Enhancing Data Reliability in TOMAHAQ for Large‐Scale Protein Quantification

The analytical scale of most mass‐spectrometry‐based targeted proteomics assays is usually limited by assay performance and instrument utilization. A recently introduced method, called triggered by offset, multiplexed, accurate mass, high resolution, and absolute quantitation (TOMAHAQ), combines both peptide and sample multiplexing to simultaneously improve analytical scale and quantitative performance. In the present work, critical technical requirements and data analysis considerations for successful implementation of the TOMAHAQ technique based on the study of a total of 185 target peptides across over 200 clinical plasma samples are discussed. Importantly, it is observed that significant interference originate from the TMTzero reporter ion used for the synthetic trigger peptides. This interference is not expected because only TMT10plex reporter ions from the target peptides should be observed under typical TOMAHAQ conditions. In order to unlock the great promise of the technique for high throughput quantification, here a post‐acquisition data correction strategy to deconvolute the reporter ion superposition and recover reliable data is proposed.     

Streptococcus pneumoniae capsule determines disease severity in experimental pneumococcal meningitis

Streptococcus pneumoniae bacteria can be characterized into over 90 serotypes according to the composition of their polysaccharide capsules. Some serotypes are common in nasopharyngeal carriage whereas others are associated with invasive disease, but when carriage serotypes do invade disease is often particularly severe. It is unknown whether disease severity is due directly to the capsule type or to other virulence factors. Here, we used a clinical pneumococcal isolate and its capsule-switch mutants to determine the effect of capsule, in isolation from the genetic background, on severity of meningitis in an infant rat model. We found that possession of a capsule was essential for causing meningitis. Serotype 6B caused significantly more mortality than 7F and this correlated with increased capsule thickness in the cerebrospinal fluid (CSF), a stronger inflammatory cytokine response in the CSF and ultimately more cortical brain damage. We conclude that capsule type has a direct effect on meningitis severity. This is an important consideration in the current era of vaccination targeting a subset of capsule types that causes serotype replacement.     

Stability ofAlternaria toxins in fruit juices and wine

Alternaria alternata is a common fungal parasite on fruits and other plants and produces a number of mycotoxins, including alternariol (3,7,9-trihydroxy-1-methyl-6H-dibenzo [b,d]pyran-6-one), alternariol monomethyl ether (3,7-dihydroxy-9-methoxy-1-methyl-6H-dibenzo[b,d]pyran-6-one), and the mutagen altertoxin I {[1S-(1α,12aβ,12bα)] 1,2,11,12,12a, 12b-hexahydro-1,4,9,12a-tetrahydroxy-3,10-perylenedione}. Alternariol and alternariol monomethyl ether have previously been detected in some samples of fruit beverages. Stability studies of these toxins as well as altertoxin I added to fruit juices and wine (10–100 ng/mL) were carried out. To include altertoxin I in the analysis, cleanup with a polymer-based Varian Abselut solid phase extraction column was used, as recoveries from C-18 columns were low. The stabilities of alternariol and alternariol monomethyl ether in a low acid apple juice containing no declared vitamin C were compared with those in the same juice containing added vitamin C (60 mg/175 ml); there were no apparent losses at room temperature over 20 days or at 80°C after 20 min. in either juice. Altertoxin I was moderately stable in pH 3 buffer (75% remaining after a two week period). Furthermore, altertoxin I was stable or moderately stable in three brands of apple juice tested over 1–27 day periods and in a sample of red grape juice over 7 days. It is concluded that altertoxin I is sufficiently stable to be found in fruit juices and should be included in methods for alternariol and alternariol monomethyl ether.     

