good mRNA expression for both of these transgenes up to 10 days p.i. with very low VLP amounts.L1 construct lacking the SP-GFP complex with the present FPL1 double recombinant, both for the differences in mRNA and protein expression and for VLP production.Therefore, before animal immunization, it will be interesting to compare a new FPL1 recombinant might represent a new prophylactic vaccine, which can putatively cross-present the antigen in association with both MHC class I and II complexes. This might contribute to a complete humoral and cellular immunity when compared to the commercially available vaccines. L1 expression was observed for almost three weeks, and this is a pre-requisite for a durable immune response. The limited amounts of assembled VLPs seen by electron microscopy make these data preliminary to the use of a modified construct for the analysis of the immune responses and protection in animal models. Although the currently available vaccines are efficient in eliciting a neutralizing immunity, the 7-year efficacy appears limited since oncogenicity is often revealed only after decades of incubation. An alive non-replicating vaccine might fit the purpose better if it can produce the L1 protein and VLPs.As L1 appears to be correctly expressed both by replication restrictive and permissive cell lines, the FPThe authors declare that they have no competing interests.CZ performed the molecular cloning and RT-PCR, prepared the primary cell cultures, revised the Figures, and analysed the data and the study results; EP performed animal immunisations, Western blotting, real-time PCR, and prepared Figures; SP performed the production of the poxvirus and the HPV-L1 recombinant; MB performed the immunofluorescence assays, and the poxvirus recombinant purification; CDGM performed electron microscopy, and conceptualised, designed, and supervised the whole study; AR performed the immunoprecipitation assays, assisted in the animal immunisations, analysed the data, interpreted the study results, and prepared the manuscript. All of the authors have read and approved the present version of the manuscript.


The results taken together with other published reports on confinement of paramagnetic metal ions within single-walled carbon nanotubes (a rolled up graphene sheet) show that confinement  of paramagnetic metal ions within graphene sheets, and not the size, shape or architecture of the graphitic carbon particles is the key determinant for increasing relaxivity, and thus, identifies nano confinement of paramagnetic ions as novel general strategy to develop paramagnetic metal-ion graphitic-carbon complexes as high relaxivity MRI contrast agents.The chemistry of high-performance magnetic resonance imaging contrast agents remains an active area of research. In this work, we demonstrate that the potassium permanganate-based oxidative chemical procedures used to synthesize graphite oxide or graphene nanoparticles leads to the confinement  of trace amounts of Mn T1 and T2 MRI CAs, and affect (decrease) the longitudinal T1 and transverse T2 relaxation times of water protons, respectively. The quantitative measure of their effectiveness to accelerate the relaxation process of the water protons is known as relaxivity; the change in relaxation rate (inverse of relaxation time) per unit concentration of the MRI CA. The widely-used clinical T1 MRI CAs are mainly synthesized as metal-ion chelate complexes, where the metal ion is the lanthanoid element gadolinium (Gd3+), or the inner-transition element manganese (Mn2+). A large body of experimental and theoretical research done in the last three decades now offers good understanding of the relaxation mechanism, and underlying structural, chemical and molecular dynamic properties that influence the relaxivity of these paramagnetic-ion chelate complexes Magnetic chemical compounds called contrast agents (CA) are widely used, and nowadays integral to improve the detection and diagnostic confidence of Magnetic resonance imaging (MRI); one of the central non-invasive imaging modalities in radiology used to provide anatomical details of various organs and tissues for improved diagnosis of pathologies and diseases. The chemistry and design of contrast agents remains an active area of research in academia and industry 3+- ion carbon nanostructure complexes have been developed as MRI CAs 3+-chelate complexes onto the external carbon sheet of carbon nanostructures such as carbon nanotubes and nanodiamonds 3+- ions within the carbon sheet of carbon nanostructures such as fullerene (a.k.a. gadofullerenes) 3+- ion carbon nanostructures show between two-fold to two-order increase in relaxivity (depending on the magnetic field) compared to Gd3+-chelate complexes with the gadonanotubes showing the highest relaxivities at low to high (0.01-3T) magnetic fields. However, the potential and efficacy of Mn2+- ion carbon nanostructure complexes as MRI CAs still has not been investigated.Over the past decade, Gd3+ ion chelates, which can be prepared at a very high level of purity and unambiguously characterized, the carbon nanostructure-Gd3+ ion systems are rather complex mainly due to their particulate nature, and intricate relationships linking their chemical, geometric, and magnetic characteristics to their properties as MRI contrast agents. Nevertheless, geometric confinement of the Gd3+ ion within nanoporous structures may be one reason 3+ ions into nanoporous structures of silicon 3+ small molecule chelate compounds (e.g. gadolinium-diethylenetriaminepentaacetic acid or Gd-DTPA), only when the Gd3+ ions are confined within single-walled carbon nanotubes 3+ ion-based complexes. Additionally, to date, there have been no studies performed to systematically investigate whether the high increase in relaxivity and unconventional NMRD profiles are unique to paramagnetic ions confined in single-walled carbon nanotubes, which are seamless cylinders formed from a graphene sheet, or in general observed for paramagnetic ions confined in other graphene or graphitic structures.The variable-magnetic field (0.01-3T) relaxivity or nuclear magnetic resonance dispersion (NMRD) profiles of the gadonanotubes are characteristically different than those obtained for any other MRI CA and their relaxation mechanisms are not well understood. A major reason for this lack of understanding is that unlike Gd4)-based oxidative chemical procedures have been used in the large scale production of graphite oxide, graphene nanoplatelets, and graphene nanoribbons using starting materials such as graphite and MWCNTs 2+ ions become confined  within the graphene sheets during the synthesis process, and that this confinement in general substantially increases the relaxivity (up to 2 order) compared to paramagnetic chelate compounds, and these materials show diverse structural, chemical and magnetic properties with NMRD profiles different than those of the paramagnetic chelates.Graphene, a two-dimensional (2-D) nanostructure of carbon, has attracted a great deal of attention, and has shown potential for various