A rapid, sensitive and economical assessment of monoclonal antibody conformational stability by intrinsic tryptophan fluorescence spectroscopy

Steady-state intrinsic tryptophan fluorescence spectroscopy is used as a rapid, robust and economic way for screening the thermal protein conformational stability in various formulations used during the early biotechnology development phase. The most important parameters affecting protein stability in a liquid formulation, e. g. during the initial purification steps or preformulation development, are the pH of the solution, ionic strength, presence of excipients and combinations thereof. A well-defined protocol is presented for the investigation of the thermal conformational stability of proteins. This allows the determination of the denaturation temperature as a function of solution conditions. Using intrinsic tryptophan fluorescence spectroscopy for monitoring the denaturation and folding of proteins, it is crucial to understand the influence of different formulation parameters on the intrinsic fluorescence probes of proteins. Therefore, we have re-evaluated and re-assessed the influence of temperature, pH, ionic strength, buffer composition on the emission spectra of tryptophan, phenylalanine and tyrosine to correctly analyse and evaluate the data obtained from thermal-induced protein denaturation as a function of the solution parameters mentioned above. The results of this study are a prerequisite for using this method as a screening assay for analysing the conformational stability of proteins in solution. The data obtained from intrinsic protein fluorescence spectroscopy are compared to data derived from calorimetry. The advantage, challenges and applicability using intrinsic tryptophan fluorescence spectroscopy as a routine development method in pharmaceutical biotechnology are discussed.     

Towards zero training for brain-computer interfacing

Electroencephalogram (EEG) signals are highly subject-specific and vary considerably even between recording sessions of the same user within the same experimental paradigm. This challenges a stable operation of Brain-Computer Interface (BCI) systems. The classical approach is to train users by neurofeedback to produce fixed stereotypical patterns of brain activity. In the machine learning approach, a widely adapted method for dealing with those variances is to record a so called calibration measurement on the beginning of each session in order to optimize spatial filters and classifiers specifically for each subject and each day. This adaptation of the system to the individual brain signature of each user relieves from the need of extensive user training. In this paper we suggest a new method that overcomes the requirement of these time-consuming calibration recordings for long-term BCI users. The method takes advantage of knowledge collected in previous sessions: By a novel technique, prototypical spatial filters are determined which have better generalization properties compared to single-session filters. In particular, they can be used in follow-up sessions without the need to recalibrate the system. This way the calibration periods can be dramatically shortened or even completely omitted for these 'experienced' BCI users. The feasibility of our novel approach is demonstrated with a series of online BCI experiments. Although performed without any calibration measurement at all, no loss of classification performance was observed.     

Discrimination of pancreatic cancer and pancreatitis by LC-MS metabolomics

Introduction

Pancreatic ductal adenocarcinoma (PDAC) is the fifth most common cause of cancer-related death in Europe with a 5-year survival rate of <5%. Chronic pancreatitis (CP) is a risk factor for PDAC development, but in the majority of cases malignancy is discovered too late for curative treatment. There is at present no reliable diagnostic marker for PDAC available.

Objectives

The aim of the study was to identify single blood-based metabolites or a panel of metabolites discriminating PDAC and CP using liquid chromatography-mass spectrometry (LC-MS).

Methods

A discovery cohort comprising PDAC (n?=?44) and CP (n?=?23) samples was analyzed by LC-MS followed by univariate (Student’s t test) and multivariate (orthogonal partial least squares-discriminant analysis (OPLS-DA)) statistics. Discriminative metabolite features were subject to raw data examination and identification to ensure high feature quality. Their discriminatory power was then confirmed in an independent validation cohort including PDAC (n?=?20) and CP (n?=?31) samples.

Results

Glycocholic acid, N-palmitoyl glutamic acid and hexanoylcarnitine were identified as single markers discriminating PDAC and CP by univariate analysis. OPLS-DA resulted in a panel of five metabolites including the aforementioned three metabolites as well as phenylacetylglutamine (PAGN) and chenodeoxyglycocholate.

Conclusion

Using LC-MS-based metabolomics we identified three single metabolites and a five-metabolite panel discriminating PDAC and CP in two independent cohorts. Although further study is needed in larger cohorts, the metabolites identified are potentially of use in PDAC diagnostics.

     

Identification of a novel protein promoting the colonization and survival of Finegoldia magna, a bacterial commensal and opportunistic pathogen

Anaerobic bacteria dominate the human normal microbiota, but strikingly little is known about these commensals. Finegoldia magna is a Gram-positive anaerobe found in the skin and at other non-sterile body surfaces, but it is also an opportunistic pathogen. This study describes a novel protein designated FAF (F. magna adhesion factor) and expressed by more than 90% of F. magna isolates. The protein is present in substantial quantities at the F. magna surface but is also released from the surface. FAF forms large protein aggregates in solution and surface-associated FAF causes bacterial clumping. In skin F. magna bacteria were localized to the epidermis, where they adhere to basement membranes. FAF was found to mediate this adhesion via interactions with BM-40, a basement membrane protein. The biological significance of FAF is further underlined by the observation that it blocks the activity of LL-37, a major human antibacterial peptide. Altogether, the data demonstrate that FAF plays an important role in colonization and survival of F. magna in the human host.     

Catabolic and anabolic periarticular bone changes in patients with rheumatoid arthritis: a computed tomography study on the role of age,disease duration and bone markers

Introduction

The aim of this study was to determine the factors, including markers of bone resorption and bone formation, which determine catabolic and anabolic periarticular bone changes in patients with rheumatoid arthritis (RA).