SNPs in DNA repair genes associated to meningitis and host immune response

In vitro and in animal models, APE1, OGG1, and PARP-1 have been proposed as being involved with inflammatory response. In this work, we have investigated if the SNPs APE1 Asn148Glu, OGG1 Ser326Cys, and PARP-1 Val762Ala are associated to meningitis. The patient genotypes were investigated by PIRA-PCR or PCR-RFLP. DNA damages were detected in genomic DNA by Fpg treatment. IgG and IgA were measured from plasma and the cytokines and chemokines were measured from cerebrospinal fluid samples using Bio-Plex assays. A higher frequency (P<0.05) of APE1 Glu allele in bacterial meningitis (BM) and aseptic meningitis (AM) patients was observed. The genotypes Asn/Asn in control group and Asn/Glu in BM group was also higher. For the SNP OGG1 Ser326Cys, the genotype Cys/Cys was more frequent (P<0.05) in BM group. The frequency of PARP-1 Val/Val genotype was higher in control group (P<0.05). The occurrence of combined SNPs is significantly higher in BM patients, indicating that these SNPs may be associated to the disease. Increasing in sensitive sites to Fpg was observed in carriers of APE1 Glu allele or OGG1 Cys allele, suggesting that SNPs affect DNA repair activity. Alterations in IgG production were observed in the presence of SNPs APE1 Asn148Glu, OGG1 Ser326Cys or PARP-1 Val762Ala. Moreover, reduction in the levels of IL-6, IL-1Ra, MCP-1/CCL2 and IL-8/CXCL8 was observed in the presence of APE1 Glu allele in BM patients. In conclusion, we obtained indications of an effect of SNPs in DNA repair genes on the regulation of immune response in meningitis.     

Induced root-secreted phenolic compounds as a belowground plant defense

Rhizosphere is the complex place of numerous interactions between plant roots, microbes and soil fauna. Whereas plant interactions with aboveground organisms are largely described, unravelling plant belowground interactions remains challenging. Plant root chemical communication can lead to positive interactions with nodulating bacteria, mycorriza or biocontrol agents or to negative interactions with pathogens or root herbivores. A recent study¹ suggested that root exudates contribute to plant pathogen resistance via secretion of antimicrobial compounds. These findings point to the importance of plant root exudates as belowground signalling molecules, particularly in defense responses. In our report,² we showed that under Fusarium attack the barley root system launched secretion of phenolic compounds with antimicrobial activity. The secretion of de novo biosynthesized t-cinnamic acid induced within 2 days illustrates the dynamic of plant defense mechanisms at the root level. We discuss the costs and benefits of induced defense responses in the rhizosphere. We suggest that plant defense through root exudation may be cultivar dependent and higher in wild or less domesticated varieties.Key words: root exudates, plant defense, t-cinnamic acid, fusarium, induced defensePlants grow and live in very complex and changing ecosystems. Because plants lack the mobility to escape from attack by pathogens or herbivores, they have developed constitutive and in addition inducible defenses that are triggered by spatiotemporally dynamic signaling mechanisms. These defenses counteract the aggressor directly via toxins or defense plant structures or indirectly by recruitment of antagonists of aggressors. Whereas induced defenses are well described in aboveground interactions, evidence of the occurrence of such mechanisms in belowground interactions remains limited. The biosynthesis of a defensive molecule could be both constitutive and inducible with a low level of a preformed pool (Fig. 1). In addition, upon encounter of an attacking organism, those levels could be induced to rise locally to a high level of active compound that is able to disarm the pathogen.^2,3 Only a few examples show that root exudates play a role in induced plant defense. Hairy roots of Ocimum basilicum secrete rosmarinic acid only when challenged by the pathogenic fungus Pythium ultimum.⁴ Wurst et al.⁵ reported on the induction of irridoid glycosides in root exudates of Plantago lanceolata in presence of nematodes. In vivo labelling experiments² with ¹³CO₂ showed the induction of phenolic compounds secreted by barley roots after Fusarium graminearum infection and the de novo biosynthesis of root secreted t-cinnamic acid within 2 days. These results show that the pool of induced t-cinnamic acid originated from both pre-formed and newly formed carbon pools (Fig. 1), highlighting a case of belowground induced defense inside and outside the root system.Open in a separate windowFigure 1Suggested mechanisms for the induction of root defense exudates in barley in response to Fusarium attack. Upon pathogen attack by Fusarium, the initial preformed pool of phenolic compounds is increased by the addition of inducible, de novo biosynthesized t-cinnamic acid. Both, the preformed pool and the de novo biosynthesized pool fuel the exudation of defense compounds from infected roots.The concept of fitness costs is frequently presented to explain the coexistence of both constitutive and induced defense.⁶ In the case of induced defense, resources are invested in defenses only when the plant is under attack. In the absence of an infection, plants can optimize allocation of their resources to reproduction and growth to compete with neighbours.⁷ Constitutive defenses are thought to be more beneficial when the probability of attack is high, whereas adjustable, induced defenses are more valuable to fight against an unpredictable pathogen. Non disturbed soil is a heterogeneous matrix where biodiversity is very high and patchy^8,9 and organism motility is rather restricted.¹⁰ As a consequence of the patchiness, belowground environment is expected to be favourable to selection for induced responses.¹¹ The absence of defense root exudates between two infections may form an unpredictable environment for soil pathogens and reduce the chance for adaptation of root attackers. Plants may also use escape strategies to reduce the effect of belowground pathogens. Henkes et al. (unpublished) showed that Fusarium-infected barley plants reduced carbon allocation towards infected roots within a day and increased allocation carbon to uninfected roots. These results illustrate how reallocation of carbon toward non infected root parts represents a way to limit the negative impact of root infection.We have demonstrated the potential of barley plants to defend themselves against soil pathogen by root exudation.² Even the barley cultivar ‘Barke’ used in our study, a modern cultivated variety, was able to launch defense machinery via exudation of antimicrobial compounds when infected by F. graminearum. We suggest that plant defense through root exudation might be cultivar dependent and perhaps higher in wild or less domesticated varieties. Taddei et al.¹² reported that constitutivelyproduced root exudates from a resistant Gladiolus cultivar inhibit spore germination of Fusarium oxysporum whereas root exudates from a susceptible cultivar do not affect F. oxysporum germination. Root exudates from the resistant cultivar contained higher amounts of aromaticphenolic compounds compared to the susceptible cultivar and these compounds may be responsible for the inhibition of spore germination. Metabolic profiling of wheat cultivars, ‘Roblin’ and ‘Sumai3’, respectively, susceptible and resistant to Fusarium Head Blight, showed that t-cinnamic acid was a discriminating factor responsible for resistance/defense function.¹³ Therefore it is likely that wild barley varieties hold higher defense capacities compare to cultivated varieties selected for high yield. In the future, plant breeders in organic and low-input farming could use root-system defense ability as new trait in varietal variation.     