material and biomedical science applications A total of 5 batches of graphene nanoplalets and nanoribbons were prepared and characterized. All the results presented except the relaxivity results are representative data of a single batch. Oxidized micro-graphite was prepared from analytical grade micro-graphite  by modified Hummer’s method 2SO4 for 2 h. KMnO4  was added, and the mixture was allowed to stir for 1 h. The reaction was then heated in an oil bath at 55–70°C for an additional 1 h, until completion. It was cooled to room temperature, and the product was washed with water, ethanol and ether, and subsequently isolated by centrifugation.Graphene nanoribbons were prepared from MWCNTs  in a procedure similar to the one previously described The solid and liquid graphene nanoplatelets and nanoribbon samples were analyzed by Inductively-coupled plasma optical emission spectroscopy (ICP-OES)  magnetometer with a sensitivity of about 10All the EPR spectra were measured at room temperature (∼296 K) under similar experimental conditions on a Bruker X-band EPR Spectrometer operating at ∼9.8 GHz microwave frequency with high 100 KHz magnetic field modulation frequency. The magnetic fields and g-values were calibrated with a standard solid sample of diphenyl picrylhydrazyl . The EPR of blank quartz tube was measured to calibrate EPR baseline for the EPR spectra. All EPR spectra were measured twice, first with 1 k Gauss sweep width, and next with 6 k Gauss sweep width. The solid samples of graphite, graphene and controls were loaded into Wilmad Quartz EPR tubes. The quartz EPR sample tubes were washed thoroughly with deionized water, and dried prior to loading of the samples. The EPR measurements on the aqueous samples were done by using a quartz flat tube designed for aqueous and other solvents with high dielectric constants. Before loading the liquid samples, the quartz EPR flat tube was washed thoroughly with deionized water and dried. The loading of aqueous samples into the quartz flat tube was done carefully into the flat portion of the tube for maximum sensitivity.2+ ions. This was achieved by first flocculating the graphene nanoparticles with HCl, and then testing the clear solution with sodium bismuthate (NaBiO3) in HNO3. In this reaction, manganese is oxidized from the +2 oxidation state (Mn+2) to the +7 oxidation state (MnO4-) which has distinctive purple or pink color. No such color change was observed indicating that no free Mn2+ions (limit of detection 1 ppm or 1 µg/ml) were present in the supernatant solution.For relaxivity measurements, 1 mg of oxidized micro-graphite, oxidized graphene nanoplatelets, reduced graphene nanoplatelets or graphene nanoribbon samples were dispersed in 2 ml of biologically compatible 1% Pluronic F127 surfactant solution, bath sonicated at 30 W for 10 min, and finally centrifuged at 5000 rpm for 1 h. The centrifugation allowed the non-water-solubilized large and dense graphene nanoparticles to settle to the bottom, and allowed the separation of soluble graphene nanoparticles in the supernatant. The supernatant solutions were also checked for the presence of any free MnT1, T2) were measured at 20 MHz (0.47 T) on a Minispec NMR spectrometer . Each sample was prepared at five known concentrations by serial dilution. The temperature was maintained at 40°C during the measurements. T1 and T2 relaxation times of each experimental sample and the control (1% Pluronic 127 solution) were measured using inversion recovery, and CPMG methods, respectively. The inverse of the relaxation times represent the respective relaxation rates, R1 and R2. A plot of relaxation rate (y-axis) versus concentration (x-axis) was created, and was fit to a linear curve. The slope of this linear fit gave the value of relaxivity. Single point relaxivity (1r) was obtained during NMRD measurements. The relaxivity values (1r), were calculated using the formula 1r = (1R–0R)/[Mn2+]; where 1, 2R and 0R are the longitudinal or transverse relaxation rates of the samples, and 1% Pluronic F127 surfactant solution respectively, and [Mn2+] is the concentration of Manganese in the volume of solution used for relaxation measurements. The 1/1T NMR dispersion (NMRD) profiles at magnetic fields corresponding to a proton Larmor frequency range 0.01–40 MHz were obtained using a fast field cycling relaxometer . A High Field Superconducting Dipole (HTS) electromagnet was used to acquire the relaxation data from 25 to 80 MHz range of proton Larmor frequency. The temperature was fixed to 27°C, and was controlled by a Stelar VTC-91 airflow heater, equipped with a copper-constantan thermocouple; the temperature calibration in the probe head was done with a Delta OHM digital thermometer, with an absolute accuracy of 0.5°C.The supernatants solutions containing the soluble graphene nanoparticles were used for relaxometry measurements. The longitudinal and transverse relaxation times  to determine the concentration of manganese. Additionally, iron content analysis was carried out for the aqueous graphene nanoribbons sample (see 2+ ions present in the sample. B) of 40 K indicating a transition between ferromagnetic and superparamagnetic states. The remnant magnetization of the hysteresis curve at 30 K is 12.47 emu/g and the coercivity is 6298.68 Oe and could be attributed to the single domain nature, and high shape anisotropy of the sample −3–10−6 emu/g The structural, chemical and elemental analysis of oxidized graphite, oxidized graphene nanoplatelets, reduced graphene nanoplatelets and graphene nanoribbons are presented in the B) is 300 K. 3O4 clusters with Fe concentration of 1–500 µg Fe per gram (1 ppm) graphite contribute 2.2×10−5 to 4×10−3 emu/g to the magnetization 1/2, Gauss) and electron relaxation time (T2e) of each EPR spectra are listed in 1/2) at their respective g values. However, graphene nanoribbons show ΔH1/2 values 2.6 times greater than oxidized micrographite, oxidized graphene nanoplatelets and reduced graphene nanoplatelets, which have similar ΔH1/2 values. The large line width indicates short electron relaxation time (2eT), and the calculated 2eT values were between 0.19–21 nanoseconds for oxidized micrographite, oxidized graphene nanoplatelets, and reduced graphene nanoplatelets. Graphene nanoribbons have 2eT values 0.072 nanoseconds; at least 2.9 times shorter than the other compounds. The EPR spectra of the graphene nanoribbons samples also shows a narrow peak in the center, which indicates presence of free radical species, possibly due to defect centers in the nanoribbon structures as reported by Tour et al. 2eT) of 88.2 nanoseconds. The large line broadening in all the compounds indicates significant manganese-to-manganese dipolar interaction. A reduction in the amount of manganese in the sample should decrease the line broadening, and resolve the 6-line manganese hyperfine structure in the EPR spectrum, and consequently, decrease the electron relaxation time.1/2, Gauss), hyperfine coupling constant, and electron relaxation time (T2e) of each EPR spectra are listed in g values are very close to