Long-term potentiation (LTP) at synapses of nociceptive nerve fibres is a proposed cellular mechanism underlying some forms of hyperalgesia. In this review fundamental properties of LTP in nociceptive pathways are described. The following topics are specifically addressed: A concise definition of LTP is given and a differentiation is made between LTP and "central sensitisation". How to (and how not to) measure and how to induce LTP in pain pathways is specified. The signal transduction pathways leading to LTP at C-fibre synapses are highlighted and means of how to pre-empt and how to reverse LTP are delineated. The potential functional roles of LTP are evaluated at the cellular level and at the behavioural level in experimental animals. Finally, the impact of LTP on the perception of pain in human subjects is discussed. Late phase LTP involves protein synthesis and lasts longer than three hours, up to the life span of an animal and may involve structural changes at synapses ..12].In conclusion, HFS and LFS may have fundamentally different effects on LTP induction at different C-fibre synapses. This finding is in line with previous reports also illustrating that the frequency of afferent barrage in C-fibres may have qualitatively different effects in spinal cord. For example, brain-derived neurotrophic factor is released from primary afferents in spinal cord slices in an activity-dependent manner by HFS at 100 Hz but not by 1 Hz LFS of primary afferent nerve fibres . FurtherAt synapses in the brain LTP induction requires synchronous, high-frequency pre-synaptic activity or pairing of low-level pre-synaptic activity with strong post-synaptic depolarization. At least some of the C-fibre synapses are apparently unique in that LTP can be induced by LFS and by natural, low or high frequency, asynchronous and irregular discharge patterns in sensory nerve fibres. In animals with spinal cord and descending pathways intact, intraplantar, s.c. injections of capsaicin 100 μl, 1%) or formalin  induce slowly rising LTP  CH 23390 . With spCH 23390 .A receptor antagonist bicuculline (1 mg/kg i.p.) the same conditioning stimulus now produces LTP rather than LTD [A receptor agonist to spinal cord prevents tetanus-induced LTP of A-fibre-evoked field potentials in animals with a CCI [A-fibre-evoked spinal field potentials are depressed by conditioning 50 Hz stimulation of sciatic nerve fibres. After GABAthan LTD . Similarthan LTD . Topicalth a CCI . This ag2+-dependent form of LTP induction in spinal cord lamina I neurons. Induction of LTP at C-fibre-synapses requires co-activation of NK1 and NK2 receptors [2+]i  receptors is required for LTP induction in pain pathways. Some indirect evidence suggests, however, that this might be the case i is essential for LTP induction and the magnitude in [Ca2+]i rise is linearly correlated with the magnitude of LTP in vitro [2+]i in lamina I neurons not only in slice preparations, but also in intact animals [2+-dependent pathways including activation of protein kinase C, calcium-calmodulin-dependent protein kinase II (CaMKII), protein kinase A (PKA) phospholipase C (PLC), inositoltriphosphate-3 (IP3) receptors, nitric oxide synthase (NOS) and members of the mitogen-activated protein kinase family (MAPK), including extracellular signal-regulated kinase (ERK) , a blocker of glial cell metabolisms (MFA), and an mGluR group I antagonist (LY367385) .Inhibition of protein synthesis in spinal cord by either cycloheximide or anisomycin selectively inhibits the maintenance of the late-phase of spinal LTP but does not affect either LTP induction or baseline responses of C-fibre evoked field potentials .Importantly, the very same signal transduction pathways are required for full expression of hyperalgesia in animal models of inflammatory and neuropathic pain e.g. -41.2-adrenoreceptors by clonidine [in vivo.LTP induction can be prevented by blockade of any of the above mentioned essential elements of signal transduction for LTP. In mature rats deep  level of anaesthesia with either urethane, isoflurane or sevoflurane is, however, insufficient to pre-empt LTP induction of C-fibre-evoked field potentials . LTP is lonidine  or spinalonidine  also preFunctional blockade of glial cells by i.t. administration of fluorocitrate changes the polarity of HFS induced synaptic plasticity. When HFS is given 1 h, but not 3 h after fluorocitrate LTD but no LTP of C-fibre-evoked field potentials is induced .LTP of C-fibre-evoked field potentials can be reversed by brief, high frequency conditioning electrical stimulation of sciatic nerve fibres at Aδ-fibre intensity . ReversaSpinal application of either, NK1 or NK2 receptor antagonists one to three hours after HFS, i.e., after LTP is established, does not affect maintenance of LTP , suggestModulation of synaptic strength is a powerful mechanism to control signal flow in selected pathways. A typical consequence of LTP at excitatory synapses would be an increase in action potential firing of the same and perhaps also of downstream neurons in response to a given stimulus. And indeed, LTP-inducing conditioning stimuli have been found to facilitate action potential firing of multireceptive neurons in deep dorsal horn e.g. -48. ThisHFS of sciatic nerve fibres which induces LTP at synapses of C-fibres in spinal cord has behavioural consequences in rats and causes thermal hyperalgesia at the ipsilateral hind paw for six days . This suAn indispensable proof for any proposed mechanism of hyperalgesia is an appropriate correlate in the human. And indeed, conditioning HFS of cutaneous peptidergic afferents in humans cause increased pain perception in response to electrical test stimuli applied through the same stimulation electrode . NoxiousInterestingly, all thermal modalities comprising cold and warm detection thresholds, cold and heat pain thresholds as well as pain summation  remain unaltered after conditioning HFS of peptidergic skin nerve fibres .When verbal pain descriptors are used to evaluate pain in addition to its perceived intensity after HFS, a significant long-term increase in scores for sensory but not for affective descriptors of pain is detected . Within In humans subjects conditioning LFS causes also an increased pain sensitivity in the area around the LFS conditioned skin site but a depression of pain evoked by stimulation through the same electrode .LTP at synapses between primary afferent C-fibres and a group of nociceptive neurons in spinal cord lamina I which express the NK1 receptor for substance P is a potential mechanism underlying some forms of pain amplification in behaving animals and perhaps human subjects. Both, LTP and hyperalgesia involve the same essential elements, i.e. primary afferent C-fibres and lamina I neurons which express the NK1 receptor. Further, induction protocols, pharmacological profile and signal transduction pathways are virtually identical.AMPA α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acidCaMKII Calcium-calmodulin-dependent protein kinase IICNS Central nervous systemHFS High frequency stimulationIASP International Association for the Study of Pain3 Inositol 1,4,5-trisphosphateIPLFS Low frequency stimulationLTP Long-term potentiationMAPK Mitogen-activated protein kinaseNK1 Neurokinin 1NK2 Neurokinin 2NMDA N-methyl-D-aspartateNOS Nitric oxide synthasePKA Protein kinase APKC Protein kinase CPLC Phospholipase CThe author declares that he has no competing interests.