Selection of a Rare Neutralization-Resistant Variant following Passive Transfer of Convalescent Immune Plasma in Equine Infectious Anemia Virus-Challenged SCID Horses

Vaccines preventing HIV-1 infection will likely elicit antibodies that neutralize diverse strains. However, the capacity for lentiviruses to escape broadly neutralizing antibodies (NAbs) is not completely understood, nor is it known whether NAbs alone can control heterologous infection. Here, we determined that convalescent immune plasma from a horse persistently infected with equine infectious anemia virus (EIAV) neutralized homologous virus and several envelope variants containing heterologous principal neutralizing domains (PND). Plasma was infused into young horses (foals) affected with severe combined immunodeficiency (SCID), followed by challenge with a homologous EIAV stock. Treated SCID foals were protected against clinical disease, with complete prevention of infection occurring in one foal. In three SCID foals, a novel neutralization-resistant variant arose that was found to preexist at a low frequency in the challenge inoculum. In contrast, SCID foals infused with nonimmune plasma developed acute disease associated with high levels of the predominant challenge virus. Following transfer to an immunocompetent horse, the neutralization-resistant variant induced a single febrile episode and was subsequently controlled in the absence of type-specific NAb. Long-term control was associated with the presence of cytotoxic T lymphocytes (CTL). Our results demonstrate that immune plasma with neutralizing activity against heterologous PND variants can prevent lentivirus infection and clinical disease in the complete absence of T cells. Importantly, however, rare neutralization-resistant envelope variants can replicate in vivo under relatively broad selection pressure, highlighting the need for protective lentivirus vaccines to elicit NAb responses with increased breadth and potency and/or CTL that target conserved epitopes.Development of an effective vaccine will be critical in the efforts to control the human immunodeficiency virus type 1 (HIV-1) pandemic. Unfortunately, vaccines evaluated in completed human efficacy trials have shown moderate to no protective effects, and, clearly, much more work is needed to define the correlates of lentivirus immune protection. Although these correlates are still not entirely known, vaccine strategies that elicit antibodies with broad neutralizing activity are currently of considerable interest, and it is widely believed that HIV-1 envelope glycoproteins that induce broadly neutralizing antibodies (NAbs) will be critical components of a protective vaccine (21, 28, 53, 63).Equine infectious anemia virus (EIAV) is a macrophage-tropic lentivirus that causes persistent infection in horses worldwide and serves as an important large-animal translational model in which to dissect basic correlates of protective lentiviral immunity (9, 31, 33, 38, 57). EIAV is a naturally occurring lentivirus, and infection results in a predictable course of recurrent episodes of plasma viremia and clinical disease. As with HIV-1 and simian immunodeficiency virus (SIV), EIAV infection is not cleared. However, infected horses eventually control viral replication and clinical disease to remain persistently infected inapparent carriers. Adaptive immune responses, including NAbs, are required for EIAV control since young horses (foals) with severe combined immunodeficiency (SCID), unlike normal foals, fail to eliminate the initial viremia following challenge (46). Equine SCID is caused by a frameshift mutation in the gene encoding the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) (55, 60) and has an autosomal recessive mode of inheritance (47). The equine SCID defect is more severe than its murine counterpart in that SCID foals are incapable of forming either coding or signal joints (55). Adoptive transfer of EIAV-specific T and B lymphocytes to a SCID foal results in functional cytotoxic T lymphocytes (CTL) and NAb activity and is protective against homologous EIAV challenge (33).During acute EIAV infection, each recurrent episode coincides with the emergence of an antigenically distinct EIAV variant as defined by type-specific NAb, which neutralizes virus isolated during early disease episodes but not virus isolated during subsequent disease episodes (2, 20, 22, 43, 52). Amino acid variation primarily occurs within hypervariable regions V1 to V8 of the envelope gp90 surface unit (SU) and particularly within the V3/principal neutralizing domain (PND) region (1, 19, 24, 25, 57). Our work with EIAV-infected SCID foals indicates that significant envelope diversification does not occur in the absence of NAbs but that rapid envelope diversification occurs when adaptive immune responses are reconstituted (35). Thus, adaptive immunity, including NAb, drives selection of EIAV envelope variants during acute infection. Amino acid changes occur primarily within the V3 to V7 hypervariable SU regions, and many changes affect potential N-linked glycosylation sites (PNLGS) (35). Importantly, however, CTL also target the SU, and