the free electron spin value, and suggest the absence of spin-orbit coupling in the ground state of manganese ions present in all four samples. The manganese hyperfine coupling (AMn) of approximately 95 Gauss in these samples are very close to that of aqua ions of manganese, Mn(H2O)6. The large hyperfine coupling indicates octahedral coordination in the manganese species of all four samples. The four aqueous samples also show similar narrow line width (ΔH1/2) values between 29.2–31.5 Gauss indicative of long electron relaxation time (2eT). The calculated 2eT values were between 2.08–2.25 ns. The free radical species present in the graphene nanoribbons have an order of magnitude longer electron relaxation time (2eT) of 55 ns. It should be noted that the EPR spectra only shows the Mn(II) ions. The spectra did not show presence of Mn(III) ions or other oxidation states of manganese even though, the Raman spectrum of at least reduced graphene nanoplatelets show the presence of Mn(III) ions. A possible reason of this non-detection could be that all the EPR measurements were done at room temperature. Mn(III) ions or other oxidation states of Manganese have very short electron relaxation times, and require very low sample temperatures (∼77 K) to obtain an EPR spectra. Thus, low temperature measurements were also carried out on all the four samples. However, the EPR spectra (results not shown) was dominated by Mn (II) contributions, and the presence of other oxidation states of manganese could not be confirmed, suggesting that most of the manganese ions present in the four samples are present in Mn(II) state.1, 2r) is an important measure of the efficacy of an MRI contrast agent. 3+-based and Mn2+ based chelate complexes 1r and 2r relaxivities compared to paramagnetic chelate complexes. At 0.47 T, the 1r and 2r values for the graphite and graphene samples are ∼8–10 times, and 19–60 times greater than paramagnetic chelate complexes. Among the graphitic and graphene samples, at 0.47 T, graphene nanoribbons, and oxidized graphite showed higher (∼20%) 1r values than oxidized graphene nanoplatelets and reduced graphene nanoplatelets. However, the trend for 2r:1r ratio was reduced graphene nanoplatelets >graphene nanoribbons >oxidized micro-graphite >oxidized graphene nanoplatelets. This trend is along expected lines since, the magnetism results show that graphene nanoplatelets and graphene nanoribbons are superparamagnetic at 40°C. It well-known that superparamagnetic materials mainly affect transverse 2T relaxation and thus, increase the 2r/1r ratio. However the 2r/1r ratio is lower than iron-based 2T contrast agents that have ratios of 10 or more. 1T contrast agents have 2r/1r ratios about 1∼2 1T contrast agents even though at higher fields (3 T or above), the reduced graphene nanoplatelets and graphene nanoribbons would give rise to 2*T effects. In case of the graphene nanoribbons, iron  could not be detected (limit of detection 1 ppb) in the aqueous samples used for relaxivity measurements (see 1T relaxation measurements at 60 MHz on 1 ppb iron chloride solution showed that the presence of iron at this low concentration show negligible change (within the error of the instrument) in the 1T relaxation time compared to deionized water. Additionally, relaxivity measurements at 60 MHz of metal free (no paramagnetic ions) graphene solutions (unpublished results) also indicate that the presence of free radicals do not affect the relaxation time. Thus, in this study, for the graphene nanoribbons, the presence of additional components such a metal catalysts or free radicals do not shorten the 1T relaxation. However, it should be mentioned higher amounts of iron catalyst (in the ppm range) could potentially confound the relaxivity values, and the interpretation of the NMRD data, and thus, extra precaution should be taken during the preparation, and purification of the graphene nanoribbons to ensure the complete removal of the iron catalyst.Relaxivity (1r relaxivities for these compounds over such a large magnetic field range (0.01–80). While oxidized micro-graphite and reduced graphene nanoplatelets show similar NMRD profiles, oxidized graphene nanoplatelets, and graphene nanoribbons show distinctly different profiles than these two samples. At mid-to-high magnetic field (<10 MHz), oxidized micro-graphite shows a smaller increase (50–66 mM−1s−1) with decrease in magnetic field, and a greater increase with decrease to lower magnetic fields (70–222 mM−1s−1). Oxidized graphene nanoplatelets shows bell shaped distribution at mid-to-high magnetic fields with a maximum of 55 mM−1s−1 at 30 MHz, and a gradual increase up to 86 mM1s−1 as the magnetic fields decrease below 10 MHz. Reduced graphene nanoplatelets shows a small increase (44–59 mM−1s−1) with decrease in magnetic field between 80–10 MHz, and the relaxivity increases at lower magnetic fields with a maximum value of 258 mM−1s−1 at 0.01 MHz. Graphene nanoribbons show a linear increase (relaxivity between 65–100 mM−1s−1) with decrease in magnetic fields up to 10 MHz, and then a continuous steep increase below 10 MHz reaching values of 724 mM−1s−1 at 0.01 MHz.The NMRD profiles between 0.01–80 MHz of aqueous solutions of oxidized graphite, oxidized graphene nanoplatelets, reduced graphene nanoplatelets and graphene nanoribbons is presented in 2+ complexes such as Mn-DTPA (DTPA = diethylene triamine penta-acetic acid) show a constant values of ∼1.9 mM−1s−1 at fields greater than 10 MHz, and marginal increase at fields less than 10 MHz. Macromolecular complexes Mn2+-DTPA-BSA  show a bell-shaped relaxivity distribution at magnetic field between 10–80 MHz with a peak value of 26 mM−1s−1 at 20 MHz −1s−1. Similar profiles have been reported for small and large molecule complexes of Gd3+ ions Gd3+@C60 (gadofullerenes) which show profiles similar to those of Mn2+- or Gd3+ macromolecular complexes Gd3+@ultrashort-single-walled carbon tubes (gadonanotubes) 2+ intercalated graphitic and graphene compounds, i.e. increase in relaxivity with decrease in magnetic field with a greater increase at magnetic fields below 10 MHz. The profile of the gadonanotubes at lower magnetic fields (<10 MHz) is most similar to that of graphene nanoribbons.The NMRD profiles of these compounds are different than the profiles of other manganese-based small molecular or macromolecular complexes 1r relaxivity , oxidized graphene nanoplatelets, reduced graphene nanoplatelets, and graphene nanoribbons, respectively. Also included are the corresponding best-fit, physically reasonable values  and Mn(II)-based compounds) for the various inner-sphere parameters. (A discussion of our fitting approach is presented in the The Solomon-Bloembergan-Morgan (SBM) set of equations  is raised to the 6th power in the SBM equations. Thus, it has a very large influence on relaxivity, with shorter the distance, larger the influence. In this work, we found that allowing the parameter to vary slightly, rather than hold it fixed at the most commonly reported value of 2.9 angstroms The parameter bons See . The sep2+-based