Stabilization of antibody structure upon association to a human carbonic anhydrase IX epitope studied by X-ray crystallography, microcalorimetry, and molecular dynamics simulations

Specific antibodies interfere with the function of human tumor-associated carbonic anhydrase IX (CA IX), and show potential as tools for anticancer interventions. In this work, a correlation between structural elements and thermodynamic parameters of the association of antibody fragment Fab M75 to a peptide corresponding to its epitope in the proteoglycan-like domain of CA IX, is presented. Comparisons of the crystal structures of free Fab M75 and its complex with the epitope peptide reveal major readjustments of CDR-H1 and CDR-H3. In contrast, the overall conformations and positions of CDR-H2 and CDR-L2 remain unaltered, and their positively charged residues may thus present a fixed frame for epitope recognition. Adoption of the altered CDR-H3 conformation in the structure of the complex is accompanied by an apparent local stabilization. Analysis of domain mobility with translation-libration-screw (TLS) method shows that librations of the entire heavy chain variable domain (V(H)) decrease and reorient in the complex, which correlates well with participation of the heavy chain in ligand binding. Isothermal titration microcalorimetry (ITC) experiments revealed a highly unfavorable entropy term, which can be attributed mainly to the decrease in the degrees of freedom of the system, the loss of conformational freedom of peptide and partially to a local stabilization of CDR-H3. Moreover, it was observed that one proton is transferred from the environment to the protein-ligand complex upon binding. Molecular dynamics simulations followed by molecular mechanics/generalized Born surface area (MM-GBSA) calculations of the ligand (epitope peptide) binding energy yielded energy values that were in agreement with the ITC measurements and indicated that the charged residues play crucial role in the epitope binding. Theoretical arguments presented in this work indicate that two adjacent arginine residues (ArgH50 and ArgH52) are responsible for the observed proton transfer.     

Objectives versus realities: Spatial,temporal, financial and social deficiencies in Australia’s public revegetation investment model

Past and continuing fragmentation and modification of ecosystems, as well as other threatening processes, cause ongoing biodiversity losses and species extinctions in Australia. At the same time as biodiversity declines, government funding for conservation and restoration is diminishing, leading to reduced action and greater reliance on private investment and community groups. In order to maintain and restore biodiverse ecosystems and the essential services they provide, both conservation of existing vegetation and habitat reconstruction are required. In this paper, we summarise the available data on planting area and cost from the Australian Government’s 20 Million Trees programme (2014–2020), the largest recent national‐scale revegetation incentives programme in Australia. We find that the current spatial scale of effort and investment in habitat reconstruction is insufficient to match the scale required to meet national conservation objectives. Furthermore, the funding rate ($/ha) and contracting arrangements are inadequate for the establishment of high‐quality self‐sustaining vegetation needed for the recovery of Australia’s threatened species and ecological communities. We estimate that the minimum amount of funding required for habitat reconstruction is at least five times higher than is provided for current national flagship programmes such as 20 Million Trees. We provide recommendations, designed to assist future habitat reconstruction programmes achieve their long‐term biodiversity objectives.     

T-helper 17 cell cytokines and interferon type I: partners in crime in systemic lupus erythematosus?

Introduction

A hallmark of systemic autoimmune diseases like systemic lupus erythematosus (SLE) is the increased expression of interferon (IFN) type I inducible genes, so-called IFN type I signature. Recently, T-helper 17 subset (Th17 cells), which produces IL-17A, IL-17F, IL-21, and IL-22, has been implicated in SLE. As CCR6 enriches for Th17 cells, we used this approach to investigate whether CCR6⁺ memory T-helper cells producing IL-17A, IL-17F, IL-21, and/or IL-22 are increased in SLE patients and whether this increase is related to the presence of IFN type I signature.

Methods

In total, 25 SLE patients and 15 healthy controls (HCs) were included. SLE patients were divided into IFN type I signature-positive (IFN⁺) (n = 16) and negative (IFN^-) (n = 9) patients, as assessed by mRNA expression of IFN-inducible genes (IFIGs) in monocytes. Expression of IL-17A, IL-17F, IL-21, and IL-22 by CD4⁺CD45RO⁺CCR6⁺ T cells (CCR6⁺ cells) was measured with flow cytometry and compared between IFN⁺, IFN^- patients and HCs.

Results

Increased percentages of IL-17A and IL-17A/IL-17F double-producing CCR6⁺ cells were observed in IFN⁺ patients compared with IFN^- patients and HCs. IL-17A and IL-17F expression within CCR6⁺ cells correlated significantly with IFIG expression. In addition, we found significant correlation between B-cell activating factor of the tumor necrosis family (BAFF)–a factor strongly correlating with IFN type I - and IL-21 producing CCR6⁺ cells.

Conclusions

We show for the first time higher percentages of IL-17A and IL-17A/IL-17F double-producing CCR6⁺ memory T-helper cells in IFN⁺ SLE patients, supporting the hypothesis that IFN type I co-acts with Th17 cytokines in SLE pathogenesis.     

