Size‐dependent thermal preferences in a pelagic fish

Large fish often inhabit colder waters than small fish. Using a simple bioenergetic model, we found that the optimal temperature for growth should decrease with increasing body size. We predicted that this mechanism would produce an ontogenetic change in thermal preference and then tested our predictions with Pacific salmon, Oncorhynchus spp. In a laboratory experiment, the slope of a regression of growth increment on initial size became steeper with increasing temperature, so that the optimal temperature for growth decreased with increasing body size. In field observations, larger and older salmon inhabited cooler areas, whereas smaller and younger salmon inhabited warmer areas. These patterns were consistent with a size‐dependent effect of temperature on condition factor, a parameter shown experimentally to be a measure of the most recent growth performance. Temperatures for maximising condition factor were lower for larger fish. Thus, an ontogenetic change in individual thermal preference toward cooler areas maximises the growth performance of fish, and the negative effects of climate warming on growth are hypothesised to be more severe for larger fish.     

Mechanism of enzymatic degradation of the azo dye Orange II determined by ex situ 1H nuclear magnetic resonance and electrospray ionization-ion trap mass spectrometry

Removal of azo dye effluents generated by textile photography industries is a main issue in wastewater treatment. Enzymatic treatment of dyes appears to be one of the most efficient processes for their degradation. The elucidation of degradation pathways is of special interest considering health and environmental priorities. Ex situ nuclear magnetic resonance (NMR) spectroscopy and electrospray ionization (ESI)-ion trap mass spectrometry performed directly on incubation medium have been used for the first time to follow kinetics of sulfonated azo dye Orange II enzymatic degradation. Nine transformation products were identified using these complementary analyses performed ex situ without any prior treatment. Three types of cleavage are proposed for the degradation pathway: (i) a symmetrical splitting of the azo linkage that leads to the formation of 4-aminobenzenesulfonate (and 1-amino-2-naphthol, not detected); (ii) an asymmetrical cleavage on the naphthalene side that generates 1,2-naphthoquinone and 4-diazoniumbenzenesulfonate as products, with the latter one being transformed into 4-hydroxybenzensulfonate; and (iii) a third degradation pathway that leads to 2-naphthol and 4-hydroxybenzenesulfonate. Moreover, three other intermediates have been identified. This study, which constitutes the first concomitant use of (1)H NMR spectroscopy and ESI-ion trap mass spectrometry in this field, illustrates the indubitable interest of the ex situ approach.     

Functional and structural study on chelator-induced suppression of S2/S1 FTIR spectrum in photosynthetic oxygen-evolving complex

Chelating agents have been shown to induce characteristic changes in the light-minus-dark Fourier transform infrared (FTIR) difference spectrum for the S(2)/S(1) difference in the oxygen-evolving complex (OEC). Addition of various ethylenediamine-N,N,N',N'-tetraacetic acid (EDTA)-type chelators, such as EDTA, O,O'-bis(2-aminoethyl)ethyleneglycol-N,N,N',N'-tetraacetic acid (EGTA), trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CyDTA), or N-(2-hydroxyethyl)ethylenediamine-N,N',N'-triacetic acid (HEDTA), to Ca(2+)-depleted PS II membranes resulted in the suppression of typical S(2)/S(1) vibrational features, including the symmetric (1365(+)/1404(-) cm(-1)) and the asymmetric (1587(+)/1566(-) cm(-1)) carboxylate stretching vibrations, as well as the amide I and II modes of the backbone polypeptides. In contrast, the addition of ethylenediamine-N,N'-diacetic acid (EDDA) showed less inhibitory effects. The effects of the chelators depended on the number of the carboxylate groups; chelators with more than two carboxymethyl groups were effective in altering the FTIR spectrum. The bridging structure that connects the two nitrogen atoms also influenced the inhibitory effects. However, the effects were not necessarily correlated with the stability constants of the chelators to Mn(2+). The vibrational modes that were suppressed by EDTA were almost completely restored by subsequent washing with Chelex-treated Ca(2+)-free buffer medium, indicating that the spectral changes are attributable to the reversible association of chelators with the Ca(2+)-depleted OEC. Nevertheless, prolonged incubation with chelators led to the impairment of the O(2)-evolving capability, with differences in the effectiveness, in the order that is consistent with that for the suppression effects on FTIR spectra. Chelators with carboxylate and/or carboxymethyl groups bound to a single nitrogen [nitrilotriacetic acid (NTA) and iminodiacetic acid (IDA)] or carbon (citric acid) were relatively ineffective for the suppression. A chelator that includes four phosphate groups, ethylenediamine-N,N,N',N'-tetrakis(methylenephosphonic) acid (EDTPO), also showed suppression effects on both the carboxylate and amide modes. Based on these findings, a possible mode of interaction between the chelators and the Mn cluster is discussed.     