or Gd3+-based T1 MRI CA (q); (2) decreasing the inner-sphere water residence lifetime  ion-based T1 paramagnetic CAs, that enhance MR signals to generate bright positive contrast. The recent discovery of nephrogenic systemic fibrosis (NSF) in some patients with severe renal disease or following liver transplant has generated concern leading to Food and Drug Administration (FDA) restrictions on clinical use of the Gd3+- ion based MRI CA 2+, and often functions as a regulatory cofactor for enzymes and receptors. Normal daily dietary requirement for manganese is 0.1–0.4 milligrams, while normal serum levels are 1 nano-molar. Manganese toxicity has only been reported following long-term exposure or at high concentrations resulting in neurological symptoms 2+-carbon nanostructure complexes as high-efficacy MRI CAs.The physiochemical characterization, and the promising relaxivity results of the graphitic, and graphene structures reported in the work opens avenues for Figure S1Representative SEM image of (a) oxidized micro-graphite and TEM images of  reduced graphene nanoplatelets and  graphene nanoribbons.(TIF)Click here for additional data file.Figure S2Representative TEM and AFM images. Arrows in (a) show the multiple layers of graphene nanoribbon sheets. (b) TEM images at 200 kV for reduced graphene nanoplatelets Shows ∼20 nm wide few layered and multilayered reduced graphene nanoplatelets. (c) AFM Section analysis of graphene nanoplatelets dispersed on silicon substrate, showing a uniform thickness of ∼1.137 nm.(TIF)Click here for additional data file.Figure S33O4), oxidized graphite and reduced graphene nanoplatelets at 532 nm showing spectral peaks at 657, 370 and 320 cm−1.Raman spectrum with the D and G bands peaks for (a) graphite, oxidized graphite, oxidized graphene nanoplatelets and reduced graphene nanoplatelets, and (b) MWCNTs and graphene nanoribbons (c) Comparison of Raman spectra between Hausmannite (Mn(TIF)Click here for additional data file.Figure S4EELS spectrum for (a) reduced graphene nanoplatelets and (b) oxidized graphene nanoplatelets showing a oxygen peak at 530 eV.(TIF)Click here for additional data file.Figure S5EPR spectrum of the (a) Wilmad quartz EPR tubes used for the measurement of the solid samples, (b) quartz EPR flat tube used for the aqueous samples, (c) DPPH standard (solid) and (d) DPPH standard (aqueous).(TIF)Click here for additional data file.Figure S6Curves obtained with all SBM parameters floating. A) Oxidized Graphite, B) Oxidized Graphene Nanoplatelets, C) Reduced Graphene Nanoplatelets, D) Graphene Nanoribbons.(TIF)Click here for additional data file.Figure S7Curves obtained for fixed Q = 2 with remaining SBM parameters allowed to float. A) Oxidized Graphite, B) Oxidized Graphene Nanoplatelets, C) Reduced Graphene Nanoplatelets, D) Graphene Nanoribbons.(TIF)Click here for additional data file.Figure S8Curves obtained for fixed Q = 4 with remaining SBM parameters allowed to float. A) Oxidized Graphite, B) Oxidized Graphene Nanoplatelets, C) Reduced Graphene Nanoplatelets, D) Graphene Nanoribbons.(TIF)Click here for additional data file.Figure S9Curves obtained for fixed Q = 6 with remaining SBM parameters allowed to float. A) Oxidized Graphite, B) Oxidized Graphene Nanoplatelets, C) Reduced Graphene Nanoplatelets, D) Graphene Nanoribbons.(TIF)Click here for additional data file.Figure S10Curves obtained for fixed Q = 8 with remaining SBM parameters allowed to float. A) Oxidized Graphite, B) Oxidized Graphene Nanoplatelets, C) Reduced Graphene Nanoplatelets, D) Graphene Nanoribbons.(TIF)Click here for additional data file.Figure S11m at values shown in Curves obtained for fixed Q = 8 and Fixed T A) Oxidized Graphite, B) Oxidized Graphene Nanoplatelets, C) Reduced Graphene Nanoplatelets, D) Graphene Nanoribbons. The fit for the Graphene Nanoribbons in D is surprisingly worse than expected.(TIF)Click here for additional data file.Table S1Trace elemental analysis of solid samples of the oxidize micro-graphite, oxidized graphene nanoplatelets, reduced graphene nanoplatelets and graphene nanoribbons. The standard deviation among the various batches was 10%.(DOCX)Click here for additional data file.Table S2Trace elemental analysis of aqueous samples of the oxidized micrographite, oxidized graphene nanoplatelets, reduced graphene nanoplatelets and graphene nanoribbons. The values presented are for one batch of samples.(DOCX)Click here for additional data file.Table S3List of parameter values in SBM equations that are fixed constants, or independently established physical quantities.(DOCX)Click here for additional data file.Table S4SBM Parameters obtained from the curve fit with all parameter values floating.(DOCX)Click here for additional data file.Table S5SBM Parameters obtained from the curve fit for fixed Q = 2 and remaining SBM parameters allowed to float.(DOCX)Click here for additional data file.Table S6SBM Parameters obtained from the curve fit for fixed Q = 4 and remaining SBM parameters allowed to float.(DOCX)Click here for additional data file.Table S7SBM Parameters obtained from the curve fit for fixed Q = 6 and remaining SBM parameters allowed to float.(DOCX)Click here for additional data file.Table S8SBM Parameters obtained from the curve fit for fixed Q = 8 and remaining SBM parameters allowed to float.(DOCX)Click here for additional data file.Table S9SBM Parameters used to obtain curve fit for fixed Q = 8 and fixed Tm values.(DOCX)Click here for additional data file.Text S117O-transverse relaxation rate measurements on oxidized micro graphite, graphene nanoplatelet and nanoribbons.Information on structural characterization, elemental and Raman analysis, Solomon-Bloembergan-Morgan Theory of Relaxivity and (DOCX)Click here for additional data file.


Drosophila, and C. elegans. In mammals, the multi-function TDP-43 encoded by the TARDBP gene is a signature protein of the ubiquitin-positive inclusions (UBIs) in the diseased neuronal/glial cells of a range of neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-U).TDP-43 is an RNA- and DNA-binding protein well conserved in animals including the mammals, Drosophila ortholog of the mammalian TARDBP gene, dTDP, by genetic, behavioral, molecular, and cytological analyses. It was found that depletion of dTDP expression caused locomotion defect accompanied with an increase of the number of boutons at the neuromuscular junctions (NMJ). These phenotypes could be rescued by overexpression of Drosophila dTDP in the motor neurons. In contrast, overexpression of dTDP in the motor neurons also resulted in reduced larval and adult locomotor activities, but this was accompanied by a decrease of the number of boutons and axon branches at NMJ. Significantly, constitutive overexpression of dTDP in the mushroom bodies caused smaller axonal lobes as well as severe learning deficiency. On the other hand, constitutive mushroom body-specific knockdown of dTDP expression did not affect the structure of the mushroom bodies, but it impaired the learning ability of the flies, albeit moderately. Overexpression of dTDP also