A marker-assisted backcross approach for developing submergence-tolerant rice cultivars

Submergence stress regularly affects 15 million hectares or more of rainfed lowland rice areas in South and Southeast Asia. A major QTL on chromosome 9, Sub1, has provided the opportunity to apply marker assisted backcrossing (MAB) to develop submergence tolerant versions of rice cultivars that are widely grown in the region. In the present study, molecular markers that were tightly linked with Sub1, flanking Sub1, and unlinked to Sub1 were used to apply foreground, recombinant, and background selection, respectively, in backcrosses between a submergence-tolerant donor and the widely grown recurrent parent Swarna. By the BC₂F₂ generation a submergence tolerant plant was identified that possessed Swarna type simple sequence repeat (SSR) alleles on all fragments analyzed except the tip segment of rice chromosome 9 that possessed the Sub1 locus. A BC₃F₂ double recombinant plant was identified that was homozygous for all Swarna type alleles except for an approximately 2.3–3.4 Mb region surrounding the Sub1 locus. The results showed that the mega variety Swarna could be efficiently converted to a submergence tolerant variety in three backcross generations, involving a time of two to three years. Polymorphic markers for foreground and recombinant selection were identified for four other mega varieties to develop a wider range of submergence tolerant varieties to meet the needs of farmers in the flood-prone regions. This approach demonstrates the effective use of marker assisted selection for a major QTL in a molecular breeding program. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A bacterial population study of commercialized wastewater inoculants

AIMS: To assess the bacterial diversity and safety of wastewater inoculants, which are commercially available products used to improve the aerobic digestion processes of the domestic waste compost in the septic tank. METHODS AND RESULTS: Eighteen wastewater inoculants were analysed on nonselective and selective media and the cultivable bacteria were identified. In all wastewater inoculants, the number of CFUs were between 10(4) and 10(7) g(-1) powder on nonselective media and Bacillus was the predominant cultivable genus. Culture-independent molecular methods such as sequencing of 16S rRNA clone libraries and denaturating gradient gel electrophoresis demonstrated the high prevalence of interfering chloroplast 16S rRNA from plant material and the presence of Bacillus spp. Only after selective enrichments and cultivation, the presence of one pathogenic strain (Klebsiella pneumoniae subsp. pneumoniae) and one opportunistic strain of (Enterobacter cloacae) bacteria were detected in six different products. CONCLUSION: The predominant cultivable species of the wastewater inoculants were Bacillus spp. and after enrichment six products were found to contain opportunistic or pathogenic strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of opportunistic pathogenic strains in the inoculants might represent a risk for immunocompromised, the elderly or children. A clear labelling should therefore be displayed on the product.     

Risk of resource failure and toolkit variation in small-scale farmers and herders

Recent work suggests that global variation in toolkit structure among hunter-gatherers is driven by risk of resource failure such that as risk of resource failure increases, toolkits become more diverse and complex. Here we report a study in which we investigated whether the toolkits of small-scale farmers and herders are influenced by risk of resource failure in the same way. In the study, we applied simple linear and multiple regression analysis to data from 45 small-scale food-producing groups to test the risk hypothesis. Our results were not consistent with the hypothesis; none of the risk variables we examined had a significant impact on toolkit diversity or on toolkit complexity. It appears, therefore, that the drivers of toolkit structure differ between hunter-gatherers and small-scale food-producers.     

Estimating surface area in early hominins

Height and weight-based methods of estimating surface area have played an important role in the development of the current consensus regarding the role of thermoregulation in human evolution. However, such methods may not be reliable when applied to early hominins because their limb proportions differ markedly from those of humans. Here, we report a study in which this possibility was evaluated by comparing surface area estimates generated with the best-known height and weight-based method to estimates generated with a method that is sensitive to proportional differences. We found that the two methods yield indistinguishable estimates when applied to taxa whose limb proportions are similar to those of humans, but significantly different results when applied to taxa whose proportions differ from those of humans. We also found that the discrepancy between the estimates generated by the two methods is almost entirely attributable to inter-taxa differences in limb proportions. One corollary of these findings is that we need to reassess hypotheses about the role of thermoregulation in human evolution that have been developed with the aid of height and weight-based methods of estimating body surface area. Another is that we need to use other methods in future work on fossil hominin body surface areas.     

Mannose-binding lectin does not explain the course and outcome of pregnancy in rheumatoid arthritis

Introduction  

Rheumatoid arthritis (RA) improves during pregnancy and flares after delivery. It has been hypothesized that high levels of the complement factor mannose-binding lectin (MBL) are associated with a favourable disease course of RA by facilitating the clearance of pathogenic immunoglobulin G (IgG) lacking galactose sugar moieties. During pregnancy, increased galactosylation of IgG and simultaneously increased MBL levels can be observed, with the latter being strictly related to maternal MBL genotypes. Therefore, increased MBL levels in concert with increased IgG galactosylation may be associated with pregnancy-induced improvement of RA. The objective of this study was to investigate whether MBL genotypes are associated with changes in RA disease activity and with changes in IgG galactosylation during pregnancy and in the postpartum period. We also studied the association between MBL genotypes and pregnancy outcomes in RA.