Removal of a C-terminal serine residue proximal to the inter-chain disulfide bond of a human IgG1 lambda light chain mediates enhanced antibody stability and antibody dependent cell-mediated cytotoxicity

Optimization of biophysical properties is a critical success factor for the developability of monoclonal antibodies with potential therapeutic applications. The inter-domain disulfide bond between light chain (Lc) and heavy chain (Hc) in human IgG1 lends structural support for antibody scaffold stability, optimal antigen binding, and normal Fc function. Recently, human IgG1λ has been suggested to exhibit significantly greater susceptibility to reduction of the inter Lc-Hc disulfide bond relative to the same disulfide bond in human IgG1κ. To understand the molecular basis for this observed difference in stability, the sequence and structure of human IgG1λ and human IgG1κ were compared. Based on this Lc comparison, three single mutations were made in the λ Lc proximal to the cysteine residue, which forms a disulfide bond with the Hc. We determined that deletion of S214 (dS) improved resistance of the association between Lc and Hc to thermal stress. In addition, deletion of this terminal serine from the Lc of IgG1λ provided further benefit, including an increase in stability at elevated pH, increased yield from transient transfection, and improved in vitro antibody dependent cell-mediated cytotoxicity (ADCC). These observations support the conclusion that the presence of the terminal serine of the λ Lc creates a weaker inter-chain disulfide bond between the Lc and Hc, leading to slightly reduced stability and a potential compromise in IgG1λ function. Our data from a human IgG1λ provide a basis for further investigation of the effects of deleting terminal serine from λLc on the stability and function of other human IgG1λ antibodies.     

Localization and Relative Quantification of Carbon Nanotubes in Cells with Multispectral Imaging Flow Cytometry

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich cell structures and de facto there is still no quantitative method to assess their distribution at cell and tissue levels. What we propose here is an innovative method allowing the detection and quantification of CNTs in cells using a multispectral imaging flow cytometer (ImageStream, Amnis). This newly developed device integrates both a high-throughput of cells and high resolution imaging, providing thus images for each cell directly in flow and therefore statistically relevant image analysis. Each cell image is acquired on bright-field (BF), dark-field (DF), and fluorescent channels, giving access respectively to the level and the distribution of light absorption, light scattered and fluorescence for each cell. The analysis consists then in a pixel-by-pixel comparison of each image, of the 7,000-10,000 cells acquired for each condition of the experiment. Localization and quantification of CNTs is made possible thanks to some particular intrinsic properties of CNTs: strong light absorbance and scattering; indeed CNTs appear as strongly absorbed dark spots on BF and bright spots on DF with a precise colocalization.This methodology could have a considerable impact on studies about interactions between nanomaterials and cells given that this protocol is applicable for a large range of nanomaterials, insofar as they are capable of absorbing (and/or scattering) strongly enough the light.     

The Chromosomal Passenger Protein Birc5b Organizes Microfilaments and Germ Plasm in the Zebrafish Embryo

Microtubule-microfilament interactions are important for cytokinesis and subcellular localization of proteins and mRNAs. In the early zebrafish embryo, astral microtubule-microfilament interactions also facilitate a stereotypic segregation pattern of germ plasm ribonucleoparticles (GP RNPs), which is critical for their eventual selective inheritance by germ cells. The precise mechanisms and molecular mediators for both cytoskeletal interactions and GP RNPs segregation are the focus of intense research. Here, we report the molecular identification of a zebrafish maternal-effect mutation motley as Birc5b, a homolog of the mammalian Chromosomal Passenger Complex (CPC) component Survivin. The meiosis and mitosis defects in motley/birc5b mutant embryos are consistent with failed CPC function, and additional defects in astral microtubule remodeling contribute to failures in the initiation of cytokinesis furrow ingression. Unexpectedly, the motley/birc5b mutation also disrupts cortical microfilaments and GP RNP aggregation during early cell divisions. Birc5b localizes to the tips of astral microtubules along with polymerizing cortical F-actin and the GP RNPs. Mutant Birc5b co-localizes with cortical F-actin and GP RNPs, but fails to associate with astral microtubule tips, leading to disorganized microfilaments and GP RNP aggregation defects. Thus, maternal Birc5b localizes to astral microtubule tips and associates with cortical F-actin and GP RNPs, potentially linking the two cytoskeletons to mediate microtubule-microfilament reorganization and GP RNP aggregation during early embryonic cell cycles in zebrafish. In addition to the known mitotic function of CPC components, our analyses reveal a non-canonical role for an evolutionarily conserved CPC protein in microfilament reorganization and germ plasm aggregation.     