led to the formation of cytosolic dTDP (+) aggregates.We have studied the function and dysfunction of the Drosophila NMJ suggest that eukaryotic TDP-43 guards against over development of the synapses. The conservation of the regulatory pathways of functions and dysfunctions of Drosophila dTDP and mammalian TDP-43 also shows the feasibility of using the flies as a model system for studying the normal TDP-43 function and TDP-43 proteinopathies in the vertebrates including human.These data together demonstrate the neuronal functions of dTDP, and by implication the mammalian TDP-43, in learning and locomotion. The effects of mis-expression of dTDP on  TDP-43, or the HIV TAR DNA-binding protein 43, is an evolutionarily conserved, 43 kD DNA/RNA-binding protein that functions in transcriptional repression Interestingly, dysfunction of TDP-43 has been implicated in the pathogenesis of a range of human neurodegenerative diseases, in particular the amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-U). Specifically, the diseased neurons/glial cells of most of the FTLD-U brains and the spinal cord motor neurons of most ALS cases are characterized by the presence of TDP-43-containing, polyubiquitin-positive aggregates or inclusion bodies (UBIs) in the cytoplasm or nuclei. Also, the TDP-43 molecules in the UBIs consist of phosphorylated 45 kD species, high molecular weight polyubiquinated species, and C-terminal fragments of the molecular weights 25 kD and 35 kD, respectively Drosophila dTDP in the whole bodies of the fruit flies impairs the adult locomotor activities Drosophila peripheral sensory neurons also decreases their dendritic branching Drosophila motor neurons also causes motor dysfunction The pathogenesis of the neurodegenerative diseases with TDP-43 (+) UBIs could be due to toxic gain-of function, loss-of-function of TDP-43, or a combination of both. With respect to this, several studies have implied TDP-43 being a factor important for various neuronal functions. In mouse, mTDP-43 molecules reside in the postsynaptic density (PSD) areas of the dendritic spines. They also form dendritic RNA granules colocalized with the neuronal activity-regulating factors FMRP and Staufen. The above pattern in cultured hippocampal neurons changes upon treatment with various neuronal activity modulating reagents, suggesting the involvement of TDP-43 in the regulation of neuronal plasticity Drosophila as the model system. As seen later, we show that while depletion of the Drosophila dTDP and overexpression of dTDP in the Drosophila motor neurons both affect the locomotor activity of the flies, the two genetic manipulations exert opposite effects on the development of the boutons at the neuromuscular junctions (NMJ). This suggests a role of dTDP/TDP-43 in the regulation of neuron development. Furthermore, mushroom body-specific depletion or overexpression of dTDP leads to the impairment of the fly learning ability. The latter result together with our recent study of mTDP-43 overexpression in the forebrain of mice The above studies have revealed important insights into the development of FTLD-U-like and ALS-like symptoms by aberrant regulation of TDP-43. In particular, they together have suggested that ALS with TDP-43 (+) UBIs may be in part caused by loss-of-function of TDP-43. In the following, we report our study on the pathogenesis of FTLD-U and ALS using the dTDP in Drosophila development was examined by analysis of fly mutants, with null-expression of dTDP. For this, deletion mutations of the dTDP gene in the KG08578 insertion line were generated by imprecise excision. Twelve out of the ninety stocks with excision of the P element from KG08578 could not survive to the adult stage. One of these recessive lethal dTDP lines, ex26dTDP, was analyzed in details. ex26dTDP consisted of an imprecise excision of the P-element leading to a 932 bp-deletion in the 5′ end of dTDP gene, and no dTDP expression could be detected by RT-PCR analysis at all developmental stages of the homozygous mutant ex26dTDP flies obtained with use of the green balancer (ex26dTDP mutant did not affect the RNA level from the gene CG4585 at upstream of dTDP (RT-PCR data not shown). The homozygous ex26dTDP line was semi-lethal with most of the flies viable from embryonic to early pupal stage but few of them (approximately 10%) eclosed to the adult stage. Most of the flies were trapped in the pupal cases and the survived ones showed weakness in their legs and severe movement defects.The function of balancer . The delex26dTDP mutant. As shown in yw control showed that dTDP was indeed absent in the homozygous ex26dTDP mutant. This loss of dTDP expression strongly impaired the locomotive behavior of the larvae by 90%. Quantitatively, the ex26dTDP mutant displayed significantly shorter moving distance than the control (p<0.0001) (yw control and the homozygous ex26dTDP mutant. The larvae were immunostained with horseradish peroxidase (HRP), which labeled the neuronal membranes, and antibody against synapsin (Syn), a presynaptic protein located in the bouton  . To exame bouton . As seene bouton . The ince bouton  and fulle bouton , respectDrosophila CNS, in particular the mushroom body supporting the olfactory learning of the fruit flies OK107-GAL4 to mark the mushroom bodies, we found that dTDP, similar to the mammalian TDP-43, was broadly distributed in the adult brain with most dTDP molecules located in the cell body, especially in the nucleus (OK107>38377 and OK107>38379) and with overexpression of dTDP in the mushroom bodies (OK107>dTDP#5-1 and OK107>dTDP#18-1), respectively, with use of the GAL4-UAS system  of the ALS and the FTLD-U patients  nucleus . We thenS system .dTDP-overexpressing lines were increased by 7 and 3 folds .As seen, Western blot analysis showed that the amounts of dTDP in the adult heads of the two RNAi-knockdown lines were decreased by approximately 46% and 23%  aggregates similar to those observed in FTLD-U brains, the heads of 3 day-old  lane 6,  but not  lane 6, . Thus, tdTDP mutant lines were assayed by the odor avoidance learning test in comparison to the control flies. As shown in OK107>38377 by constitutive RNAi knockdown did not cause obvious defective phenotype of the mushroom bodies (p<0.05) when compared to the control flies. On the other hand, the olfactory learning abilities of the two dTDP-overexpressing lines were severely impaired when compared to the control. Furthermore, parallel to the structural defects of the mushroom bodies (OK107>dTDP#5-1) showed approximately ∼80% reduction in the performance score when compared to the control flies; on the other hand, the lower level of dTDP overexpression, as in OK107>dTDP#18-1, caused only 30% decrease of the learning ability  to knockdown the endogenous dTDP in the motor neurons. However, the resulting flies did not exhibit locomotion defect, possibly due to inefficient knockdown of the dTDP. We then used the pan-neuron driver elav-GAL4 to knockdown dTDP. As shown in elav>38377 were lower than the control larvae (elav>+). Furthermore, similar to the