SM/HIV-2 lineage that is believed to have originated by duplication of the common vpr gene present in primate lentiviruses. Vpx is incorporated into virion particles and is thus present during the early steps of viral infection, where it is thought to drive nuclear import of viral nucleoprotein complexes. We have previously shown that Vpx is required for SIVMAC-derived lentiviral vectors (LVs) infection of human monocyte-derived dendritic cells (DCs). However, since the requirement for Vpx is specific for DCs and not for other non-dividing cell types, this suggests that Vpx may play a role other than nuclear import.Vpx is a non-structural protein coded by members of the SIVSM/HIV-2 lineage. At a molecular level, Vpx acts by promoting the accumulation of full length viral DNA. Furthermore, when supplied in target cells prior to infection, Vpx exerts a similar effect following infection of DCs with retroviruses as divergent as primate and feline lentiviruses and gammaretroviruses. Lastly, the effect of Vpx overlaps with that of the proteasome inhibitor MG132 in DCs.Here, we show that the function of Vpx in the infection of DCs is conserved exclusively within the SIVOverall, our results support the notion that Vpx modifies the intracellular milieu of target DCs to facilitate lentiviral infection. The data suggest that this is achieved by promoting viral escape from a proteasome-dependent pathway especially detrimental to viral infection in DCs. SM/HIV-2 lineage, but absent in HIV-1 and in most SIV lineages [Vpx is a non-structural protein coded by members of the SIVlineages -12. Howelineages -19. It iMAC LVs, while it is largely dispensable for the infection of other non-dividing cell types [in trans by pre-incubation of DCs with non-infectious SIVMAC VLPs. In this setting, VLPs composed of viral structural and accessory proteins but devoid of viral genome are simply used as carriers of those viral proteins that are normally delivered in target cells upon viral infection. By analyzing VLPs of different composition, we have determined that Vpx is the sole viral protein required for the positive effect of SIVMAC VLPs . Upon ppx-VLPs, . This efSM/HIV-2 lineage act by promoting the rapid accumulation of full length viral DNA following infection with a wide variety of retroviruses. More importantly, we discovered that MG132, a known proteasome inhibitor, partially rescues the defect of Vpx-deficient SIVMAC LVs and displays effects similar and non-additive to Vpx in the infection of DCs by HIV-1.Here, we investigated Vpx functions at a molecular level and showed that Vpx proteins derived from different strains of the SIVSM/HIV-2 lineage, DCs were infected with SIVMAC and HIV-2 LVs coding or lacking Vpx. Cells were analyzed 3 days later by flow cytometry to score GFP positive infected cells . On the contrary, pre-incubation completely rescued the defect of Vpx-deficient HIV-2 LVs, demonstrating that HIV-2 and SIVMAC Vpx proteins have conserved functions.Given that Vpx rescues the infectivity defect of Vpx-deficient SIVLVs Fig. . Pre-incMAC and HIV-2 were compared with the one derived from the red capped mangabey SIV (SIVRCM) with which they share only 30% sequence identity. Proteins were flag-tagged at their N-terminus, as no available antibody allowed SIVRCM Vpx detection. The ability of these proteins to functionally replace SIVMAC Vpx was first assayed in the context of SIVMAC LVs infection  and virion particles were purified and normalized. DCs were then infected at high and low viral inputs , these results suggest that the function of Vpx in the infection of DCs is unique to members of this lineage.Given that Vpx proteins derived from other strains of the SIVMAC LVs containing or not Vpx  were readily detected, although a minor defect in MSSS accumulation was observed at 24 hrs post infection in the absence of Vpx . In contrast, accumulation of full length (FL) viral DNAs was drastically reduced in the absence of Vpx (at least 100 fold). Not surprisingly, no episomal 2LTRs forms were detected in this case.MAC infection.These results strongly suggest that Vpx is required in DCs for the accumulation of full length viral DNA during the early steps of SIVTo explain the positive effect of Vpx on the efficiency of infection of an heterologous virus , two hyTo characterize the effect of Vpx on heterologous viruses infection, the accumulation of viral DNA products was examined Fig.  and 3D. Overall, these results indicate that Vpx promotes the accumulation of full length viral DNA in DCs following homologous as well as heterologous retrovirus infection. Given the low sequence conservation between viral elements of MLV, FIV and HIV, we believe these results strongly argue that Vpx modifies the intracellular environment of DCs to the virus advantage.Viral DNA accumulation most likely relies on multiple factors such as RT synthesis rates and viral nucleoprotein complexes stability or trafficking to favorable intracellular locations. To gain further insights into Vpx function, a more detailed time course analysis of complete reverse transcripts accumulation was carried out on DCs infected with HIV-1 LVs with or without Vpx-VLPs pre-incubation Fig. . HIV-1 w+ cells is similar if the drug is absent or added at this early time point.To prove that viral genomes synthesized early in presence of Vpx are truly infectious and that they have an advantage over DNA produced at later times, DCs were infected with HIV-1 LVs (at MOI 1 and 10) in presence or absence of Vpx-VLPs pre-incubation. Infections were blocked after 7 hrs with the nonnucleoside inhibitor Nevirapine and compared to untreated samples 5 days post-infection by flow cytometry Fig. . NevirapMAC LVs lacking Vpx or HIV-1 LVs  when compared to the effect of Vpx. These results suggest that Vpx promotes viral DNA accumulation via a separate mechanism.Arsenic acid is a drug known to enhance reverse transcription efficiency in certain cell types by an unknown mechanism . As Vpx MAC LVs, DCs were infected in presence or absence of the drug for 7 hrs prior to media replacement and drug and virus removal. If cells were analyzed by flow cytometry 2 days later, MG132 treatment didn't consistently rescue the infectivity defect caused by the absence of Vpx (data not shown). We hypothesized this could be due to the drastic defect of Vpx-deficient SIVMAC LVs and to the short time of treatment to which the experiments were constrained. However, if infections were analyzed by PCR 24 hrs post-infection, MG132 partially relieved the block in FL viral DNA accumulation . Lastly, the positive effect of Vpx is maintained if Vpx-VLPs and infectious LVs enter DCs via distinct entry pathways , supporting the notion that Vpx targets cellular rather than viral components.We believe that multiple evidences support the hypothesis that Vpx modifies DCs by counteracting a specific restriction mechanism. Vpx provided The kinetic analysis of full length viral DNA accumulation following HIV-1 infection revealed that Vpx speeds up the completion of the RT process, a reaction that seems relatively slow in DCs. Indeed, the majority of viral DNA is synthesized by 7 hrs in presence of Vpx as opposed to 48 hrs in its absence. Despite the fact that at 48 hrs post-infection equivalent amounts of viral DNA accumulated in both conditions, the viral DNA synthesized in presence of Vpx is by far more infectious. This suggests that viral genomes that are not completed in a short time are more likely to be targeted by anti-viral cellular defense mechanisms that diminish their infectivity. Given that the viral DNA is contained within a nucleoprotein complex that chaperones it through its life cycle, such defenses may act at multiple steps. In this respect, by promoting the completion of viral DNA synthesis by RT, Vpx may drive structural rearrangements in viral complexes that alter their stability or their trafficking within the cytoplasm with the result of protecting them.MAC LVs lacking Vpx. MG132 had an effect of the same order of magnitude of Vpx on HIV-1 infection but the two effects were not additive. Lastly, the positive effect of proteasome inhibitors on viral infection of most cell types appears much milder than the one observed here in DCs  are unaffected by Vpx.A Vif-insensitive restriction block specified by APOBEC3G molecules present in the form of low molecular weight complexes has been described in cells resistant to HIV-1 infection, such as quiescent lymphocytes, monocytes and more recently DCs ,30. HoweMAC LVs is not relieved with increasing amounts of viral targets and human TRIM5α is not known to target HIV-1 nor SIVMAC infection. This suggests that Vpx may act by counteracting a distinct restriction pathway that remains to be identified.Our data may be reminiscent of the tripartite motif protein 5alpha-induced restriction (TRIM5α) and of its negative impact on lentiviral infection ,32. HoweSM in macaques. Our data indicates that Vpx exerts a unique function in lentiviral infection of DCs by promoting a rapid accumulation of complete viral DNA forms and in mediating the escape of viral genomes from a proteasome-dependent pathway that restricts viral infection in such cells. Given the central role of DCs in viral spread, these results may partly explain the drastic phenotype of Vpx mutants in vivo.Vpx is required for viral spread and dissemination of SIVHuman primary lymphocytes and monocytes were obtained from peripheral blood mononuclear cells (PBMCs) of healthy donors at the Etablissement Français du Sang de Lyon . MonocytMAC251 (SIVMAC in the text), HIV-2 and FIV-based lentiviral vectors, as well as the murine leukemia virus (MLV) retroviral vector have been described elsewhere [gag-pro-pol and viral accessory proteins; a miniviral genome bearing a CMV-GFP expression cassette; and a vesicular stomatitis virus G envelope protein (VSVg) that confers them ample cellular tropism [The HIV-1, SIVlsewhere ,35-38. T tropism ,35,38,39 tropism ). VirionMAC Vpx-deficient packaging construct has been described previously . Unless otherwise specified, non-infectious SIVMAC Vpx containing VLPs were produced from complete packaging vectors and VSVg pseudotyped (Vpx-VLPs in the text), as we had shown that Vpx is the only protein of SIVMAC required for their positive effect [The SIV. The HI, and Sigma .MAC251, HIV-1, FIV or MLV sequences; acc. n°D01065, M38432, NC_001482 and Z1118, respectively): minus-strand strong-stop, MSSS, PE103-AGTCGCTCTGCGGAGAGGCTG (nt 507–527) and PE83-TGCTAGGGATTTTCCTGC (nt 789–807) for SIVMAC251, AC35-GCCTCAATAAAGC TTGCCTTG (nt 522–542) and AC117-GCATG CTGCTAGAGATTTTCCACAC (nt 616–635) for HIV-1, AC373-GAGTCTCTTTGTTGAG GACTTTTG (nt 217–240) and AC374-TGCG AAGTTCTCGGCCCGGATTCCG (nt 331–355) for FIV, AC311-GTCCTCCGATAGACTGAGT C and AC312-GTAGTCAATCACTCTGAG for MLV; full length, FL, 39-CCGTCGTGGTTGG TTCCTGCCG (nt 878–899) and 40-GCTAGA TACCCAGAAGAGTTGGAAG (nt 294–309) for SIVMAC251, AC37-CACTCCCAACGAAGAC AAG (nt 9100–9120) and AC38-CAGCAAGCC GAGTCCTGCGT for HIV-1 (nt 699–708), AC375-TGGGATGAGTATTGGAACCCTGAA G (nt 1–25) and AC376-TTTCTATTGCTCTAG CTTCACTTCC (nt 394–419) for FIV, AC310-CTCAGCAGTTTCTTAAGACCC (nt 8094–8114) and AC267-GATCTGAGCCTATTGATC GATC (nt 44–65) for MLV; 2LTRs circles, PE107-AGCTGCCATTTTAGAAGTAAGCC (nt 664–686) and PE151-TCTGACAGGCCTGA CTTGC (nt 318–336) for SIVMAC251, AC34-TCC CAGGCTCAGATCTGGTCTAAC (nt 465–489) and AC35 for HIV-1, AC377-TGTCGAGTAT CTGTGTAATCTTTTTTACC (nt 292–320) and AC378-AAAAGTCCTCAACAAAGAGACTC (nt 217–239) for FIV, AC292-GCTGTTGCAT CCGACTCGTG (nt 60–79) and AC293-CACC GCAGATATCCTGTTTG (nt 7975–7994) for MLV ; mitochondrial DNA, 98-GAATGTCTG CACAGCCACTTTCCAC and 99-GATCGTGG TGATTTAGAGGGTGAAC; actinup-CGAGA AGATGACCCAGATC, actindown-TGCCGCC AGACAGCACTGTG. Probe sequences were: primer PE107 for SIVMAC251 MSSS and FL; primer 40 for SIVMAC251 2LTRs; primer AC36-TAGAGATCCCTCAGACCCTT (nt 589–608) for HIV-1 MSSS, FL and 2LTRs; primer AC292 for MLV MSSS and FL, AC 312 for MLV 2LTRs; mitoprobe100-TGGGGTTTGGC AGAGATGT; actinprobe-GGAGAAGAGCTA CGAGCTGC.Infections were generally carried out at MOIs comprised between 2 and 10 and PCR analysis carried out on serial five-fold dilutions of cellular lysate, as previously described . Primer The author(s) declare that they have no competing interests.CG: carried out experiments, data analysis and contributed to writing of the manuscriptLR: carried out initial cloning of Vpx proteins from different SIV strainsLJW: purified blood material, tested proteasome inhibitors cytotoxicity and contributed to virion preparationsJB: provided blood materialDR: provided blood materialJLD: data analysis and study designAC: study design, data interpretation and supervision


The melanosome is a secretory organelle unique to the melanocyte and its neoplastic counterpart, malignant melanoma. The synthesis and assembly of these intracytoplasmic organelles is not yet fully understood. We have developed a murine monoclonal antibody (MoAb) against melanosomes isolated from human melanocytes (newborn foreskin) cultured in the presence of 12-O tetradecanoyl phorbol-13-acetate (TPA). This MoAb, designated HMSA-5 (Human Melanosome-Specific Antigen-5) (IgG1), recognised a cytoplasmic antigen in both normal human melanocytes and neoplastic cells, such as common and dysplastic melanocytic nevi, and malignant melanoma. None of the carcinoma or sarcoma specimens tested showed positive reactivity with MoAb HMSA-5. Under immunoelectron microscopy, immuno-gold deposition was seen on microvesicles associated with melanosomes, and a portion of the ER-Golgi complexes. Radioimmunoprecipitation analysis showed that the HMSA-5 reactive antigen was a glycoprotein of M(r) 69 to 73 kDa. A pulse-chase time course study showed that the amount of antigen detected by MoAb HMSA-5 decreased over a 24 h period without significant expression on the cell surface, or corresponding appearance of the antigen in the culture supernatant. This glycoprotein appears to play a role in the early stages of melanosomal development, and the HMSA-5 reactive epitope may be lost during subsequent maturation processes. Importantly, HMSA-5 can be identified in all forms of human melanocytes, hence it can be considered a new common melanocytic marker even on routine paraffin sections.