Molecular cloning and characterization of triterpene synthases from Medicago truncatula and Lotus japonicus

Cloning of OSCs required for triterpene synthesis from legume species that are amenable to molecular genetics will provide tools to address the importance of triterpenes and their derivatives during normal plant growth and development and also in interactions with symbionts and pathogens. Here we report the cloning and characterization of a total of three triterpene synthases from the legume species Medicago truncatula and Lotus japonicus. These include a -amyrin synthase from M. truncatula (MtAMYI) and a mixed function triterpene synthase from Lotus japonicus (LjAMY2). A partial cDNA predicted to encode a -amyrin synthase (LjAMY1) was also isolated from L. japonicus. The expression patterns of MtAMY1, LjAMY1 and LjAMY2 and of additional triterpene synthases previously characterised from M. truncatula and pea differ in different plant tissues and during nodulation, suggesting that these enzymes may have distinct roles in plant physiology and development.     

Cloning and functional characterization of a homoglutathione synthetase from pea nodules

The thiol tripeptide glutathione (GSH; γ Glu-Cys-Gly) is very abundant in legume nodules where it performs multiple functions that are critical for optimal nitrogen fixation. Some legume nodules contain another tripeptide, homoglutathione (hGSH; γ Glu-Cys- β Ala), in addition to or instead of GSH. We have isolated from a pea ( Pisum sativum L.) nodule library a cDNA, GSHS2 , that is expressed in nodules but not in leaves. This cDNA was overexpressed in insect cells and its protein product was identified as a highly active and specific hGSH synthetase. The enzyme, the first of this type to be completely purified, is predicted to be a homodimeric cytosolic protein. It shows a specific activity of 3400 nmol hGSH min⁻¹ mg⁻¹ protein with a standard substrate concentration (5 m M β -alanine) and K_m values of 1.9 m M for β -alanine and 104 m M for glycine. The specificity constant (V_max/K_m) shows that the pure enzyme is 57.3-fold more specific for β -alanine than for glycine. Southern blot analysis revealed that the gene is present as a single copy in the pea genome and that there are homologous genes in other legumes. We conclude that the synthesis of hGSH in pea nodules is catalysed by a specific hGSH synthetase and not by a GSH synthetase with broad substrate specificity.     

Lupus IgG VH4.34 antibodies bind to a 220-kDa glycoform of CD45/B220 on the surface of human B lymphocytes

Anti-lymphocyte autoantibodies are a well-recognized component of the autoimmune repertoire in human systemic lupus erythematosus (SLE) and have been postulated to have pathogenic consequences. Early studies indicated that IgM anti-lymphocyte autoantibodies mainly recognized T cells and identified CD45, a protein tyrosine phosphatase of central significance in the modulation of lymphocyte function, as the main antigenic target on T cells. However, more recent work indicates that lupus autoantibodies can also recognize B cells and that CD45 may also represent their antigenic target. In particular, IgM Abs encoded by V(H)4.34 appear to have special tropism for B cells, and strong, but indirect evidence suggests that they may recognize a B cell-specific CD45 isoform. Because V(H)4.34 Abs are greatly expanded in SLE, in the present study we investigated the antigenic reactivity of lupus sera V(H)4.34 IgG Abs and addressed their contribution to the anti-lymphocyte autoantibody repertoire in this disease. Our biochemical studies conclusively demonstrate that lupus IgG V(H)4.34 Abs target a developmentally regulated B220-specific glycoform of CD45, and more specifically, an N-linked N-acetyllactosamine determinant preferentially expressed on naive B cells that is sterically masked by sialic acid on B220-positive memory B cells. Strikingly, our data also indicate that this reactivity in SLE sera is restricted to V(H)4.34 Abs and can be eliminated by depleting these Abs. Overall, our data indicate that V(H)4.34 Abs represent a major component of the lupus IgG autoantibody repertoire and suggest that the carbohydrate moiety they recognize may act as a selecting Ag in SLE.     

Dissection of the HLA-DR4 peptide repertoire in endocrine epithelial cells: strong influence of invariant chain and HLA-DM expression on the nature of ligands

Class II MHC (MHC II) expression is restricted to professional APCs and thymic epithelium but it also occurs in the epithelial cells of autoimmune organs which are the unique targets of the CD4 autoreactive T cells in endocrine autoimmune diseases. This specificity is presumably conditioned by an epithelium-specific peptide repertoire associated to MHC II at the cell surface. MHC II expression and function is dependent on the action of two main chaperones, invariant chain (Ii) and DM, whose expression is coregulated with MHC II. However, there is limited information about the in vivo expression levels of these molecules and uncoordinated expression has been demonstrated in class II-positive epithelial cells that may influence the MHC-associated peptide repertoires and the outcome of the autoimmune response. We have examined the pool of peptides associated to DR4 molecules expressed by a neuroendocrine epithelial cell and the consequences of Ii and DM coexpression. The RINm5F rat insulinoma cell line was transfected with HLA-DRB1*0401, Ii, and DM molecules in four different combinations: RIN-DR4, -DR4Ii, -DR4DM, and -DR4IiDM. The analysis of the peptide repertoire and the identification of the DR4 naturally processed ligands in each transfected cell were achieved by mass spectrometry. The results demonstrate that 1) the expression of Ii and DM affected the DR4 peptide repertoires by producing important variations in their content and in the origin of peptides; 2) these restrictions affected the stability and sequence of the peptides of each repertoire; and 3) Ii and DM had both independent and coordinate effects on these repertoires.     

