Semi-Automated,Occupationally Safe Immunofluorescence Microtip Sensor for Rapid Detection of Mycobacterium Cells in Sputum

An occupationally safe (biosafe) sputum liquefaction protocol was developed for use with a semi-automated antibody-based microtip immunofluorescence sensor. The protocol effectively liquefied sputum and inactivated microorganisms including Mycobacterium tuberculosis, while preserving the antibody-binding activity of Mycobacterium cell surface antigens. Sputum was treated with a synergistic chemical-thermal protocol that included moderate concentrations of NaOH and detergent at 60°C for 5 to 10 min. Samples spiked with M. tuberculosis complex cells showed approximately 10⁶-fold inactivation of the pathogen after treatment. Antibody binding was retained post-treatment, as determined by analysis with a microtip immunosensor. The sensor correctly distinguished between Mycobacterium species and other cell types naturally present in biosafe-treated sputum, with a detection limit of 100 CFU/mL for M. tuberculosis, in a 30-minute sample-to-result process. The microtip device was also semi-automated and shown to be compatible with low-cost, LED-powered fluorescence microscopy. The device and biosafe sputum liquefaction method opens the door to rapid detection of tuberculosis in settings with limited laboratory infrastructure.     

Distribution and function of TrkB receptors in the developing brain of the opossum Monodelphis domestica

The expression, development pattern, spatiotemporal distribution, and function of TrkB receptors were investigated during the postnatal brain development of the opossum. Full‐length TrkB receptor expression was detectable in the newborn opossum, whereas three different short forms that are expressed in the adult brain were almost undetectable in the newborn opossum brain. The highest level of full‐length TrkB receptor expression was observed at P35, which corresponds to the time of eye opening. We found that in different brain structures, TrkB receptors were localized in various compartments of cells. The hypothalamus was distinguished by the presence of TrkB receptors not only in cell bodies but also in the neuropil. Double immunofluroscent staining for TrkB and a marker for the identification of the cell phenotype in several brain regions such as the olfactory bulb, hippocampus, thalamus, and cerebellum showed that unlike in eutherians, in the opossum, TrkB receptors were predominantly expressed in neurons. A lack of TrkB receptors in glial cells, particularly astrocytes and oligodendrocytes, provides evidence that TrkB receptors can play a functionally different role in marsupials than in eutherians. The effects of TrkB signaling on the development of cortical progenitor cells were examined in vitro using shRNAs. Blockade of the endogenous TrkB receptor expression induced a decrease in the number of progenitor cells proliferation, whereas the number of apoptotic progenitor cells increased. These changes were statistically significant but relatively small. In contrast, TrkB signaling was strongly involved in regulation of the cortical progenitor cell differentiation process. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 74: 707–722, 2014     

Dietary Flavanols Modulate the Transcription of Genes Associated with Cardiovascular Pathology without Changes in Their DNA Methylation State

Background

In a recent intervention study, the daily supplementation with 200 mg monomeric and oligomeric flavanols (MOF) from grape seeds for 8 weeks revealed a vascular health benefit in male smokers. The objective of the present study was to determine the impact of MOF consumption on the gene expression profile of leukocytes and to assess changes in DNA methylation.

Methodology/Principal Findings

Gene expression profiles were determined using whole genome microarrays (Agilent) and DNA methylation was assessed using HumanMethylation450 BeadChips (Illumina). MOF significantly modulated the expression of 864 genes. The majority of the affected genes are involved in chemotaxis, cell adhesion, cell infiltration or cytoskeleton organisation, suggesting lower immune cell adhesion to endothelial cells. This was corroborated by in vitro experiments showing that MOF exposure of monocytes attenuates their adhesion to TNF-α-stimulated endothelial cells. Nuclear factor kappa B (NF-κB) reporter gene assays confirmed that MOF decrease the activity of NF-κB. Strong inter-individual variability in the leukocytes'' DNA methylation was observed. As a consequence, on group level, changes due to MOF supplementation could not be found.

Conclusion

Our study revealed that an 8 week daily supplementation with 200 mg MOF modulates the expression of genes associated with cardiovascular disease pathways without major changes of their DNA methylation state. However, strong inter-individual variation in leukocyte DNA methylation may obscure the subtle epigenetic response to dietary flavanols. Despite the lack of significant changes in DNA methylation, the modulation of gene expression appears to contribute to the observed vascular health effect of MOF in humans.     