Sixty-eight patients with advanced breast cancer were treated with mitoxantrone and clinical responses assessed. Expression of c-erbB-2 protein and cytosolic glutathione S-transferase (GST) isoenzymes pi, alpha and mu by the primary tumours of these patients was determined immunohistochemically, and correlated with treatment response. Tumours overexpressing c-erbB-2  showed a lower response rate (50% vs 58%) and shorter duration of response to treatment, compared with c-erbB-2 negative tumours. These associations were not statistically significant but survival following start of treatment was significantly shorter in the c-erbB-2 positive group. For each GST isoenzyme, the response rate and duration of response of the group showing enzyme expression did not differ significantly from those with negatively staining tumours. These data do not support a role for expression of GSTs alone in resistance to mitoxantrone monotherapy in advanced breast cancer. The poorer post treatment survival of patients with c-erbB-2 positive tumours suggests they could be selected for more intensive treatment regimens.


A study has been carried out in which a comparison was made between EL4 lymphoma (assumed to be an insulin-producing secreting tumour) and thymoma (an insulin-dependent tumour). Tumour development and incidence, 3H-thymidine incorporation and insulin content in tumours, the host's food intake, blood insulin, glucose and cholesterol were determined in non-diabetic and alloxan-diabetic mice. Whereas no significant differences were observed between the diabetic and non-diabetic EL4 tumour-bearing mice, the diabetic, thymoma tumour-bearing mice showed reduced tumour growth and lower tumour incidence as compared with their non-diabetic counterparts. Insulin administration to diabetic tumour bearing mice, enhanced 3H-thymidine incorporation in the thymoma tumour cells only, and the insulin content of the EL4 tumours was found to be higher than that of the thymoma tumours. Rapid diabetes remission was observed in the diabetic, EL4 tumour-bearing mice as compared with the thymoma tumour-bearing mice.


C) into strain dependent isoforms of scrapie associated protease resistant isoform (PrPSc) of prion protein (PrP). These processes are determined by similarities as well as strain dependent variations in the PrP structure. Selective self-interaction between PrP molecules is the most probable basis for initiation of these processes, potentially influenced by chaperone molecules, however the mechanisms behind these processes are far from understood. We previously determined that polymorphisms do not affect initial PrPC to PrPSc binding but rather modulate a subsequent step in the conversion process. Determining possible sites of self-interaction could elucidate which amino acid(s) or amino acid sequences contribute to binding and further conversion into other isoforms. To this end, ovine – and bovine PrP peptide-arrays consisting of 15-mer overlapping peptides were probed with recombinant sheep PrPC fused to maltose binding protein (MBP-PrP).The common event in transmissible spongiform encephalopathies (TSEs) or prion diseases is the conversion of host-encoded protease sensitive cellular prion protein . The first high binding area comprises sheep-PrP peptides 43–102 (AA 43–116), including the N-terminal octarepeats. The second high binding area of sheep-PrP peptides 134–177 (AA 134–191), encompasses most of the scrapie susceptibility-associated polymorphisms in sheep. This concurs with previous studies showing that scrapie associated-polymorphisms do not modulate the initial binding of PrPC to PrPSc. Comparison of ovine – and bovine peptide-array binding patterns revealed that amino acid specific differences can influence the MBP-PrP binding pattern. PrP-specific antibodies were capable to completely block interaction between the peptide-array and MBP-PrP. MBP-PrP was also capable to specifically bind to PrP in a Western blot approach. The octarepeat region of PrP seems primarily important for this interaction because proteinase K pre-treatment of PrPSc completely abolished binding.The peptide-arrays revealed two distinct high binding areas as well as some regions of lower affinity in PrPC-PrPC as well as PrPC-PrPSc may be possible, which ultimately lead to variations in species barrier and strain differences.Binding of MBP-PrP to PrP-specific sequences indicate that several specific self-interactions between individual PrP molecules can occur and suggest that an array of interactions between PrP Sc) mainly in tissues of the central nervous system. TSEs (or prion diseases) include (among others) familial, sporadic and variant Creutzfeldt-Jacob disease in humans, bovine spongiform encephalopathy (BSE) in cattle, and scrapie in sheep. Formation of PrPSc is a posttranslational process and involves refolding (conversion) of the host-encoded prion protein (PrPC) into partially protease resistant forms (PrPSc)  is repeated five times in the ovine peptide-array and six times in the bovine peptide-array , capable of selective immuno-precipitation of prion infectivity  sequence occurs twice in the PrP sequence. AA [152-YYR-154]  have also been shown to be a determinant of conversion [YYR-154] (together with [140-PLIHFGNDY-148]) is part of the of the epitope of the motif-grafted antibody containing mouse AA 136–158, capable of selective immuno-precipitation of prion infectivity [YYR] domain has also been described as the epitope of an antibody that selectively recognizes PrPSc [NFV-179] is the common AA sequence of three consecutive peaks of which the fist two peaks encompass amino acids of a peptide (corresponding to AA 163–176) that inhibits in vitro conversion [The second high binding area contains 3 different interaction domain consensuses. This area also includes most of the polymorphisms found in sheep PrP48] Fig.  is situa48] Fig. , S1 and ectivity . This doectivity  by affecr prions . The YY54] Fig.  is situa4 Fig.  i67] Fig.  is situaectivity . The , but may be a result of a cross-interaction with WN, instead of binding to domain [102-WNK-104] within the first high binding area. Binding with domain consensus [42-PGQGSPGG-49]  is part of the non-linear epitope of the PrPSc specific monoclonal antibody 15B3 [The remaining lower binding areas are more difficult to interpret. The domain consensus for the peptides 22–33 Fig.  seems to49] Fig.  is relat93] Fig.  and [19202] Fig.  is also . C Fig. , H2 whicSeveral studies identified antibodies ,19,23-26et al.  only when these AA were no longer present in the peptides. The supposedly neutral substitution of glycine for serine at AA 100 is most likely responsible for the observed differences in binding and may be attributed to the greater conformational flexibility of G, affecting availability of other AA's for interaction with MBP-PrP. Taking this in account, binding with peptides 90–92 should be a result of cross-binding with two consecutive Glycines present in these peptides in stead of binding with the consensus octarepeat domain. Glutamatic acid only contains an oxygen in place of the amido group in glutamine and therefore these AA's are considered readily interchangeable ) for and subsequent heating 10 min. at 96°C. This aliquot was de-glycosylated in the presence of 1000 U PNGase F/ml for at least 36 hours at 37°. Untreated, PK treated – and PNGase F treated brain homogenates samples were analyzed by SDS-PAGE and Western blot. As positive controls His-PrP (positive PrP control) and MBP-PrP (positive detection control) were included. Reverse detection of PrP was accomplished by incubating the Western blot for 1 hour at room temperature with approximately 2 ng/μl MBP-PrP followed by immunodetection using mouse anti-MBP monoclonal antibody and rabbit anti-mouse-IgG-peroxidase (RAMPO). PrP was also detected on Western blot using the PrP-specific monoclonal antibody 9A2 and RAMPO. To determine if detection is PrP specific, a Western blot was carried out with MBP alone instead of MBP-PrP.TSE: transmissible spongiform encephalopathy, PrP: general denotation for prion protein, CPrP: host-encoded cellular prion protein (protease sensitive), ScPrP: scrapie associated isoform of the prion protein , MBP-PrP: fusion protein of maltose binding protein linked to the N-terminus of the mature sheep prion protein. Non-Polar Amino Acids; I: isoleucine, V: valine, L: leucine, F: phenylalanine, M: methionine, A: alanine, G: glycine, W: tryptophane, P: proline (ordered from most hydrophobic to most hydrophilic). Neutral Polar Amino Acids; C: cysteine, T: threonine, S: serine, Y: tyrosine, N: asparagine, Q: glutamine (ordered from most hydrophobic to most hydrophilic). Charged Polar Amino Acids; H: histidine, D: aspartate, E: glutamatic acid, K: lysine, R: arginine (ordered in accordance to increasing hydrophilicity)AR and AB conceived the study and together with JPML were responsible for study design and coordination. PLJMM was involved in later stage study design and responsible for development and testing of MBP-PrP. JGJ was responsible for the production and testing of monoclonal antibody 6C2. All peptide-arrays were performed at Pepscan B.V. by DPT and all other experiments were performed by AR. JPML and AR were responsible for choice of – and analyses of peptide-arrays. AB is project manager of the NWO-project under which this study was performed. AR and AB were responsible for data-analysis. AR drafted the manuscript and AB and JPML critically read the manuscript before submission. All authors have read and approved the final manuscript.