Application of factorial design to the optimization of medium composition in batch cultures of Streptomyces lividans TK21 producing a hybrid antibiotic

Summary The composition of the liquid medium employed to obtain a hybrid antibiotic in batch cultures of a recombinant strain of Streptomyces lividans TK21 has been studied. Starch and glutamic acid are the most appropriate carbon and nitrogen sources to support respectively cell growth and antibiotic production. A central composite experimental design has been employed to derive a statistical model of the effect of phosphate and glutamic acid on growth and antibiotic production, and an initial concentration of 10 mM phosphate and 52.8 mM glutamic acid have been found optimal to maximize the final antibiotic concentration in batch cultures.     

TLR-9 agonist and CD40-targeting vaccination induces HIV-1 envelope-specific B cells with a diversified immunoglobulin repertoire in humanized mice

The development of HIV-1 vaccines is challenged by the lack of relevant models to accurately induce human B- and T-cell responses in lymphoid organs. In humanized mice reconstituted with human hematopoietic stem cells (hu-mice), human B cell-development and function are impaired and cells fail to efficiently transition from IgM B cells to IgG B cells. Here, we found that CD40-targeted vaccination combined with CpG-B adjuvant overcomes the usual defect of human B-cell switch and maturation in hu-mice. We further dissected hu-B cell responses directed against the HIV-1 Env protein elicited by targeting Env gp140 clade C to the CD40 receptor of antigen-presenting cells. The anti-CD40.Env gp140 vaccine was injected with CpG-B in a homologous prime/boost regimen or as a boost of a NYVAC-KC pox vector encoding Env gp140 clade C. Both regimens elicited Env-specific IgG-switched memory hu-B cells at a greater magnitude in hu-mice primed with NYVAC-KC. Single-cell RNA-seq analysis showed gp140-specific hu-B cells to express polyclonal IgG1 and IgG3 isotypes and a broad Ig VH/VL repertoire, with predominant VH3 family gene usage. These cells exhibited a higher rate of somatic hypermutation than the non-specific IgG⁺ hu-B-cell counterpart. Both vaccine regimens induced splenic GC-like structures containing hu-B and hu-Tfh-like cells expressing PD-1 and BCL-6. We confirmed in this model that circulating ICOS⁺ memory hu-Tfh cells correlated with the magnitude of gp140-specific B-cell responses. Finally, the NYVAC-KC heterologous prime led to a more diverse clonal expansion of specific hu-B cells. Thus, this study shows that CD40-targeted vaccination induces human IgG production in hu-mice and provides insights for the development of a CD40-targeting vaccine to prevent HIV-1 infection in humans.     

Onchocerciasis Transmission in Ghana: Persistence under Different Control Strategies and the Role of the Simuliid Vectors

Background

The World Health Organization (WHO) aims at eliminating onchocerciasis by 2020 in selected African countries. Current control focuses on community-directed treatment with ivermectin (CDTI). In Ghana, persistent transmission has been reported despite long-term control. We present spatial and temporal patterns of onchocerciasis transmission in relation to ivermectin treatment history.

Methodology/Principal Findings

Host-seeking and ovipositing blackflies were collected from seven villages in four regions of Ghana with 3–24 years of CDTI at the time of sampling. A total of 16,443 flies was analysed for infection; 5,812 (35.3%) were dissected for parity (26.9% parous). Heads and thoraces of 12,196 flies were dissected for Onchocerca spp. and DNA from 11,122 abdomens was amplified using Onchocerca primers. A total of 463 larvae (0.03 larvae/fly) from 97 (0.6%) infected and 62 (0.4%) infective flies was recorded; 258 abdomens (2.3%) were positive for Onchocerca DNA. Infections (all were O. volvulus) were more likely to be detected in ovipositing flies. Transmission occurred, mostly in the wet season, at Gyankobaa and Bosomase, with transmission potentials of, respectively, 86 and 422 L3/person/month after 3 and 6 years of CDTI. The numbers of L3/1,000 parous flies at these villages were over 100 times the WHO threshold of one L3/1,000 for transmission control. Vector species influenced transmission parameters. At Asubende, the number of L3/1,000 ovipositing flies (1.4, 95% CI = 0–4) also just exceeded the threshold despite extensive vector control and 24 years of ivermectin distribution, but there were no infective larvae in host-seeking flies.

Conclusions/Significance

Despite repeated ivermectin treatment, evidence of O. volvulus transmission was documented in all seven villages and above the WHO threshold in two. Vector species influences transmission through biting and parous rates and vector competence, and should be included in transmission models. Oviposition traps could augment vector collector methods for monitoring and surveillance.