Steered Molecular Dynamics Simulations of a Type IV Pilus Probe Initial Stages of a Force-Induced Conformational Transition

Type IV pili are long, protein filaments built from a repeating subunit that protrudes from the surface of a wide variety of infectious bacteria. They are implicated in a vast array of functions, ranging from bacterial motility to microcolony formation to infection. One of the most well-studied type IV filaments is the gonococcal type IV pilus (GC-T4P) from Neisseria gonorrhoeae, the causative agent of gonorrhea. Cryo-electron microscopy has been used to construct a model of this filament, offering insights into the structure of type IV pili. In addition, experiments have demonstrated that GC-T4P can withstand very large tension forces, and transition to a force-induced conformation. However, the details of force-generation, and the atomic-level characteristics of the force-induced conformation, are unknown. Here, steered molecular dynamics (SMD) simulation was used to exert a force in silico on an 18 subunit segment of GC-T4P to address questions regarding the nature of the interactions that lead to the extraordinary strength of bacterial pili. SMD simulations revealed that the buried pilin α1 domains maintain hydrophobic contacts with one another within the core of the filament, leading to GC-T4P''s structural stability. At the filament surface, gaps between pilin globular head domains in both the native and pulled states provide water accessible routes between the external environment and the interior of the filament, allowing water to access the pilin α1 domains as reported for VC-T4P in deuterium exchange experiments. Results were also compared to the experimentally observed force-induced conformation. In particular, an exposed amino acid sequence in the experimentally stretched filament was also found to become exposed during the SMD simulations, suggesting that initial stages of the force induced transition are well captured. Furthermore, a second sequence was shown to be initially hidden in the native filament and became exposed upon stretching.     

The Integrator Complex Subunit 6 (Ints6) Confines the Dorsal Organizer in Vertebrate Embryogenesis

Dorsoventral patterning of the embryonic axis relies upon the mutual antagonism of competing signaling pathways to establish a balance between ventralizing BMP signaling and dorsal cell fate specification mediated by the organizer. In zebrafish, the initial embryo-wide domain of BMP signaling is refined into a morphogenetic gradient following activation dorsally of a maternal Wnt pathway. The accumulation of β-catenin in nuclei on the dorsal side of the embryo then leads to repression of BMP signaling dorsally and the induction of dorsal cell fates mediated by Nodal and FGF signaling. A separate Wnt pathway operates zygotically via Wnt8a to limit dorsal cell fate specification and maintain the expression of ventralizing genes in ventrolateral domains. We have isolated a recessive dorsalizing maternal-effect mutation disrupting the gene encoding Integrator Complex Subunit 6 (Ints6). Due to widespread de-repression of dorsal organizer genes, embryos from mutant mothers fail to maintain expression of BMP ligands, fail to fully express vox and ved, two mediators of Wnt8a, display delayed cell movements during gastrulation, and severe dorsalization. Consistent with radial dorsalization, affected embryos display multiple independent axial domains along with ectopic dorsal forerunner cells. Limiting Nodal signaling or restoring BMP signaling restores wild-type patterning to affected embryos. Our results are consistent with a novel role for Ints6 in restricting the vertebrate organizer to a dorsal domain in embryonic patterning.     