Anti-cyclic citrullinated peptide (CCP) antibody is a useful marker for thediagnosis and prognosis of rheumatoid arthritis (RA). Recently, clinicalsignificance of follow-up in anti-CCP antibody titer has been pointedout. Thus, we investigated the serial determination in anti-CCP antibodiestiter in RA patients. Six patients with RA, who were followed up for longer than 5 years, wereassessed in anti-CCP antibodies and radiographs (Larsen score). Anti-CCPantibodies in frozen sera were measured using ELISA. As a result, 6 patientswith RA were divided into two groups: one possessed high titers withoutvariation, and the other was without high titers. Joint damage progressedduring observation in 2 out of 3 patients with high anti-CCP titers in aretrospective assessment. In contrast, the RA patient, whose anti-CCP titerdecreases although it had been high titer at baseline, did not show increasein the Larsen score. These findings suggest that it might be necessary toanalyze changes in anti-CCP to predict the prognosis of joint destruction. Rheumatoid arthritis (RA) is a chronic inflammatory autoimmunedisease characterized by progressive cartilage erosion anddestruction. Previous investigations have shown thatautoantibodies to a cyclic citrullinated peptide (anti-CCP) arehighly specific for RA, including the early form, and that theseantibodies may be of prognostic value as markers predictingprogression to more serious disease –3. RecenFirstly, to confirm the specificity and sensitivity of anti-CCPantibodies in our hospital, we assessed anti-CCP titer in RA orother various autoimmune diseases see before sIn the longitudinal observation, anti-CCP antibody was detected insera obtained from 6 RA patients who were followed upfor 5 years. Each serum sample was frozen at −80°C andstored. The 6-patient profiles at the start of follow-up aredemonstrated in Anti-CCP titers in patients with RA or other autoimmune diseasesare shown in Anti-CCP titers changes in the 6 patients are shown inWe show the variations in radiographic damage in each patientaccording to Larsen and Dale  and LindFinally, the decrease in titers of anti-CCP autoantibodies, evenif high titer at baseline, may predict the suppression of jointdamage. Serially determined anti-CCP antibodies as well asbaseline determination should be considered from the predictableviewpoint.


Objective: Bacterial vaginosis (BV) is characterized by a shift in vaginal tract ecology, which includes a decreasein the concentration and/or prevalence of facultative lactobacilli. Currently, mechanisms which could accountfor the disappearance of lactobacilli are not well understood. The objective of this study was to determine whethervaginal streptococci/enterococci can produce bacteriocin-like inhibitors antagonistic to vaginal lactobacilli.Methods: Seventy strains of vaginal streptococci or enterococci were tested for antagonistic activities againstvaginal lactobacilli using the deferred antagonism technique.Results: One strain, Enterococcus faecium 62-6, which strongly inhibited growth of lactobacilli was selected forfurther characterization. The spectrum of inhibitory activity of strain 62-6 included Gram-positive organismsfrom the vaginal environment, although native lactobacilli from the same host were resistant to inhibitor action.Following growth inMRSbroth the strain 62-6 inhibitor was shown to be heat- , cold-  and pH- (4–7) stable. The sensitivity of inhibitor-containing supernatants to pepsin andα-chymotrypsin suggested an essential proteinaceous component. The inhibitor was sensitive to lipase but resistantto lysozyme. Dialysis of inhibitor-containing culture supernatants suggested a molecular mass greater than12 000 Da. All physicochemical properties were consistent with its classification as a bacteriocin-like inhibitor.Kinetic assays demonstrated a sharp onset of inhibitor production coinciding with a concentration of 62-6 of107 cfu/ml, suggesting that production may be regulated by quorum sensing.Conclusions: These results may have clinical significance as a novel mechanism to account for the decline of vaginalLactobacillus populations and contribute to both the establishment and recurrence of BV.


The biochemical basis of multidrug-resistant (MDR) phenotype has been investigated in drug-resistant sublines independently obtained in our laboratories by single step doxorubicin (DOX) selection of LoVo, DLD1, and SW948 human colon carcinoma (HCC) cell lines. All the chemoresistant sublines have been found to be cross-resistant to DOX, actinomycin-D (ACT-D) and vincristine (VCR) but not to cis-diamminedichloroplatinum (CDDP), and have exhibited an increased expression level of mdr1 mRNA and gp170 glycoprotein. Comparative analyses in drug-resistant and sensitive cells of resistance index, extracellular and intracellular equitoxic DOX concentrations, and mdr1 gene products expression have indicated that MDR phenotype is a multifactorial phenomenon due to different and possibly independent biochemical mechanisms which cooperate, in varying degrees from cell line to cell line, in conferring cellular chemoresistance.


Activated macrophages express a cell surface receptor for the vitamin folic acid. Because this receptor is inaccessible or not measurably expressed on other normal cells, folic acid has been recently exploited to selectively deliver attached radio-emitters to sites of activated macrophage accumulation, allowing scintigraphic imaging of inflamed joints and organs of arthritic rats. We demonstrate here that folate-linked haptens can also be targeted to activated macrophages, decorating their cell surfaces with highly immunogenic molecules. Under conditions in which the rodent has already been immunized against keyhole limpet hemocyanine-(fluorescein isothiocyanate) FITC, activated macrophages are eliminated. Administration of folate-FITC conjugates to rodents with experimental arthritis attenuates (a) systemic and peri-articular inflammation, (b) bone and cartilage degradation, and (c) arthritis-related body weight loss. Treatment with folate-hapten conjugates is comparable to methotrexate, etanercept, anakinra, and celecoxib at alleviating the symptoms of arthritis. We conclude that reduction of activated macrophages by folate-targeted immunotherapy can ameliorate the symptoms of arthritis in two rodent models of the disease. Rheumatoid arthritis (RA) is an autoimmune disease characterized by inflammation of the joints and destruction of cartilage and bone. Activated macrophages have been identified as a key mediator of the disease, as numbers and level of macrophage activation correlate with the extent of joint inflammation and bone degradation -4. In adMany therapies for RA are specifically designed to eliminate pro-inflammatory byproducts of activated macrophages . RemicadEfforts have been made to eliminate the entire population of macrophages -19, but D approximately 10-10 M) [Activated macrophages from patients and rodents with arthritis over-express a cell surface receptor for folic acid that also binds folate-linked compounds with high affinity (K10-10 M) -24. Rece10-10 M) -25. We hMycoplasma butyricum ; light mineral oil, methotrexate (MTX), clodronate, bovine serum albumin, keyhole limpet hemocyanine (KLH), anti-rat immunoglobulin G (IgG)-horseradish peroxidase (HRP), IgG-phycoerythrin conjugates, and alum ; biotin-conjugated goat anti-rat IgG1, IgG2a, and IgG (H+L), and streptavidin-conjugated HRP ; aminofluorescein (single isomer) and fluorescein isothiocyanate (FITC) ; Microcon-30 membranes ; TiterMax Gold® adjuvant ; Celecoxib ; entanercept and anakinra ; EC20 (a folate-linked chelator of 99mTc) and folate-FITC  were obtained from commercial sources. The studies were approved by the Purdue University Animal Care and Use Committee.Heat-killed ® (two injections each on days 0 and 28) or Alum . Ten days after the last boost, the serum was analyzed for anti-FITC antibodies by enzyme-linked immunosorbent assay [Anti-FITC antibodies were induced in rodent models with experimental arthritis by vaccination with KLH-FITC (KLH-FITC molar ratio of 1:13), using a modification of a published procedure . Rodentsnt assay .LacJ mice (approximately 7 weeks old) . The arthritic rodents were weighed weekly. Arthritis scores were determined using a weighted criterion  and scored by a trained investigator blinded to the treatment groups. When the arthritis score reached 7, mice were randomly assigned to different treatment groups. Rodents were maintained on a folate-deficient diet (Harlan Tec) for 3 weeks prior to each study to lower serum folate levels to their physiologic range (approximately 25 nM) [Adjuvant-induced arthritis (AIA) was promoted in 200-g female Lewis rats  via either the footpad method  or the by 25 nM) .+ cells but cannot mediate anti-FITC antibody binding in the absence of KLH-FITC immunization. Folate-targeted immunotherapy (FTI) was compared with other therapies by treating AIA rats (base-of-tail method) 7 days after arthritis induction with one of the following: FTI , MTX  [Folate-FITC was administered i.p. to KLH-FITC-immunized rodents according to the doses/schedules described in each figure legend. For negative controls, KLH-FITC-immunized rodents were treated with the non-targeted aminofluorescein, which displays no affinity for folate receptors. Alternatively, non-immunized rodents were treated with folate-FITC, which binds readily to FRk, i.p.) , celecoxk, i.p.) , etanerck, i.p.) , clodronk, i.p.) , or anakk, i.p.) -10.To determine whether FTI could ameliorate the symptoms of experimental arthritis in rodent models, disease status was assessed by monitoring changes in limb volume/ankle diameter, radiological score (RAD score), and systemic inflammation. Limb volume was determined by calculating the product of the measured length, width, and height of the limb . To determine the impact of the therapies on bone/cartilage degradation, lateral radiographic projections of the tarsus of each rat were scored at the end of each study. Radiographs were taken with direct exposure (1:1) on un-screen KODAK X-OMAT TL film  using a Faxitron X-ray system with a 0.5-mm focal spot and beryllium window . Radiographs were scored by a board-certified veterinary radiologist blinded to the treatment groups. All radiographs were evaluated by a board-certified radiologist without knowledge of the assignment of treatment groups. RAD scores were assigned according to a modification of a previously described method . The rad+ activated macrophages, scintigraphy and the biodistribution of a folate-99mTc radioimaging agent (EC20) in relevant tissues were also evaluated [To compare the abundance of macrophages in the ankle joints of arthritic rats that were treated with FTI versus phosphate-buffered saline (PBS), sections were analyzed by immunohistochemistry for ED1 antigen using a protocol described previously . Brieflyvaluated .To obtain an indication of the toxicity associated with treatment with FTI, KLH-FITC-immunized rats were injected three times per week for 15 weeks with PBS or folate-FITC (300 nmole/kg). Four rats per group were sacrificed every 3 weeks, and blood was analyzed for indicators of organ function . Solid tissue samples were obtained from each animal, and organs were examined histologically for signs of tissue damage .As documented elsewhere , one dia+ tumors. Thus, it has been demonstrated that some cancer cells over-express a folate receptor and that the surfaces of cancer cells can be decorated with highly immunogenic haptens by targeting their FRs with folate-hapten conjugates [+ tumors, it seemed logical to explore whether a similar approach  that was previously shown to be taken into activated macrophages by FR-mediated endocytosis [99mTc complex (EC20) in spleen, liver, kidneys, and limbs is substantial in non-immunized rats treated with folate-FITC, but largely limited to the kidneys  in KLH-99mTc agent (EC20) in hind limbs was more intense in non-immunized than in KLH-FITC-immunized AIA rats treated with folate-FITC (FTI)  Figure . Taken tp < 0.001). Similar suppression of EC20 uptake was also observed in arthritic rats treated with clodronate liposomes (CL), a bisphosphonate commonly used to deplete all mononuclear phagocytes [To quantitatively assess the degree of systemic depletion of activated macrophages by FTI, uptake of EC20 was analyzed in the spleen and liver of AIA rats by scintillation counting. EC20 content was approximately seven times greater in the organs of both non-immunized rats treated with folate-FITC and KLH-FITC-immunized rats left untreated than in healthy rats or KLH-FITC-immunized animals treated with folate-FITC (FTI) , a considerable reduction of ED1+ (a macrophage-specific marker) cells was also demonstrated by immunohistochemical staining of the inflamed soft tissue of the ankle joint of the FTI-treated rats   and then treated with folate-FITC (FTI) experienced a >60% reduction in swelling of non-injected limbs when compared with untreated arthritic rats Figure . Evidencp < 0.05) , however, the degree of cartilage and bone damage was markedly reduced relative to that of KLH-FITC-immunized rats not treated with folate-FITC  Figure . A similBecause therapeutic data in rodent models of arthritis can be model-specific, the efficacy of FTI was also evaluated in established CIA. All therapies were initiated seven days after onset of CIA, when disease symptoms had already progressed to a severe state. Visual inspection and arthritis scores Figure  revealedFor comparison with the potencies of other common arthritis therapies, a more stringent base-of-tail adjuvant-induced arthritis model was used . The disAIA rats treated with FTI showed a reduction in ankle diameter similar to that promoted by MTX, celecoxib, etanercept, anakinra, and CL Figure . CompariTo assess the long-term toxicity of FTI, both serum and tissues from non-immunized and KLH-FITC-immunized healthy rats treated three times per week for up to 15 weeks with folate-FITC were evaluated by a commercial pathologist . Indicators of kidney (creatinine) and liver (aspartate aminotransferase) function were within normal ranges for the duration of the study Figure . Also, nWe have explored a novel approach for the treatment of experimental arthritis  -5,32-34.+ macrophages. As noted above, activated macrophages constitute a likely orchestrator of inflammation and joint destruction in experimental arthritis [We hypothesize that the efficacy of the FTI is related to its ability to promote systemic depletion of FRrthritis -5,32-34.rthritis . Severalrthritis ,23,25 harthritis , and we Comparative experiments revealed that FTI was as effective as CL, MTX, etanercept, anakinra, and celecoxib at alleviating the symptoms of experimental arthritis. Furthermore, FTI appeared to be better than the above therapies at suppressing systemic inflammation, as measured by both accumulation of EC20 in liver and spleen and by reduction of inflammation-mediated spleen enlargement. This capability to reduce systemic inflammation may be important, because patients with RA have a reduced life expectancy that is caused by systemic, rather than articular, inflammation .An attractive feature of the FTI approach lies in its apparent lack of toxicity. Because FRs are also expressed on the apical surfaces of a few normal epithelia , it might have been anticipated that immune responses would have been elicited against these tissues also. However, fluorescence micrographs show that sites of FR expression on normal epithelia are inaccessible to circulating antibodies , becauseOne concern that must accompany systemic depletion of activated macrophages relates to the possible decline in defense against pathogens. Although no infections/diseases were observed in rats maintained on the therapy for 15 continuous weeks, nor in RA dogs treated for more than 2.5 years , unpublished observations), sufficient time has not yet elapsed to conclude that long-term defects will not emerge during continuous treatment. Although resting macrophages and other immune cells might compensate for elimination of most activated macrophages, it will be important to examine chronically treated animals for deficiencies in tasks normally performed by activated macrophages. Where temporary deficiencies do arise, it might be sufficient to remove the patient from the therapy until the deficiency subsides.A second potential concern might arise from the possible formation of multimeric immune complexes in circulation after injection of the folate-hapten conjugate. However, because folate-FITC contains only one antibody-binding site, it cannot mediate formation of multimeric immune complexes, even though monomeric IgG-folate-FITC complexes are readily observed in circulation. Thus, deposition of macroscopic immune complexes in non-targeted tissues has not constituted a source of observed toxicity.In summary, we have demonstrated that removal of folate-binding macrophages suppresses established arthritis in two animal models of the disease. Because little measurable toxicity accompanied the therapy, these data suggest that removal or inactivation of folate receptor-expressing macrophages might constitute an alternate approach for treatment of the pathology. Thus, in addition to the existing pantheon of drugs aimed at neutralizing secretion products of activated macrophages , examination of folate-targeted drugs that can block the inflammatory activity of the same cell type would seem to warrant further scrutiny .AIA = adjuvant-induced arthritis; CIA = collagen-induced arthritis; CL = clodronate liposomes; FITC = fluorescein isothiocyanate; FR = folate receptor; FTI = folate-targeted immunotherapy; HRP = horseradish peroxidase; Ig = immunoglobulin; IL = interleukin; KLH = keyhole limpet hemocyanine; MTX = methotrexate; PBS = phosphate-buffered saline; RA = rheumatoid arthritis; RAD score = radiographic score; TNF-α = tumor necrosis factor alpha.PSL has shares in Endocyte, Inc.CMP designed the project, carried out the animal studies, and drafted the manuscript. BV designed the project, carried out the biodistribution studies, and drafted the manuscript. WRW blindly scored radiographs and contributed intellectually to the project. GJB carried out scintigraphic imaging of the rodents and contributed intellectually to the project. EV carried out the biodistribution and whole-body scintigraphic studies. PSL supported and contributed intellectually to the project and carefully edited the manuscript. All authors read and approved the final manuscript.