Using Ion Mobility Spectrometry–Mass Spectrometry to Decipher the Conformational and Assembly Characteristics of the Hepatitis B Capsid Protein

The structural and functional analysis of the core protein of hepatitis B virus is important for a full understanding of the viral life cycle and the development of novel therapeutic agents. The majority of the core protein (CP149) comprises the capsid assembly domain, and the C-terminal region (residues 150–183) is responsible for nucleic acid binding. Protein monomers associate to form dimeric structural subunits, and helices 3 and 4 (residues 50–111 of the assembly domain) have been shown to be important for this as they constitute the interdimer interface. Here, using mass spectrometry coupled with ion mobility spectrometry, we demonstrate the conformational flexibility of the CP149 dimer. Limited proteolysis was used to locate involvement in this feature to the C-terminal region. A genetically fused CP dimer was found to show decreased disorder, consistent with a more restricted C-terminus at the fusion junction. Incubation of CP149 dimer with heteroaryldihydropyrimidine-1, a small molecule known to interfere with the assembly process, was shown to result in oligomers different in shape to the capsid assembly-competent oligomers of the fused CP dimer. We suggest that heteroaryldihydropyrimidine-1 affects the dynamics of CP149 dimer in solution, likely affecting the ratio between assembly active and inactive states. Therefore, assembly of the less dynamic fused dimer is less readily misdirected by heteroaryldihydropyrimidine-1. These studies of the flexibility and oligomerization properties of hepatitis B virus core protein illustrate both the importance of C-terminal dynamics in function and the utility of gas-phase techniques for structural and dynamical biomolecular analysis.     

Using Ion Mobility Spectrometry–Mass Spectrometry to Decipher the Conformational and Assembly Characteristics of the Hepatitis B Capsid Protein

The structural and functional analysis of the core protein of hepatitis B virus is important for a full understanding of the viral life cycle and the development of novel therapeutic agents. The majority of the core protein (CP149) comprises the capsid assembly domain, and the C-terminal region (residues 150–183) is responsible for nucleic acid binding. Protein monomers associate to form dimeric structural subunits, and helices 3 and 4 (residues 50–111 of the assembly domain) have been shown to be important for this as they constitute the interdimer interface. Here, using mass spectrometry coupled with ion mobility spectrometry, we demonstrate the conformational flexibility of the CP149 dimer. Limited proteolysis was used to locate involvement in this feature to the C-terminal region. A genetically fused CP dimer was found to show decreased disorder, consistent with a more restricted C-terminus at the fusion junction. Incubation of CP149 dimer with heteroaryldihydropyrimidine-1, a small molecule known to interfere with the assembly process, was shown to result in oligomers different in shape to the capsid assembly-competent oligomers of the fused CP dimer. We suggest that heteroaryldihydropyrimidine-1 affects the dynamics of CP149 dimer in solution, likely affecting the ratio between assembly active and inactive states. Therefore, assembly of the less dynamic fused dimer is less readily misdirected by heteroaryldihydropyrimidine-1. These studies of the flexibility and oligomerization properties of hepatitis B virus core protein illustrate both the importance of C-terminal dynamics in function and the utility of gas-phase techniques for structural and dynamical biomolecular analysis.     

Development of a novel and efficient cell culture flocculation process using a stimulus responsive polymer to streamline antibody purification processes

Recent advances in mammalian cell culture processes have significantly increased product titers, but have also resulted in substantial increases in cell density and cellular debris as well as process and product related impurities. As such, with improvements in titer, corresponding improvements in downstream processing are essential. In this study we have developed an alternative antibody harvest process that incorporates flocculation using a novel stimulus responsive polymer, benzylated poly(allylamine), followed by depth filtration. As tested on multiple antibodies, this process demonstrates high process yield, improved clearance of cells and cell debris, and efficient reduction of aggregates, host cell proteins (HCP) and DNA. A wide operating window was established for this novel flocculation process through design of experiments condition screening and optimization. Residual levels of impurities in the Protein A eluate were achieved that potentially meet requirements of drug substance and thus alleviate the burden for further impurities removal in subsequent chromatography steps. In addition, efficient clearance of residual polymer was demonstrated using a fluorescence tagged polymer in the presence of a stimulus reagent. The mechanism of HCP and aggregates removal during flocculation was also explored. This novel and efficient process can be easily integrated into current mAb purification platforms, and may overcome downstream processing challenges. Biotechnol. Bioeng. 2013;110: 2928–2937. © 2013 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Nutrient input from hemiparasitic litter favors plant species with a fast-growth strategy