Methods

Forty RA patients received high-resolution peripheral quantitative computed tomography (HR-pQCT) analysis of the metacarpophalangeal joints II and III of the dominantly affected hand at two sequential time points (baseline, one year follow-up). Erosion counts and scores as well as osteophyte counts and scores were recorded. Simultaneously, serum markers of bone resorption (C-terminal telopeptide of type I collagen (CTX I), tartrate-resistant acid phosphatase 5b (TRAP5b)), bone formation (bone alkaline phosphatase (BAP), osteocalcin (OC)) and calcium homeostasis (parathyroid hormone (PTH), 25-hydroxyvitamin D3 (Vit D)) were assessed. Bone biomarkers were correlated to imaging data by partial correlation adjusting for various demographic and disease-specific parameters. Additionally, imaging data were analyzed by mixed linear model regression.

Results

Partial correlation analysis showed that TRAP5b levels correlate significantly with bone erosions, whereas BAP levels correlate with osteophytes at both time points. In the mixed linear model with erosions as the dependent variable, disease duration (P <0.001) was the key determinant for these catabolic bone changes. In contrast, BAP (P = 0.001) as well as age (P = 0.018), but not disease duration (P = 0.762), were the main determinants for the anabolic changes (osteophytes) of the periarticular bone in patients with RA.

Conclusions

This study shows that structural bone changes assessed with HR-pQCT are accompanied by alterations in systemic markers of bone resorption and bone formation. Besides, it can be shown that bone erosions in RA patients depend on disease duration, whereas osteophytes are associated with age as well as serum level of BAP. Therefore, these data not only suggest that different variables are involved in formation of bone erosions and osteophytes in RA patients, but also that periarticular bone changes correlate with alterations in systemic markers of bone metabolism, pointing out BAP as an important parameter.     

B Cell Activation by Outer Membrane Vesicles—A Novel Virulence Mechanism

Secretion of outer membrane vesicles (OMV) is an intriguing phenomenon of Gram-negative bacteria and has been suggested to play a role as virulence factors. The respiratory pathogens Moraxella catarrhalis reside in tonsils adjacent to B cells, and we have previously shown that M. catarrhalis induce a T cell independent B cell response by the immunoglobulin (Ig) D-binding superantigen MID. Here we demonstrate that Moraxella are endocytosed and killed by human tonsillar B cells, whereas OMV have the potential to interact and activate B cells leading to bacterial rescue. The B cell response induced by OMV begins with IgD B cell receptor (BCR) clustering and Ca²⁺ mobilization followed by BCR internalization. In addition to IgD BCR, TLR9 and TLR2 were found to colocalize in lipid raft motifs after exposure to OMV. Two components of the OMV, i.e., MID and unmethylated CpG-DNA motifs, were found to be critical for B cell activation. OMV containing MID bound to and activated tonsillar CD19⁺ IgD⁺ lymphocytes resulting in IL-6 and IgM production in addition to increased surface marker density (HLA-DR, CD45, CD64, and CD86), whereas MID-deficient OMV failed to induce B cell activation. DNA associated with OMV induced full B cell activation by signaling through TLR9. Importantly, this concept was verified in vivo, as OMV equipped with MID and DNA were found in a 9-year old patient suffering from Moraxella sinusitis. In conclusion, Moraxella avoid direct interaction with host B cells by redirecting the adaptive humoral immune response using its superantigen-bearing OMV as decoys.     

Chlamydia-like bacteria in respiratory samples of community-acquired pneumonia patients

Chlamydia-like bacteria, obligate intracellular relatives of Chlamydia trachomatis and Chlamydophila pneumoniae, are widely distributed in nature. Using a two-step nested and semi-nested PCR approach targeting the 16S rRNA gene, we found DNA of Chlamydia-like bacteria in respiratory samples from patients with community-acquired pneumonia. Of 387 cases tested, four (1.03%) tested positive if only sequences showing less than 99.9% 16S rRNA gene sequence similarity to known Chlamydiae were considered. These included for the first time Protochlamydia amoebophila, Waddlia chondrophila, and 'Candidatus Rhabdochlamydia porcellionis'-related sequences. This study extends previous findings suggesting an association of Chlamydia-like bacteria with respiratory disease, but a causal link between these microorganisms and respiratory tract infections has yet to be established.     

Siscowet lake charr (Salvelinus namaycush siscowet) visual foraging habitat in relation to daily and seasonal light cycles

Hydrobiologia - Using siscowet lake charr (Salvelinus namaycush siscowet) as an example organism, we modeled visual foraging habitat in relation to: (i) daily solar and lunar intensity, (ii)...     

Mixed Phase Solid‐State Plastic Crystal Electrolytes Based on a Phosphonium Cation for Sodium Devices

Na batteries are seen as a feasible alternative technology to lithium ion batteries due to the greater abundance of sodium and potentially similar electrochemical behavior. In this work, mixed phase electrolyte materials based on solid‐state compositions of a tri methylisobutylphosphonium (P_111i4) bis(tri fluromethanesulphonyl)amide (NTf₂) organic ionic plastic crystal (OIPC) and high concentration of NaNTf₂ that support safe, sodium metal electrochemistry are demonstrated. A Na symmetric cell can be cycled efficiently, even in the solid state (at 50 °C and 60 °C), for a 25 mol% (P_111i4NTf₂)–75 mol% NaNTf₂ composition at 0.1 mA cm^?2 for 100 cycles. Thus, these mixed phase materials can be potentially used in Na‐based devices under moderate temperature conditions. It is also investigated that the phase behavior, conductivity, and electrochemical properties of mixtures of NaNTf₂ with this OIPC. It is observed that these mixtures have complex phase behavior. For high compositions of the Na salt, the materials are solid at room temperature and retain a soft solid consistency even at 50 °C with remarkably high conductivity, approaching that of the pure ionic liquid at 50 °C, i.e., 10^?3–10^?2 S cm^?1.