ATM  is an important cancer susceptibility gene that encodes a key apical kinase in the DNA damage response pathway. ATM mutations in the germ line result in ataxia-telangiectasia (A-T), a rare genetic syndrome associated with hypersensitivity to double-strand DNA breaks and predisposition to lymphoid malignancies. ATM expression is limited by a tightly regulated nonsense-mediated RNA decay (NMD) switch exon (termed NSE) located in intron 28. In this study, we identify antisense oligonucleotides that modulate NSE inclusion in mature transcripts by systematically targeting the entire 3.1-kb-long intron. Their identification was assisted by a segmental deletion analysis of transposed elements, revealing NSE repression upon removal of a distant antisense Alu and NSE activation upon elimination of a long terminal repeat transposon MER51A. Efficient NSE repression was achieved by delivering optimized splice-switching oligonucleotides to embryonic and lymphoblastoid cells using chitosan-based nanoparticles. Together, these results provide a basis for possible sequence-specific radiosensitization of cancer cells, highlight the power of intronic antisense oligonucleotides to modify gene expression, and demonstrate transposon-mediated regulation of NSEs. ATM inhThe average length of SSOs employed in our screening was close to the minimum for unique targets . ShorterAlthough SSO-NSE3 and other SSOs can repress endogenous NSE-containing mRNAs  18] and and18] a