dTDP-null fly line ex26dTDP described in elav>38377 larvae were also higher than the control larvae (elav>+) and the dTDP-knockdown larvae (elav>38377) were significant (p<0.05), it was less than those between the control (yw) and the dTDP-null mutant ex26dTDP as shown in ex26dTDP  with dTDP overexpression in the motor neurons were also generated with use of the GAL4-UAS system. Immunostaining was then used to examine the levels and distribution patterns of dTDP in the larval ventral nerve cords of these two mutant lines in comparison to the D42>+ control flies. As seen in dTDP-overexpressing lines, the dTDP molecules appeared to be translocated from nucleus to the cytoplasm  UBIs. It should be mentioned that overexpression of human hTDP-43 in Drosophila has been carried out previously by others Drosophila. They also indicate that Drosophila with overexpression of dTDP could be developed into working disease models if the dose of the exogeneous dTDP is appropriately controlled. Finally, the analysis of Drosophila lines with knockdown and overexpression of dTDP suggests that phenotypes of FTLD-U and ALS with TDP-43 (+) UBIs are caused by a combination of loss-of-TDP-43 function and gain-of-negative function of TDP-43.In this study, we have investigated the functions of the dTDP-null mutant ex26dTDP using P-element excision, which exhibits early lethal phenotype with locomotion defects in the larvae as well as the adult flies  have normal life span and apparently normal mushroom bodies even at the age of 30-day old (dTDP-overexpressing lines is apparent (In view of the impairment of learning and memory in FTLD-U patients with TDP-43 (+) UBIs m bodies . Remarka-day old . On the apparent . These dex26dTDP null mutant  aggregates and dTDP-depleted nuclei , and abeDrosophila dTDP in appropriate tissues/cells has also been shown. Thus, in addition to the protein sequences and general structure of the hTDP-43/dTDP Drosophila. Fly models with overexpression of the homologous dTDP likely will have certain advantages when compared to the ones with overexpression of the heterologous hTDP-43, for the basic and translational research of neurodegenerative diseases with TDP-43 proteinopathies.In summary, this study has demonstrated the requirement of dTDP for development and for neuronal functioning, in learning as well as in locomotion, of the fruit flies. The feasibility of the generation of fly models of neurodegeneration diseases with TDP-43 (+) UBIs by overexpression of the homologous elav-GAL4) flies, motor neuron-specific GAL4 (D42-GAL4) flies, mushroom body-specific GAL4 (OK107-GAL4) flies and P-element insertion mutation stock of dTDP: 1 w67c23; P{SUPor-P}TBPHKG08578y (KG08578) were obtained from the Bloomington Drosophila Stock Center. The muscle-specific GAL4 flies, MHC-GAL4, were kindly provided by Dr. Chien's lab at Institute of Molecular Biology (IMB), Academia Sinica. The transgenic RNAi stocks VDRC38377 and VDRC38379 were obtained from the Vienna Drosophila RNAi Center. The P-element excision of KG08578 was carried out by the standard P-element mobilization approach. Homologous mutants at the early development stage were created with use of a green balancer.Fly stocks were maintained in standard medium at 25°C and 60% humidity under a 12-h light-dark cycle. The pan-neuron GAL4 CyO:TM6B,Tb1dTDP were crossed with the males of ex26,D42-GAL4/TCyO:TM6B,Tb1dTDP. The excision line ex26dTDP was crossed with yw for five generations and all the other stocks described above, were equilibrated by six generations of out-cross to the w background.Drosophila melanogaster at different stages by using the Trizol reagent (Invitrogen). For analysis of the expression levels of dTDP, 1 µg of the total RNAs were reverse transcribed with SuperScript™ II Reverse Transcriptase (Invitrogen) using oligo (dT) primers. The primers used for PCR reactions are: dTDP-F, 5′-CCATGGATTTCGTTCAAGTGTCG-3′; and dTDP-R, 5′-TTAAAGAAAGTTTGA CTTCTCCGC-3′.Total RNAs were isolated from A cDNA fragment encoding the C-terminus of dTDP (amino acid 307–531) was amplified by PCR reaction and cloned into BamHI/XhoI sites of pRSET vector. Expression of the protein and immunization of the rabbits were done by the commercial supplier .For muscle staining and analysis of the NMJ phenotypes, wandering late third instar larvae were dissected and the tissues were incubated in a fixative solution  for 20 minutes. The tissues were then blocked with 10% normal goat serum at room temperature for 1h. The primary antibodies used were against dTDP (1∶200), synapsin  and HRP , respectively. For secondary antibody, goat anti-rabbit conjugated to Alex Fluor 647 or goat anti-mouse conjugated to Alex Fluor 647 was used (Molecular Probes). FITC-conjugated Phalloidin was used for muscle staining.For staining of the larval ventral nerve cords, adult brains, and adult thoracic ganglia, larvae or adult females were dissected in PBS, fixed with 4% paraformaldehyde in PBS with 0.25% Triton-X-100, and then irradiated using a microwave oven . After blocking with 10% normal goat serum at room temperature for 1 h, the tissues were incubated overnight at room temperature with rabbit polyclonal anti-dTDP (1∶200). After washing, the tissues were incubated overnight at room temperature with biotinylated anti-rabbit antibody  and rat anti-elav antibody . They were then washed again and incubated overnight with streptavidin-Alexa-647 antibody  and Alexa-546 conjugated goat anti-rat antibody .The confocal images were acquired using a Zeiss LSM 510 Meta or LSM710. The images for quantification of the NMJ bouton numbers were from a projection of the z-sections. To quantify the NMJ muscle area, the images were analyzed using Zeiss LSM Image Examiner.The locomotor activity of the adult flies was measured in a dark room using a countercurrent apparatus as described To measure the larval locomotor activity, 20 larvae per genotype were transferred to a Petri dish coated with 1% agarose, and their behaviors were monitored for 2 minutes. The average distance of the larval trails of each group was then determined with use of an ImageJ software.The abilities of the olfactory associative learning of the flies were measured by training 2∼5-day old adult flies in a T-maze following the classical conditioning procedure OK107>+ and OK107>dTDP#5-1, 100 Drosophila heads from 50 males and 50 females of the age of 3 days were collected. For comparison of D42>+ and D42>dTDP#18-1, the heads and thoraces from 20 males and 20 females of the age of 13 days were collected. All samples were lysed in cold RIPA buffer  containing protease and phosphatase inhibitors , and then sonicated. The protein lysates were first cleared by centrifugation to generate the RIPA-soluble samples. The pellets were then washed four times with RIPA buffer and extracted with the urea buffer , sonicated, and centrifuged. For preparation of the total extracts, the Drosophila samples were lysed directly in the urea buffer.To examine the solubility profiles of dTDP, sequential extractions were performed as described by Winton et al.