variants that escape CTL recognizing an EIAV V3/PND epitope have been identified (37, 38). Thus, both NAbs and CTL are capable of contributing to the selection of EIAV SU variants, but the relative contributions of each to such selection are not known.Recently, SU variation was evaluated in an immunocompetent pony experimentally inoculated with the virulent wild-type Wyoming strain of EIAV (57). Seventy-one distinct V3 variants that partitioned into five major nonoverlapping groups were identified and designated PND1 to PND5. Neutralization assays using chimeric infectious molecular clones containing these PNDs suggested a transition from type-specific NAb responses toward more broadly reactive immune responses during the course of infection and indicated that genetic changes conferring resistance to broadly NAbs lead to recrudescence of clinical disease following a lengthy clinically quiescent period (57). Thus, the NAb response broadens significantly during long-term persistent EIAV infection, and broadly NAbs play a critical role in EIAV immune control.Studies of nonhuman primates have provided important information regarding the protective effects of NAbs. Passive immunization of macaques with purified immunoglobulin from chimpanzees infected with several different HIV-1 isolates results in complete protection from homologous chimeric simian/human immunodeficiency virus (SHIV) infection when the immunoglobulin is given 24 h prior to challenge (54). Passive transfer of a triple combination of broadly neutralizing human monoclonal antibodies directed against the envelope of a primary HIV-1 isolate results in complete protection against SHIV infection in some macaques while others become infected but exhibit decreased plasma viremia (29). The contribution of T cells to partial protection in these studies is not clear, and the presence or absence of viral escape variants in the unprotected macaques has not been evaluated. In neonatal macaques, various combinations of broadly neutralizing human monoclonal antibodies directed against conserved HIV envelope epitopes administered before and after SHIV challenge result in protection against persistent systemic infection in some animals, but clinical disease develops in others (12-14). Virus-specific T-cell proliferative responses are detected in some of the protected animals, indicating that cellular immune responses occur and likely contribute to protection by eliminating infected cells (13).Despite the fact that NAbs can block experimental SHIV infection, selection pressure exerted by NAbs plays a critical role in HIV-1 and SIV envelope evolution during infection, and evasion of NAb responses is an important mechanism of HIV-1 and SIV persistence (11, 16, 27, 48, 59). The maturation of a type-specific NAb response in SIV-infected rhesus macaques significantly correlates with diversification in the V1/V2 region of the SIV envelope (50). In HIV-1, NAbs are detectable within the first 2 months postinfection and result in an early and significant selection force on the virus population (49). Escape from NAbs involves many amino acid substitutions with little cross-neutralization between closely related strains, and NAb responses drive the diversification of the HIV-1 envelope during the early stages of infection (16). The early appearance of NAbs in patients with acute HIV-1 infection results in the replacement of neutralization-sensitive virus by successive populations of resistant virus, and virus escape primarily involves changes in N-linked glycosylation (59). Thus, overcoming neutralization escape constitutes a significant barrier to the ultimate efficacy of any NAb-eliciting HIV-1 vaccine.Because the SCID defect occurs naturally in the horse, it provides a powerful and unique opportunity to finely dissect the protective effects of immune interventions against a naturally occurring lentivirus independent of other de novo adaptive immune responses. This level of dissection is not possible in other lentivirus model systems. The goal of the current study was to determine if broadly NAbs could protect against lentivirus challenge in the complete absence of T lymphocytes and other adaptive immune responses. We hypothesized that convalescent immune plasma from a long-term persistently infected inapparent carrier horse containing antibodies capable of neutralizing homologous and several heterologous EIAV SU PND variants would provide complete protection when infused into SCID foals before experimental virus inoculation. This plasma was administered to four SCID foals 24 h prior to challenge, and four control SCID foals received normal horse plasma. Clinical outcome, plasma viral load, and serum neutralization activity were analyzed in all foals. Although complete protection was achieved in one treated foal, infection occurred in the others. In foals that became viremic, the SU sequence and neutralization phenotype of the breakthrough virus were determined. As part of these experiments, blood containing this virus was inoculated into a naive immunocompetent horse, and the adaptive immune responses associated with its control were further evaluated.     