These computational simulations show that cross-bridge binding increased during slow-velocity concentric and eccentric contractions, compared to isometric contractions. Over the full ranges of velocities that we simulated, cross-bridge cycling and energy utilization (i.e. ATPase rates) increased during shortening, and decreased during lengthening. These findings are consistent with the Fenn effect, but arise from a complicated relationship between velocity-dependent cross-bridge recruitment and cross-bridge cycling kinetics. We also investigated how force production, power output, and energy utilization varied with cross-bridge and myofilament compliance, which is impossible to address under typical experimental conditions. These important simulations show that increasing cross-bridge compliance resulted in greater cross-bridge binding and ATPase activity, but less force was generated per cross-bridge and throughout the sarcomere. These data indicate that the efficiency of force production decreases in a velocity-dependent manner, and that this behavior is sensitive to cross-bridge compliance. In contrast, significant effects of myofilament compliance on force production were only observed during isometric contractions, suggesting that changes in myofilament compliance may not influence power output during non-isometric contractions as greatly as changes in cross-bridge compliance. These findings advance our understanding of how cross-bridge and myofilament properties underlie velocity-dependent changes in contractile efficiency during muscle movement.Muscles produce force and power by utilizing chemical energy through ATP hydrolysis. During concentric contractions (shortening), muscles generate less force compared to isometric contractions, but consume greater amounts of energy as shortening velocity increases. Conversely, more force is generated and less energy is consumed during eccentric muscle contractions (lengthening). This relationship between force, energy use, and the velocity of contraction has important implications for understanding muscle efficiency, but the molecular mechanisms underlying this behavior remain poorly understood. Here we used spatially-explicit, multi-filament models of Ca Vmax). Conversely, forced-lengthening of a contracting muscle (i.e. eccentric contraction) at loads that are greater than that of isometric contraction results in a rapid rise in force [Vmax, the continued consumption of chemical energy means that the system efficiency approaches zero [During concentric muscle contraction, the force generated during shortening decreases non-linearly as the shortening velocity increases. Reciprocally, shortening velocity of a concentrically-contracting muscle increases as the resistive load decreases, until the muscle reaches its maximal unloaded shortening velocity  of myosin may be regulated through phosphorylation of the regulatory light chain, which results in a prolonged myosin duty cycle and slower contractile velocity ,32. Thes-1 , though  pN nm-1 –37. HereVopt) around -1 ML/s . Increasing XB stiffness to 10 pN/nm or decreasing XB stiffness to 1 pN/nm . Decreasing the stiffness of the actin filament by an order of magnitude resulted in decreased force production during isometric contractions, though these differences were negligible during sarcomere shortening . There wWe performed this series of numerical experiments to investigate how force development, cross-bridge kinetics, and ATP utilization are influenced by sarcomere shortening and lengthening across a range of velocities. In addition, we examined how these parameters were affected by changes in cross-bridge and myofilament compliance. Our data show that the typical force-velocity relationship  involvesVmax) is reached. Our simulated force-velocity curves had profiles which were comparable to those obtained from empirical measurements, including a rapid rise in force during lengthening transients that plateaued at a value roughly 50% greater than isometric conditions (Vmax measured with default myofilament stiffness (3 pN/nm) was within the range of empirical values measured in mammalian skeletal muscle [Vopt toward a slower velocity, and the opposite trend was observed when cross-bridge stiffness was decreased  phosphorylation status represents one of the principal modulators of actomyosin cross-bridge compliance in empirical studies , where RAccording to recent empirical studies, the myosin ATPase rate may be reduced when the lever arm is strained ,49, and We were surprised to see that the force-velocity relationship was relatively insensitive to myofilament compliance. Increasing or decreasing thick-filament compliance had little or no observable effect on the force- or power-velocity relationships (data not shown), and while some variance was observed with altered thin-filament compliance, these changes disappeared during shortening transients . In addiBoth the amount of force produced and the energy consumed during normal physiological muscle movement fluctuates with muscle length ,50. ThisThe efficiency of muscle force production decreases as shortening velocity increases, an observation first described by Wallace Fenn in 1923 using frog sartorius muscle. However, the molecular mechanisms that contribute to this velocity-dependent change in contractile efficiency are difficult to measure empirically. We used spatially-explicit, multi-filament models of muscle contraction within a half-sarcomere to investigate potential mechanisms that coordinate myosin force production and energy utilization during lengthening and shortening. We found that the fraction of bound cross-bridges increased during slow-velocity concentric and eccentric contractions, but that ATP utilization increased during shortening transients due to faster cross-bridge cycling. The fraction of cross-bridges bound and ATPase activity also both increased in models with reduced cross-bridge stiffness while the force per cross-bridge decreased, resulting in reduced economy of force production within the sarcomere. These findings improve our understanding of how contractile efficiency and power output are modulated throughout dynamic contractions to facilitate muscle movement and locomotion.2+-activated muscle contraction during lengthening, shortening, and isometric conditions. Prior model development and validation primarily focused on matching isometric tension values, Ca2+-sensitivity of contraction, and the associated rates of tension development and ATPase from rabbit fast-twitch muscle [Building upon a spatially-explicit computational model of the half-sarcomere , we simuh muscle –53.2+-activation of troponin and subsequent tropomyosin movement to make thin-filament sites available for myosin cross-bridge binding [In brief, these models comprise 4 thick- and 8 thin-filaments represented as a network of linear elastic springs. Each thick-filament is composed of 61 spring elements linked together in series at ‘nodes’. At each node three myosin head-pairs extend radially from each thick-filament node with 120° between each myosin. From one node to the next the trio of heads rotates 40° to reflect the three-start helical pitch of myosin crowns in vertebrate striated muscle –56. Each binding ). Period2+ is not bound to troponin and the actin-target sites along the thin-filament are unavailable for myosin binding, TF2) Ca2+ is bound to troponin, but the there is no movement of tropomyosin so the binding sites remain unavailable, and TF3) Ca2+ is bound to troponin and the movement of tropomyosin reveals an available myosin biding site. Cross-bridge cycling is also represented as a three-state model: XB1) Myosin is unbound from actin, XB2) myosin is bound to actin as a low-force bearing, pre-power stroke cross-bridge, and 3) myosin is bound to actin as a high-force bearing, post-power stroke cross-bridge. This kinetic scheme includes cooperative activation of thin-filaments from neighboring thin-filament regulatory units and bound cross-bridges and distortion-dependent cross-bridge transition rates identical to our previous models [Thin-filament activation represents three states : TF1) Cas models ,58. Mont-1 for resting (unstrained) elements of length 14.3 and 12.3 nm, respectively. Default cross-bridge stiffness was set at 3 pN nm-1, consistent with the parameter value used in our previous studies and estimates from cellular experiments [pas is the passive force produced by a length change (xoffset) of a half-sarcomere with a given length (xhs). Values for σ (0.4037 N m-2) and L (200.2 nm) were found by curve fitting. At each time-step, titin stiffness was updated to provide the appropriate amount of passive force for a given change in sarcomere length. Potential interactions between titin and the myofilaments were not modeled.Unless otherwise noted, spring elements for the thick- and thin-filaments were 6060 and 5230 pN nmeriments –61. We aeriments :Fpas=σ*0 is isometric tension.Lengthening and shortening transients were achieved by changing the M-line position with respect to the Z-line . Concentric and eccentric contractions were simulated by shortening 100 nm, then held for 250 ms to achieve a stable isometric contraction, followed by re-lengthening to the original half-sarcomere length . This pr