H1-69) and variable kappa light chain (VK3-20) gene families as the prototype broadly neutralizing anti-MPER mAb, 4E10 (residues 672–680). These data indicate that there are multiple immunogenic targets in the C-terminus of the MPER of HIV-1 gp41 envelope and suggests that gp41 neutralizing epitopes may interact with a restricted set of naive B cells during HIV-1 infection.Broadly neutralizing antibodies are not commonly produced in HIV-1 infected individuals nor by experimental HIV-1 vaccines. When these antibodies do occur, it is important to be able to isolate and characterize them to provide clues for vaccine design. CAP206 is a South African subtype C HIV-1-infected individual previously shown to have broadly neutralizing plasma antibodies targeting the envelope gp41 distal membrane proximal external region (MPER). We have now used a fluoresceinated peptide tetramer antigen with specific cell sorting to isolate a human neutralizing monoclonal antibody (mAb) against the HIV-1 envelope gp41 MPER. The isolated recombinant mAb, CAP206-CH12, utilized a portion of the distal MPER  and neutralized a subset of HIV-1 pseudoviruses sensitive to CAP206 plasma antibodies. Interestingly, this mAb was polyreactive and used the same germ-line variable heavy (V The isolation of new anti-HIV-1 envelope neutralizing human monoclonal antibodies (mAbs) is a high priority since they may identify potential targets for vaccine design. Until recently only a handful of such mAbs were available, and these were isolated either through traditional EBV-transformation or phage-display libraries 664DKW666 essential for its recognition 672, F673 and W680 critical for binding 671 and D674The membrane proximal external region (MPER) in gp41 represents an important target for anti-HIV-1 neutralizing antibodies 674H and Vk Ig gene families as the 4E10 mAb and its CDRH3 sequence showed strong similarities with that of Z13e1 as a result of shared J gene usage We recently described anti-MPER antibodies in a chronically infected subtype C infected individual (CAP206) that were responsible for plasma neutralization breadth, and were targeted to the distal MPER centered around DWe have previously identified an HIV-1-infected individual from the CAPRISA 002 acute infection cohort in Durban, South Africa who developed broadly cross-reactive neutralizing antibodies targeting the MPER +, CD27+) that were dual stained with both MPR.03-AF647 and MPR.03-PacBlue were sorted into individual wells of a 96 well plate IT676 motif, which overlapped with the 4E10 672 and F673672 or F673 had a more drastic effect on 4E10 binding (<20% binding) than on CAP206-CH12 (30–40% binding) (671 and residues N-terminus to this (S668LW670), did not play a role in CAP206-CH12 binding  . A criti binding . Thus, td = 1.6 nM; koff = 1.5×10−5 s−1, 25), CAP206-CH12 binding to gp41-inter was considerably weaker (Kd = 21.4 nM) and displayed about 10-fold faster off-rates (koff = 2.2×10−4 s−1). This may explain the lower neutralization potency of CAP206-CH12 when compared to that of 4E10.Previously, 2F5 and 4E10 mAbs were shown to bind strongly with exceptionally slow off-rates to trimeric 92UG gp41-inter, a protein that mimics the pre-hairpin intermediate state of gp41 d of 120 nM (d of 265 nM and koff (koff = 1.1×10−3 s−1) which was about 20-fold faster than that of the mature CAP206-CH12 mAb  are also crucial for 4E10 neutralization and both F673 and D674 were previously shown to affect neutralization by CAP206 plasma antibodies We used the subtype C pseudovirus, COT6.15 which has been alanine-scanned across the MPER, to assess which residues were crucial for neutralization. As shown in 674 but had an arginine at position 677 possibly accounting for its lack of sensitivity. Other isolates that had D674 and either K or N at 677 were resistant suggesting that simply having the nominal epitope was not sufficient and other aspects such as exposure of the MPER are likely important in determining CAP206-CH12 sensitivity. The 2 other residues F673 and W680 identified in the COT6.15 scanning as important for neutralization    were alsH and VL families by this new MPER neutralizing mAb, CAP206-CH12, as that used by the prototype MPER mAb 4E10. Overall, this approach has significant potential for the discovery of additional novel neutralizing anti-MPER mAbs from HIV-1 infected individuals, which will allow for further interrogation of this important gp41 vaccine target.In this study we demonstrate the power of prior epitope mapping of plasma neutralizing antibodies that allowed for the rationale design of an antigen-specific memory B cell receptor ligand (bait). Furthermore, the use of single cell sorting with dual-labeled ligands facilitated the efficient and specific selection of relevant memory B cells. Remarkably, we saw utilization of the same VH1-69 heavy chains from this individual supported this notion, although this could not be proven due to the inability to express them as functional mAbs. Nonetheless, the CAP206-CH12 mAb epitope directly overlapped the epitope of plasma antibodies confirming that it comprised a component of plasma neutralizing activity. Overall, CAP206-CH12 showed modest neutralization activity and limited potency, similar to Z13e1, and reacted with both subtype C and B viruses. While the CAP206-CH12 mAb was polyreactive, unlike 2F5 and 4E10, CAP206-CH12 did not bind lipids avidly. Since both 2F5 and 4E10 rely on lipid reactivity for virion membrane binding in order to mediate neutralization While the CAP206-CH12 mAb neutralized approximately two-thirds of the viruses sensitive to CAP206 plasma antibodies, this mAb did not recapitulate all the plasma neutralizing activity. This suggests that this type of antibody was responsible for a portion of the breadth observed in plasma, and that full coverage would likely be provided by additional mAbs, possibly somatic variants of CAP206-CH12. Indeed the isolation of additional VH1-69 and Vκ3-20 similar to the gp41 antibody, 4E10. Gorny and colleagues have previously reported that non-neutralizing human antibodies that bind to epitopes in the cluster II region of gp41  frequently use a VH1-69 Ig heavy chain H1-69. Another example of restricted usage of VH1-69 has recently been reported following the isolation of influenza broadly neutralizing antibodies to the stalk of hemagglutinin H1-69 antibodies are hydrophobic and one hypothesis is that these antibodies are preferentially used for regions of virus envelopes that are in close proximity to viral membranes. Alternatively, Johnson and co-workers reported that the percentage of the blood B cell repertoire that use VH1-69 is directly related to the VH1-69 copy number H1-69 in anti-viral responses.It was striking that CAP206-CH12 utilized the VAnother interesting finding was that both CAP206-CH12 and another anti-MPER mAb, Z13e1 had the YYYYMD motif in their CDRH3 as a result of a shared J allele. In the case of Z13e1, three of the Tyr residues positioned at the base of CDRH3 make contacts with the peptide 668LWNWFDITN677670. In particular, position 674 was shown to be critical for both Z13e1 and CAP206-CH12. Residue F673, a major determinant of the 4E10 epitope was also important for the CAP206-CH12 mAb as shown in both binding and neutralization studies. For MPER mAbs that bind to overlapping residues, differences in both orientation and conformation of gp41 recognized by 4E10 and Z13e1 have been described 672/F673 accessible MPER conformation.The epitope of the CAP206-CH12 mAb overlapped that of both 4E10 and Z13e1. The Z13e1 epitope spans residues Sd of CAP206-CH12 for gp41-inter protein is consistent with its poorer neutralization activity. Both lipid reactivity and a stable binding of MPER antibodies with extremely slow off-rates have been proposed to be a requirement for the capture of the transient pre-hairpin intermediate conformation and