Association of 25-Hydroxyvitamin D status and genetic variation in the vitamin D metabolic pathway with FEV1 in the Framingham Heart Study

Background

Vitamin D is associated with lung function in cross-sectional studies, and vitamin D inadequacy is hypothesized to play a role in the pathogenesis of chronic obstructive pulmonary disease. Further data are needed to clarify the relation between vitamin D status, genetic variation in vitamin D metabolic genes, and cross-sectional and longitudinal changes in lung function in healthy adults.

Methods

We estimated the association between serum 25-hydroxyvitamin D [25(OH)D] and cross-sectional forced expiratory volume in the first second (FEV₁) in Framingham Heart Study (FHS) Offspring and Third Generation participants and the association between serum 25(OH)D and longitudinal change in FEV₁ in Third Generation participants using linear mixed-effects models. Using a gene-based approach, we investigated the association between 241 SNPs in 6 select vitamin D metabolic genes in relation to longitudinal change in FEV₁ in Offspring participants and pursued replication of these findings in a meta-analyzed set of 4 independent cohorts.

Results

We found a positive cross-sectional association between 25(OH)D and FEV₁ in FHS Offspring and Third Generation participants (P = 0.004). There was little or no association between 25(OH)D and longitudinal change in FEV₁ in Third Generation participants (P = 0.97). In Offspring participants, the CYP2R1 gene, hypothesized to influence usual serum 25(OH)D status, was associated with longitudinal change in FEV₁ (gene-based P < 0.05). The most significantly associated SNP from CYP2R1 had a consistent direction of association with FEV₁ in the meta-analyzed set of replication cohorts, but the association did not reach statistical significance thresholds (P = 0.09).

Conclusions

Serum 25(OH)D status was associated with cross-sectional FEV₁, but not longitudinal change in FEV₁. The inconsistent associations may be driven by differences in the groups studied. CYP2R1 demonstrated a gene-based association with longitudinal change in FEV₁ and is a promising candidate gene for further studies.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0238-y) contains supplementary material, which is available to authorized users.