High-throughput sequence (HTS) data exhibit position-specific nucleotide biases that obscure the intended signal and reduce the effectiveness of these data for downstream analyses. These biases are particularly evident in HTS assays for identifying regulatory regions in DNA . Biases may result from many experiment-specific factors, including selectivity of DNA restriction enzymes and fragmentation method, as well as sequencing technology-specific factors, such as choice of adapters/primers and sample amplification methods.We present a novel method to detect and correct position-specific nucleotide biases in HTS short read data. Our method calculates read-specific weights based on aligned reads to correct the over- or underrepresentation of position-specific nucleotide subsequences, both within and adjacent to the aligned read, relative to a baseline calculated in assay-specific enriched regions. Using HTS data from a variety of ChIP-seq, DNase-seq, FAIRE-seq, and ATAC-seq experiments, we show that our weight-adjusted reads reduce the position-specific nucleotide imbalance across reads and improve the utility of these data for downstream analyses, including identification and characterization of open chromatin peaks and transcription-factor binding sites.A general-purpose method to characterize and correct position-specific nucleotide sequence biases fills the need to recognize and deal with, in a systematic manner, binding-site preference for the growing number of HTS-based epigenetic assays. As the breadth and impact of these biases are better understood, the availability of a standard toolkit to correct them will be important.The online version of this article (doi:10.1186/s12859-017-1766-x) contains supplementary material, which is available to authorized users. High-throughput short-read sequencing (HTS) has enabled the genome-wide identification of functional regulatory regions including transcription factor binding sites and epigenomic features such as histone tail modifications and regions of open chromatin. HTS-based assays such as ChIP-seq, DNase-seq, FAIRE-seq, and ATAC-seq generate millions of reads per experiment that then are used to identify regions of interest. However, a combination of biases in these HTS protocols often results in a deviation from the background frequency of nucleotides present at each position in HTS reads, which we call nucleotide-specific bias. As the routine use of HTS is already widespread and increasing, it is especially important to fully understand any biases associated with HTS protocols and take these biases into account when analyzing the resulting data .There are several steps involved in preparing pools of DNA for HTS, each of which may introduce nucleotide-specific bias. All short-read HTS protocols require some form of DNA fragmentation into smaller DNA molecules to facilitate high-throughput sequencing. In many of these assays, including ChIP-seq and FAIRE-seq, this is accomplished by sonication. There is evidence that sonication breaks DNA strands between nucleotides preferentially based on their binding affinity . Most asIn addition, there are a variety of assay-specific steps that may introduce nucleotide biases. In DNase-seq , the DNaMuch of the previous work on correcting biases in HTS data has focused on RNA-seq , 9–11. SRecently, methods have been proposed for correcting nucleotide-specific biases in DNase-seq data. The accurate estimation of cut frequencies in DNase-seq is particularly important in the identification of “footprints”, which correspond to evidence of transcription-factor binding characterized by local dips in digestion within larger DNase peaks . These mSimilarly, methods have been published to address sequence bias in ChIP-seq data by taking into account the contribution of GC content, chromatin structure, and other factors . HoweverWe introduce a method that corrects nucleotide-specific bias in HTS from a variety of DNA-based sequencing assays. Our method computes an accurate baseline nucleotide distribution within the same sample data without the need for extra sequencing and corrects biases that are based on nucleotide composition within and surrounding HTS reads, regardless of the source of bias. We calculate read weights that adjust the distribution of position-specific nucleotide frequencies within the read to match the expected nucleotide frequency based on a random sampling of reads within the target region(s). We demonstrate that this adjustment improves the performance of each of the evaluated protocols for detecting genomic features, including open chromatin regions and transcription-factor binding footprints.Sequence reads from a variety of HTS assays, including DNase-seq, ChIP-seq, FAIRE-seq, and ATAC-seq show distinct position-specific nucleotide biases that differ across assays Fig. . The obsk-mer in each non-overlapping k nucleotide window as described in “fk-mer,r+ik), where fk-mer is the relative frequency of a k-mer at an offset i∗k from the aligned read location r, as our feature space to perform principal component analysis (PCA). Figure To characterize the biases within and differences between experiments, we computed the frequency of every We observed the greatest cumulative bias in DNase-seq and ATAC-seq Fig. . The biak-mers (motifs of length k) starting at each position relative to the start of the aligned reads, including genomic positions upstream and downstream of the reads. For brevity, we call the sliding k-mer windows at each relative position “tiles”, where every aligned read has a specific k-mer at the same “tile” relative to their respective aligned start position. Next, we compute expected baseline k-mer frequencies by sampling randomly from within all reads and a 50-bp margin around each read. This baseline exhibits no significant position-specific nucleotide variance while capturing the expected average nucleotide content of the sequenced feature(s), such as average GC content, in genomic regions being targeted in a particular assay. From this set of tiles, we identify those that are significantly biased - where variance is above the 95% confidence threshold of the baseline variance. The pairwise covariance between k-mer frequencies of all biased tiles is computed. The frequencies of correlated tiles are averaged together; then all independently varying tile groups are compounded to produce an overall read weight. To adjust these weights to reflect the local likelihood of observing a read at a particular locus, we normalize the overall weight by the average weight of simulated reads at every locus within a 20 bp window surrounding the observed read site.Our bias correction method is applied independently for each replicate or sequencing run, since each may have its own unique biases. Briefly, we compute the frequency of http://github.com/txje/sequence-bias-adjustment.Our method is open source and freely available at We ran and evaluated our method using whole-genome DNase-seq, ChIP-seq, FAIRE-seq, and ATAC-seq. To observe effects of biases in sample, preparation, and protocol, we used data from GM12878, K562, and H1-hESC cell lines and from several different labs and institutions. Sequence data from several open chromatin and transcription factor binding assays were selected from the Encyclopedia of DNA Elements (ENCODE) project , includiR. Nucleotide-specific bias is quantified by the variance in relative frequency of each nucleotide at a particular locus relative to the 5’ end of a read, r. We confirmed that nucleotide bias observed in aligned sequences was not a result of bias in the alignment protocol by comparing intra-read nucleotide content for all reads with the nucleotide content on the reference genome where reads align. These showed identical patterns of bias, indicating that no strong nucleotide bias is introduced during alignment. Throughout, we used the nucleotide sequence of the reference genome, S, to take into account bias outside the read boundaries.We first detect the extent of nucleotide-specific biases within and surrounding all aligned reads, k-mers across each read. For each offset from −20 to n+20 relative to the read’s alignment start position, A(r), in S, where n is the read length, we count the occurrences of each unique k-mer across all reads. Each count is then divided by the total number of reads to give the relative frequency of that k-mer; these frequencies represent the global bias signature for a single experiment . Baseline k-mer frequencies is nontrivial, largely because bias is not uniform across reads and bias values are not independent between k-mer windows, or “tiles”. We often observe high covariance between correction weights for both adjacent (abutting but non-overlapping) and non-adjacent k-mer tiles, thus they cannot simply be compounded to get an accurate whole-read weight. We use several steps to determine which tiles represent significant bias and whether tiles are covariant or independent.To compute read weights from bias and baseline k-mer tile, we determine if it is significantly biased among all reads by comparing the average nucleotide variance to the variance observed in the baseline. Average nucleotide variance (anv) is calculated by computing the relative frequency of each nucleotide at each position in the k-mer tile  outside the variance in the baseline, we mark a tile as significantly biased. The identified biased tiles vary between assay types, although there is concordance between replicates and experiments using the same protocol   to predihttp://github.com/txje/F-seq. We used this modified F-seq to predict peaks from our bias-corrected read data, using the default parameters.We identified open chromatin peaks in DNase-seq, ATAC-seq, FAIRE-seq, and ChIP-seq peaks using F-seq . For eack-mer frequencies to match the observed background frequency had the desired effect of driving the read-relative nucleotide frequencies toward the background level  generated within multiple laboratories as part of the ENCODE project  and in iDNase-seq, FAIRE-seq, and ATAC-seq are commonly used to measure chromatin accessibility where transcription factors bind. These assays can be used to identify evidence of transcription factor binding , 23. BinWe assessed the utility of bias correction to improve footprint identification by using protein interaction quantification (PIQ)  to prediTo observe the effect of bias correction on open chromatin inference as a whole, we compared the covariance between DNase-seq, FAIRE-seq, ATAC-seq, and CTCF ChIP-seq from GM12878 cells under the same condition, but prepared and sequenced in different labs. Table We have shown that aggregate nucleotide-specific biases in high-throughput sequencing reads are greatly reduced by using our bias correction model. Reads are assigned weights to better represent their likelihood of occurrence in the absence of biases, regardless of the source of the bias. When our method is applied to epigenetic assays including DNase-seq, FAIRE-seq, ATAC-seq, and ChIP-seq, true open chromatin and transcriptionally active domains are more accurately identified.Unlike previous methods focusing only on correction of DNase I restriction bias, our method is applicable to a wide range of HTS assays and conditions which may vary between lab, protocol specifics (including read length), cell type, and experimental condition. Existing methods to correct DNase-seq data apply read corrections based only on small motifs of 2-6 bp, often do not consider nucleotide biases outside the read boundaries, and/or require full sequencing of deproteinized “naked” DNA to identify DNase I and experimental biases , 14. OurWhile correlation between nucleotide-frequency-adjusted DNase-seq, ChIP-seq, FAIRE-seq, and ATAC-seq illustrates the generality of our method, there are several factors that may confound these correlations. Notably, many potentially bias-inducing steps during their respective HTS protocols are shared, particularly adapter ligation. We observe evidence of significant shared biases in the observed position-specific nucleotide frequencies, illustrated by the correlation between bias signatures under various experimental conditions Fig. . PrincipIn most cases, popular peak-detection and variant detection methods can be trivially extended to use floating-point read weights. However, since we introduce new information about these HTS data with our bias-adjusted weights, these weights are not taken into account by default. In cases where this modification is not trivial, the weight data can be implicitly represented as variable integer copies of individual reads, thus increasing the amount of data that must be processed, but allowing this information to be used by existing analysis tools without modification.Additional file 1Figure S1. GM12878 DNase-seq 5-mer tile covariance matrix. The pairwise correlation is shown between bias values of 5-mer tiles in a 160bp window surrounding the 5’ end of aligned reads. The block structure between tiles 0 and 3 indicates correlation between adjacent k-mer frequencies within DNase-seq reads. (PDF 203 kb)Additional file 2Table S1. Area under curve (AUC) values for the ROC curves representing sensitivity and specificity of footprint detection for several transcription factors. AUC values at increasing false positive rates (FPR) are computed independently for each motif before and after correction. For all factors except SP1, bias correction improved our ability to accurately predict footprints using protein interaction quantification (PIQ), especially at low to moderate FPR. SP1 motifs often appear in promoters and coincide with binding sites for other factors, which may explain it’s high AUC and the increase in false positives caused by other detectable footprints after correction. (PDF 250 kb)Additional file 3Figure S2. ChIP-seq and DNase-seq coverage in a super enhancer region :10.1016/j.cell.2013.09.053). This region is also in a DNase hypersensitivity region. We show both the ChIP-seq and DNase-seq signal before (A) and after (B) bias correction. In general, for regions with very high ChIP or DNase coverage like this and other “super enhancers”, bias correction doesn’t dramatically change the profile since peak and valley profiles are very robust. (PDF 327 kb)


Editorial on the Research TopicNew Approaches to Radiation-Therapeutic Agent Cancer Care for WomenIt is estimated that 852,000 women will be diagnosed with cancer in the United States (US), and one-third will die each year . Advanceany history of that unique (women’s cancer) subset over any given year   and the fourteenth leading cause of cancer-related death  , an inhibitor of ribonucleotide reductase (RNR). RNR is an enzyme overexpressed in uterine cervix cancer that, when inhibited, blocks production of deoxyribonucleotides required for DNA replication or repair, arrests cancer cells at their G1/S cell cycle phase restriction checkpoint, raises the number of unrepaired damaged DNA foci, and promotes cell lethal events . Althougherertz; ]. This sListeria monocytogenes that secretes the antigen HPV E7 protein fused to a non-hemolytic virulence factor listeriolysin O (LM-LLO-E7). The putative therapeutic effect of ADXS-HPV is its ability to induce E7-specific tumor-infiltrating T-cells, reduce regulatory CD4+ CD25+ T-cells and block tumor blood vessel formation (LM-LLO-E7 following standard radiochemotherapy (NCT02853604).Another example is the development of ADXS-human papillomavirus (HPV) (ADXA11-001), a recombinant live-attenuated ormation . ADXS-HPNewly diagnosed regionally advanced uterine cervix cancer affects about 4,615 women each year  or approximately 2.8 per 100,000 women in the US  women gain access to newly discovered drugs matched to the underlying biology of their disease much earlier than they otherwise would (if ever), and (b) quality, safety, and efficacy of such drugs can be addressed before widespread use. We advocate that US National Cancer Institute’s Cancer Therapy Evaluation Program programmatic collaborations leverage bench science and trials to foster better and faster oncology product development in rare orphan subsets. The example of triapine in a rare orphan subset of uterine cervix cancer highlights a potential regulatory pathway where scientific and financial risks are high, but rewards measured in potential efficacy are higher.Conception: CK, EK; writing: CK, EK; review and approval: CK, EK.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


During cardiac MR imaging (CMR), ECG traces (<10 mV) are highly distorted due to strong gradient-induced voltages (GIV). GIV reaches 1000-3000 mV in high gradient-duty-cycle [GDC = /(Cardiac cycle time)] sequences, for ECG electrodes located farther from magnet's iso-center. An approach which predicted and removed GIV overlaid on ECG traces acquired during CMR using a 19-parameter analytical equation based on Maxwell's equations  and the concomitant fields equation , was previously verified in 10 subjects at rest and following stress . Automating this method would enable its use for efficiently synchronizing CMR (not missing QRS peaks), and for detecting cardiac events  during higher-risk CMR and MR-guided interventions.To this end we created a self-triggered software pipeline, which requires minimal human intervention, in order to compute and display 12-lead ECG traces without GIV. We verify its efficiency in human subjects during high GDC sequences; SSFP and Short-TR GRE.CMR was performed at 3T . All data used in the analysis were recoded using a National Instruments Data Acquisition card controlled by MATLAB . A commercial CardioLab system  was used to amplify and filter the signal from the ECG leads.x, Gy, Gz) were simultaneously recorded directly from Siemens hardware. Once all the traces have been acquired for at least three QRS cycles, GIVs are calculated by subtracting the clean ECG template from the corrupted traces, and then fitted to the equation, yielding the coefficients for each sequence. These coefficients are automatically transferred to the real-time program-component, which calculates GIV for each ECG electrode at each time-point during CMR, using the simultaneously-acquired gradient-waveforms, and then subtracts them from the corrupted signals, thereby cleaning the traces (Figure The process is outlined in Figure s Figure .Figure 1DOI: 10.1002/mrm.25078] provides >90% efficiency in CMR synchronization.In two volunteers, we obtained >95% noise removal in 12-lead ECG traces for SSFP and GRE using this automated approach which functioned equivalently to the manual method. The approach succeeded in removing GIV even during changes in heart-rate (Figure An efficient automated procedure to remove gradient-induced voltages from ECG traces permits real-time 12-lead ECG-monitoring during high gradient-duty-cycle sequences.