Aims

Hemiparasitic plants often produce nutrient-rich litter with high decomposition rates, and thus can enhance nutrient availability. When plant species have differential affinities for this nutrient source, hemiparasitic litter might influence species composition in addition to the parasitic suppression of host species. We expected that species adapted to fertile habitats derive a higher proportion of nutrients from the hemiparasitic litter compared to other species.

Methods

¹⁵N-labeled litter of Rhinanthus angustifolius and Pedicularis sylvatica was added to experimental field plots and adjacent litter bags. We examined N release from the litter, N uptake by the vegetation 2, 4 and 12 months after litter addition and differences in the proportion of N taken up from the litter (N_L) between co-occurring species.

Results

The percentage of N in shoots of co-occurring plant species that is derived from the added hemiparasitic litter (N_L) strongly differed between the species (0.1–6.2 %). After exclusion of species with an alternative N source (legumes as well as ectomycorrhizal and ericoid mycorrhizal species), N_L was positively related (p?<?0.001) with specific leaf area (SLA) and at Pedicularis sites with leaf N concentration (LNC) and leaf phosphorus concentration (LPC) (p?<?0.05), i.e. leaf traits associated with a fast-growth strategy and adaptation to high-nutrient environments.

Conclusions

Our results suggest that nutrient release from hemiparasitic litter favors plant species with a fast-growth strategy adapted to high-nutrient environments compared to species with a slow-growth strategy. Whether continued hemiparasitic litter inputs are able to change species composition in the long term requires further research.     

A new method to analyze postural stability during a transition task from double-leg stance to single-leg stance

Time to stabilization (TTS) has been introduced as a method to analyze dynamic postural stability during jump and landing tasks, but has also been applied during the transition task from double-leg stance (DLS) to single-leg stance (SLS). However, the application of the originally described TTS technique during the latter task has some important limitations. The first goal of this study was to present an adapted version of the TTS technique to provide an effective alternative method to better analyze postural stability during the transition from DLS to SLS. The second goal was to study the influence of pathology and different speeds on postural stability outcomes. Fifteen healthy control subjects and 15 subjects with chronic ankle instability (CAI) performed the transition task on their preferred speed and as fast as possible, with eyes open and with eyes closed. Subjects with CAI performed the transition significantly slower when moving at their preferred speed with eyes closed. The time subjects needed to reach a new stability point was not discriminative between groups and largely dependent on movement speed. However, the amount of sway after this new stability point was significantly increased in the CAI group and when eyes were closed. The results of this study suggest that subjects with CAI have a decreased ability to overcome the postural perturbation created by the voluntary movement from DLS to SLS. Focusing only on TTS during the transition from DLS to SLS may lead at least in some cases to misinterpretations when assessing postural stability.     

An integrated,modular approach to data science education in microbiology

We live in an increasingly data-driven world, where high-throughput sequencing and mass spectrometry platforms are transforming biology into an information science. This has shifted major challenges in biological research from data generation and processing to interpretation and knowledge translation. However, postsecondary training in bioinformatics, or more generally data science for life scientists, lags behind current demand. In particular, development of accessible, undergraduate data science curricula has the potential to improve research and learning outcomes as well as better prepare students in the life sciences to thrive in public and private sector careers. Here, we describe the Experiential Data science for Undergraduate Cross-Disciplinary Education (EDUCE) initiative, which aims to progressively build data science competency across several years of integrated practice. Through EDUCE, students complete data science modules integrated into required and elective courses augmented with coordinated cocurricular activities. The EDUCE initiative draws on a community of practice consisting of teaching assistants (TAs), postdocs, instructors, and research faculty from multiple disciplines to overcome several reported barriers to data science for life scientists, including instructor capacity, student prior knowledge, and relevance to discipline-specific problems. Preliminary survey results indicate that even a single module improves student self-reported interest and/or experience in bioinformatics and computer science. Thus, EDUCE provides a flexible and extensible active learning framework for integration of data science curriculum into undergraduate courses and programs across the life sciences.