Clinical symptoms of ADHD and psychopathy in perpetrators

ADHD and psychopathy are both associated with poor social adaptation and antisocial behavior. Previous studies have also suggested that ADHD and psychopathy share some symptomatology. The current study attempted to further uncover the relationship between ADHD and psychopathy in perpetrators by investigating the possibility of underlying common factors of ADHD and psychopathy using standardized instruments. Correlation analyses and principal component analyses were performed on ADHD-SB self-rating questionnaires and the PCL-SV from a population of 314 offenders. Further, subjects without ADHD, full ADHD and partially remitted ADHD according to DSM-IV were compared regarding psychopathic traits. ADHD and psychopathy rating scores were found to be correlated. Only psychopathic features related to unstable and antisocial lifestyle, but not related to affective features of psychopathy accounted to this association. Findings on the principal component analysis suggest that ADHD and psychopathy are unrelated on a symptomatic level, with the exception of impulsivity, which is a common feature that seems to combine both constructs.     

Higher Risk Perception of HIV than of Chlamydia and HPV among Secondary School Students in Two German Cities

Background

Chlamydia and genital human papillomavirus (HPV) are the two most common sexually transmitted infections (STIs) among teens and young adults in industrialised countries. The majority of adolescents, however, have limited or no knowledge of these infections. Within the context of a cross-sectional survey on awareness and knowledge of sexually transmitted infections, secondary school students attending the 8th grade and above in Bremen and Bremerhaven, two cities in northern Germany, were asked to rate the risk of peers to get infected with HIV, HPV or chlamydia.

Methods

Between October and December 2011, students aged 12–20 years completed an anonymous, self-administered questionnaire at their school. In addition to answering questions on awareness and knowledge of sexually transmitted infections, all students were also asked to rate the risk of peers to get infected with HIV, HPV or chlamydia. Furthermore, those reporting ever having sexual intercourse were asked to rate their own risk of getting infected with each of the three infections.

Results

1,148 students, 55% female, completed the questionnaire. 27% of the students reported having had sexual intercourse. 68% of all students rated the risk of same-aged students to get infected with HIV/AIDS as high/medium. The corresponding proportions for HPV and chlamydia were 19 and 25% respectively. Those reporting ever having sexual intercourse generally perceived their own risk of getting infected with HIV, chlamydia or HPV as lower than that of their peers.

Conclusion

Generally, the risk of getting infected with HIV was perceived as being higher than that of getting infected with HPV or chlamydia, most likely due to the fact that the students were more aware of HIV than of the other two infections. Efforts should be made to improve awareness and knowledge of HPV and chlamydia among school going adolescents, and to make them realize that these are common infections that are preventable.     

High Levels of Asymmetric Dimethylarginine Are Strongly Associated with Low HDL in Patients with Acute Myocardial Infarction

Objectives

Low levels of high-density lipoprotein (HDL) cholesterol are associated with an increased risk of acute myocardial infarction possibly through impaired endothelial atheroprotection and decreased nitric oxide (NO) bioavailability. Asymmetric dimethylarginine (ADMA) mediates endothelial function by inhibiting nitric oxide synthase activity. In patients with acute myocardial infarction, we investigated the relationship between serum levels of HDL and ADMA.

Approach and Results

Blood samples from 612 consecutive patients hospitalized for acute MI <24 hours after symptom onset were taken on admission. Serum levels of ADMA, its stereoisomer, symmetric dimethylarginine (SDMA) and L-arginine were determined using high-performance liquid chromatography. Patients with low HDL (<40 mg/dL for men and <50 mg/dL for women) were compared with patients with higher HDL. Most patients (59%) had low HDL levels. Median ADMA levels were markedly higher in the low HDL group (0.69 vs. 0.50 µmole/L, p<0.001). In contrast, SDMA and L-arginine levels were similar for the two groups (p = 0.120 and p = 0.064). Notably, ADMA, but not SDMA or L-arginine, was inversely correlated with HDL (r = −0.311, p<0.001). In stratified analysis, this relationship was only found for low HDL levels (r = −0.265, p<0.001), but not when HDL levels were higher (r = −0.077, p = 0.225). By multivariate logistic regression analysis, ADMA level was strongly associated with low HDL levels (OR(95%CI):6.06(3.48–10.53), p<0.001), beyond traditional confounding factors.

Conclusions

Our large population-based study showed for the first time a strong inverse relationship between HDL and ADMA in myocardial infarction patients, suggesting a functional interaction between HDL and endothelium, beyond metabolic conditions associated with low HDL levels.