The prevention of mother to child transmission of HIV has resulted in reduced burden of pediatric HIV-infection, but the prevalence of maternal HIV infection remains high in sub-Saharan African countries. HIV-exposed-uninfected infants have an increased risk of morbidity and mortality due to infectious diseases than HIV-unexposed infants, particularly during the first six months of life, which in part might be due to lower levels of pathogen-specific protective antibodies acquired transplacentally from their mothers. This could be mitigated by vaccinating pregnant women to boost antibody levels; although vaccine responses among HIV-infected pregnant women might differ compared to HIV-uninfected women. We reviewed studies that compared natural and vaccine-induced antibody levels to different epitopes between HIV-infected and HIV-uninfected pregnant women.Streptococcus (GBS) vaccines. Immunogenicity studies on influenza vaccines indicated that HIV-infected pregnant women had lower vaccine induced hemagglutination inhibition antibody titers and a decreased likelihood of seroconversion compared to HIV-uninfected women; and while higher CD4+ T-lymphocyte levels were associated with better immune responses to vaccination, HIV viral load was not associated with responses. Furthermore, infants born to influenza vaccinated HIV-infected pregnant women also had lower antibody levels and a lower proportion of HIV-exposed infants had titers above the putative correlate of protection compared to HIV-unexposed infants. The immunogenicity of a CRM197-conjugated trivalent GBS vaccine was also lower in HIV-infected pregnant women compared to HIV-uninfected women, irrespective of CD4+ T-lymphocyte counts.Most studies reported lower baseline/pre-vaccination antibody levels in HIV-infected pregnant women, which may not be reversed by antiretroviral therapy during pregnancy. There were only few studies on vaccination of HIV-infected pregnant women, mainly on influenza virus and group B Poorer immunogenicity of vaccines reported in HIV-infected compared to HIV-uninfected pregnant women might compromise the potential benefits to their young infants. Alternate vaccination strategies, including vaccines with higher antigen concentration, adjuvanted vaccines or multiple doses schedules might be required in HIV-infected pregnant women to optimize antibody transferred to their fetuses. Streptococcus pneumoniae disease and 2.3-fold increased risk of invasive group B Streptococcus (GBS) disease [Advances in the prevention of mother to child transmission (PMTCT) of HIV-1 infection have substantially decreased the prevalence of pediatric HIV-infection and the burden of HIV attributable-childhood infectious diseases, including in high prevalence sub-Saharan African countries –4. Marin disease , 7, whic disease . Additio disease   exposure has been associated with mitochondrial toxicity, lower numbers of circulating T-cell lymphocytes and neutrophils in young infants [The possible mechanisms for the increased susceptibility of HEU infants to infectious diseases were recently reviewed , 4, 10. Vaccination during pregnancy could potentially improve maternal and child health , as alreThe protection of the infants could either be due to prevention of mother-to-child transmission of pathogens during close contact, or through transfer of maternal epitope-specific protective antibodies via the placenta and/or breastmilk. This is especially beneficial against diseases presenting soon after birth, or during the initial vulnerable period prior to young infants completing their immunization against vaccine preventable diseases . The effStreptococcus pneumoniae, GBS and Haemophilus influenzae type b; whereas IgG1 is induced primarily by protein epitopes [The transplacental acquired antibodies in the fetus are almost exclusively IgG antibodies, with more efficient and abundant transfer of IgG1 compared to IgG2 . IgG2 isepitopes , 18. Theepitopes . To tranepitopes . Factorsepitopes , 14, 21.Vaccination during pregnancy aimed at protecting the infants, needs to induce sufficient maternal antibody levels that will be transferred to the fetus within the remaining gestational period; and persist in the infant through early infancy. The active transfer of maternal IgG antibodies  occurs most efficiently in the third trimester of pregnancy . Infant Streptococcus pneumoniae, Haemophilus influenzae type b, GBS and tetanus compared to HIV-uninfected women [Generally, HIV-infected pregnant women have lower baseline/pre-vaccination antibody levels, including to epitopes of measles virus, influenza virus, ed women , 28–30. ed women , the neted women . An assoed women . Therefoed women  Fig. 1)StreptocoHaemophilus influenzae, pertussis and pneumococcus antibodies in HIV-infected compared to HIV-uninfected mother-newborn dyads [This could be further compounded by reduced transplacental antibody transfer in HIV-infected compared to HIV–uninfected mother-newborn dyads Fig. . HIV-infrn dyads , 21, 33.rn dyads ; high marn dyads .HIV-exposed uninfected infants less than six months have an increased risk of hospitalization and death from respiratory virus-associated lower respiratory tract infections, including influenza virus , 9. In SAfter vaccination with an unadjuvanted 2009 pandemic H1N1 monovalent influenza vaccine, HAI titers were also significantly increased in women with higher CD4+ T-lymphocyte counts , 38. In The inferior immune response to unadjuvant inactivated influenza vaccines in HIV-infected adults has led to investigation for alternate strategies to improve the immune responses to vaccination in this population. Influenza vaccines with higher antigen content elicited higher HAI titers and improved seroresponse rates in HIV-infected non-pregnant adults, however, the responses were still inferior compared to HIV-uninfected adults , 44. FurAmong the newborns of HIV-infected women, birth HAI titers correlated strongly with maternal antibody titers at delivery , 38. A cIn healthy adults, a HAI titer of 1:40 or higher is associated with 50% protection against influenza disease . HoweverIn the South African randomized-controlled trials of trivalent influenza vaccine in pregnant women, the vaccine efficacy against PCR-confirmed influenza was similar in HIV-infected  and HIV-uninfected  women despite HIV-infected women having a reduced immune response to vaccination , 35. TwoAlthough recto-vaginal GBS colonization is similar or lower in HIV-infected compared to HIV-uninfected pregnant women –56, HEU Natural serotype-specific and surface-protein IgG antibodies have been compared between HIV-infected and HIV-uninfected mother-newborn dyads , 29. In Maternal GBS vaccination is potentially cost effective, both in high income , 61 and Antibody transfer from mother to fetus is crucial in protecting young infants against various infectious diseases prior to their primary immunization or during the period that they are unable to mount a robust immune response to infection. Poor immune responses to natural infection result in lower antibody levels in HIV-infected women and may contribute to disease susceptibility in HIV-exposed infants. Although maternal vaccination reduces early infant disease, lower immune responses to vaccines have been reported in HIV-infected compared to HIV-uninfected pregnant women; this needs to be considered in vaccine development, especially in settings with high maternal HIV-infection prevalence and regardless of PMTCT or ART. Females who were born with HIV and those on long-term ART would soon be entering child-bearing age; these women need to be further studied as to whether T- and B-cell memory is preserved.Immunogenicity may be enhanced in HIV-infected women through two-dosing vaccine schedules . The fir


The pragmatic–explanatory continuum indicator summary version 2 (PRECIS-2) tool has recently been developed to classify randomized clinical trials (RCTs) as pragmatic or explanatory based on their design characteristics. Given that treatment effects in explanatory trials may be greater than those obtained in pragmatic trials, conventional meta-analytic approaches may not accurately account for the heterogeneity among the studies and may result in biased treatment effect estimates. This study investigates if the incorporation of PRECIS-2 classification of published trials can improve the estimation of overall intervention effects in meta-analysis.Using data from 31 published trials of intervention aimed at reducing obesity in children, we evaluated the utility of incorporating PRECIS-2 ratings of published trials into meta-analysis of intervention effects in clinical trials. Specifically, we compared random-effects meta-analysis, stratified meta-analysis, random-effects meta-regression, and mixture random-effects meta-regression methods for estimating overall pooled intervention effects.Our analyses revealed that mixture meta-regression models that incorporate PRECIS-2 classification as covariate resulted in a larger pooled effect size (ES) estimate  than conventional random-effects meta-analysis .In addition to the original intent of PRECIS-2 tool of aiding researchers in their choice of trial design, PRECIS-2 tool is useful for explaining between study variations in systematic review and meta-analysis of published trials. We recommend that researchers adopt mixture meta-regression methods when synthesizing evidence from explanatory and pragmatic trials. Randomized controlled trials (RCTs) are cited as the highest level of evidence that can inform clinical and policy decisions about the efficacy and/or effectiveness of an intervention –3. HowevA number of studies have explored the use of PRECIS instruments when synthesizing evidence from published trials. For example, Patsopoulos  suggestsWhile the uptake of PRECIS instruments  in systematic reviews is increasing, these instruments are mostly used descriptively but their impact in explaining heterogeneity in meta-analytic investigations has not been investigated. Given the variations in study designs, differences in study characteristics of explanatory and pragmatic trials are likely to influence both statistical heterogeneity and intervention effect estimates in meta-analyses. Aves et al.  argues tThis study aimed to assess whether the incorporation of PRECIS classification could improve the modeling of heterogeneity among published trials in meta-analytic investigations. Using data from a Cochrane systematic review of 31 trials of community-based obesity intervention in children , we compPRECIS was developed by a group of international researchers and methodologists to assist trialists in distinguishing between pragmatic and explanatory trial designs , 14. PREData were from the Cochrane systematic review of trials that investigated the efficacy or effectiveness of community-based obesity prevention interventions in children . The sysBefore rating the trials in this systematic review, three study co-authors  first read and discussed relevant papers on PRECIS –16 and p2), and Bayesian information criterion. All analyses, including kappa estimated, were conducted using R software [Descriptive statistics was used to summarize the average domain-specific and overall PRECIS-2 scores across the 31 studies included in this analysis. Fleiss kappa statistic was used to assess inter-rater reliability among the domain-specific and overall ratings of each trial on the PRECIS-2 scale . Four meκ = 0.41 and 0.48, respectively). The average overall ratings ranged between 2.44 and 4.56. Using a cut-off of 3.0, five studies were classified as explanatory while the remaining 26 studies were classified as pragmatic  even revealed substantially large and statistically significant pooled effect size  , assessed using PRECIS-2 tool, can influence the estimation of pooled intervention effects in meta-analysis of published trials when there is substantial heterogeneity attributable to pragmatism. Secondly, this study also reveals the need for meta-regression methods that, adjusts for PRECIS information as covariate, for estimating pooled intervention effects in meta-analytic investigations. This ensures that valid conclusions are derived from systematic reviews and meta-analysis of published trials. We recommend that meta-analytic investigations in systematic reviews should incorporate information about design characteristics using PRECIS-2 when synthesizing evidence from published studies.