subsequently the blocking of cell fusion The lack of binding to denatured or plate bound JR-FL or ConS gp140 and also to gp41 proteins suggests that the epitope recognized by CAP206-CH12 is conformation sensitive. Even in cases where the MPER sequences were identical, differences elsewhere in gp41 and also within gp120 might affect gp120-gp41 interactions and the overall conformation of MPER accounting for the lack of binding. Furthermore, while denaturing gp140 proteins would expose the linear sequence, it does not preserve the secondary structure which may account for some of the differences seen between peptides and denatured proteins. However, like the previously described broadly neutralizing gp41 MPER antibodies (2F5 and 4E10), CAP206-CH12 bound to gp41-inter protein. Thus CAP206-CH12 is capable of targeting the pre-hairpin intermediate conformation of gp41, which is not recognized by non-neutralizing gp41 cluster II antibodies The finding that the germline-like antibody of CAP206-CH12 bound the MPR.03 peptide and gp41-inter weakly allows us to propose a model whereby MPER epitopes on the early transmitted founder virions initiated the hypermutation process in a naive B cell during acute HIV-1 infection. The fact that the first binding antibodies in HIV-1 infection are to gp41 Finally, our studies show that epitope mapping of plasma antibodies followed by the rational design of fluoresceinated MPER peptide tetramers can successfully isolate antigen-reactive single B cells for Ig rescue. Scheid and colleagues have previously used fluoresceinated gp140 envelope for this purpose for isolation of Env-reactive B cells Stored plasma and PBMC from CAP206 an HIV-1 subtype C chronically infected individual were used for this study. This participant was part of the CAPRISA 002 Acute infection cohort whose antibody neutralization profile has been studied since the point of seroconversion KKKNEQELLELDKWASLWNWFDITNWLWYIRKKK-biotin) and a scrambled version were purchased from CPC Scientific Inc . Peptides were >98% pure as tested by HPLC. MPER656 (Biotin-NEQELLELDKWASLWNWFNITNWLW), SP62 (2F5 peptide), SP400 (peptide from gp41 immunodominant region) and 4E10 peptides have been described previously NEQELLELDKWASLWNWFNITNWLWYIK-YKRWIILGLNKIVRMYS) was used in the preparation of MPER peptide liposomes The MPR.03 biotinylated peptide containing lysines at both ends for solubility  and Pacific Blue labelled streptavidins and titered on antibody-coated beads and on antibody expressing cell lines (using the 13H11 and 2F5 mAbs which both bind the MPR.03 peptide). Briefly, excess biotinylated peptide (approximately 33∶1 molar ratio of peptide to streptavidin for fluorochrome-labeled tetramers) was incubated at 4°C overnight and isolated using gel filtration on Micro BioSpin 30 columns. Tetramers were assayed for final concentration determined using standard spectrophotometric techniques. Final titers were determined using a combination of 2F5-coated beads and 13H11- expressing cell lines. Tetramers were used in equimolar amounts in combination with a panel of mAbs to identify memory B cells in PBMC.neg, CD14neg, CD16neg, CD235aneg, CD19pos, CD27hi, CD38low and IgG+. Tetramer-stained B cells were sorted as single cells into wells of a 96-well plate, selecting those cells that were labelled by both tetramers. Cells were stored in reverse-transcriptase reaction buffer at −80°C until use Thawed PBMC were stained with a combination of the following antibodies: CD3 PE-Cy5, CD14 PE-Cy5, CD16 PE-Cy5, CD235a PE-Cy5, CD19 APC-Cy7, CD27 PE-Cy7, CD38 APC-Cy5.5 and IgG-PE . All antibodies were titered and used at optimal concentrations for flow cytometry. Memory B cells were gated as CD3H and VL were amplified by PCR using a modification of the method described by Tiller and co-workers H, VK and VL genes were amplified from this cDNA separately in a 96-well nested PCR as described and analysed on 2% agarose gels H and VL genes into functional linear Ig gene expression cassettes (see below). PCR products were purified and sequenced. The variable gene segments and the potential functionality of the Ig genes was determined using the SoDA program The genes encoding VH, VK or VL genes amplified from single B cells were assembled by overlapping PCR. PCR products containing linear full-length Ig heavy- and light-chain genes were purified and the paired Ig heavy and light-chain products co-transfected into 293T cells grown in 12-well plates using Fugene. Cultures were fed 6–12 hrs later with ∼2 mls fresh medium containing 2% FCS and incubated for 72 hours at 37°C in a 5% CO2 incubator. Thereafter culture supernatants were harvested for antibody characterization.Three linear Ig expression cassettes each containing the CMV promoter and human Ig leader as one fragment were used for small-scale expression and specificity analysis The SoDA program H and VK genes from CAP206-CH12 were cloned into human Igγ and Igκ expression vectors in pcDNA3.3 The selected Ig VSupernatants from the small scale transfections and purified mAbs were tested for reactivity using various peptides and proteins in an ELISA as described d measurements were made as described earlier MPER656, MPR.03 and a scrambled version of MPR.03 were individually anchored on a BIAcore SA sensor chip as described previously 50 value) was reported. Purified mAb was used for these experiments to avoid interference from transfection reagents. Plasma was heat inactivated prior to performing neutralization assays.The TZM-bl pseudovirus assay was used to assess the neutralization activity of CAP206-CH12 and control mAbs against viruses that were sensitive to CAP206 plasma antibodies as well as to a large panel of 26 unselected heterologous Tier 2 viruses from multiple subtypes available in Dr David Montefiori's CAVD Consortium. Mapping of residues in the MPER crucial for CAP206-CH12 neutralization made use of the COT6.15 virus which was alanine-scanned across the MPER. The mAb concentration at which 50% of virus neutralization was seen  and nucleotide sequences of CAP206-CH12 and CAP206-CH12_RUA heavy and light chains (B).Amino acid alignment of CAP206-CH12 V(PPTX)Click here for additional data file.Figure S2Mapping of a MPER amino acid residues important for CAP206-CH12 and 4E10 binding. Bar graphs show relative binding of CAP206-CH12 and 4E10 mAbs to alanine scanned MPER656 peptides. Residues highlighted in red show more than 50% reduction in binding relative to wild-type peptide. Individual SPR curves for 3 residues W672, F673, N674 (boxed in bar graph) normalized to 100% binding show the differential effect of the substitution on 4E10 (blue) and CAP206-CH12 (red) binding. While each of the substitutions drastically reduced the binding (>50%) of CAP206-CH12 relative to wild-type, the substitutions show marked differences in the off-rates of the binding of 4E10 to W672A and F673A peptides but not to N674A. Thus W672A and F673A substitutions had a more drastic effect on 4E10 binding than CAP206-CH12 binding. Alanine substituted mutants were designed by substituting single amino acids within the MPER peptide with the following sequence – Biotin-GGG-QELLELDKWASLWNWFNITNWLWYIK (MPER26). Relative binding of the mutants was measured using the wild type MPER656 peptide (Biotin-NEQELLELDKWASLWNWFNITNWLW). The binding of CAP206-CH12 mAb to all three MPER peptides  used in this study were similar. CAP206-CH12 bound to MPER26 and MPER656 peptide with Kd of 4.95 and 5.15 nM respectively.(PDF)Click here for additional data file.Figure S3MPER sequences of viruses sensitive and resistant to CAP206-CH12 mAb. Amino acids at positions 674 and 677 are highlighted in red. Residues at 673 and 680 (boxed) were conserved in all sensitive and resistant isolates.(PDF)Click here for additional data file.Table S1Neutralization sensitivity of viruses to CAP206-CH12.(PDF)Click here for additional data file.