Subspecies typing of Streptococcus agalactiae based on ribosomal subunit protein mass variation by MALDI-TOF MS

Background: A ribosomal subunit protein (rsp)-based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method was developed for fast subspecies-level typing of Streptococcus agalactiae (Group B Streptococcus, GBS), a major cause of neonatal sepsis and meningitis. Methods: A total of 796 GBS whole genome sequences, covering the genetic diversity of the global GBS population, were used to in silico predict molecular mass variability of 28 rsp and to identify unique rsp mass combinations, termed “rsp-profiles”. The in silico established GBS typing scheme was validated by MALDI-TOF MS analysis of GBS isolates at two independent research sites in Europe and South East Asia. Results: We identified in silico 62 rsp-profiles, with the majority (>80%) of the 796 GBS isolates displaying one of the six rsp-profiles 1-6. These dominant rsp-profiles classify GBS strains in high concordance with the core-genome based phylogenetic clustering. Validation of our approach by in-house MALDI-TOF MS analysis of 248 GBS isolates and external analysis of 8 GBS isolates showed that across different laboratories and MALDI-TOF MS platforms, the 28 rsp were detected reliably in the mass spectra, allowing assignment of clinical isolates to rsp-profiles at high sensitivity (99%) and specificity (97%). Our approach distinguishes the major phylogenetic GBS genotypes, identifies hyper-virulent strains, predicts the probable capsular serotype and surface protein variants and distinguishes between GBS genotypes of human and animal origin. Conclusion: We combine the information depth of whole genome sequences with the highly cost efficient, rapid and robust MALDI-TOF MS approach facilitating high-throughput, inter-laboratory, large-scale GBS epidemiological and clinical studies based on pre-defined rsp-profiles

Functional asymmetry in the lysyl-tRNA synthetase explored by molecular dynamics, free energy calculations and experiment

BACKGROUND: Charging of transfer-RNA with cognate amino acid is accomplished by the aminoacyl-tRNA synthetases, and proceeds through an aminoacyl adenylate intermediate. The lysyl-tRNA synthetase has evolved an active site that specifically binds lysine and ATP. Previous molecular dynamics simulations of the heat-inducible Escherichia coli lysyl-tRNA synthetase, LysU, have revealed differences in the binding of ATP and aspects of asymmetry between the nominally equivalent active sites of this dimeric enzyme. The possibility that this asymmetry results in different binding affinities for the ligands is addressed here by a parallel computational and biochemical study. RESULTS: Biochemical experiments employing isothermal calorimetry, steady-state fluorescence and circular dichroism are used to determine the order and stoichiometries of the lysine and nucleotide binding events, and the associated thermodynamic parameters. An ordered mechanism of substrate addition is found, with lysine having to bind prior to the nucleotide in a magnesium dependent process. Two lysines are found to bind per dimer, and trigger a large conformational change. Subsequent nucleotide binding causes little structural rearrangement and crucially only occurs at a single catalytic site, in accord with the simulations. Molecular dynamics based free energy calculations of the ATP binding process are used to determine the binding affinities of each site. Significant differences in ATP binding affinities are observed, with only one active site capable of realizing the experimental binding free energy. Half-of-the-sites models in which the nucleotide is only present at one active site achieve their full binding potential irrespective of the subunit choice. This strongly suggests the involvement of an anti-cooperative mechanism. Pathways for relaying information between the two active sites are proposed. CONCLUSIONS: The asymmetry uncovered here appears to be a common feature of oligomeric aminoacyl-tRNA synthetases, and may play an important functional role. We suggest a manner in which catalytic efficiency could be improved by LysU operating in an alternating sites mechanism

Distinct Transcriptional and Anti-Mycobacterial Profiles of Peripheral Blood Monocytes Dependent on the Ratio of Monocytes: Lymphocytes.

The ratio of monocytes and lymphocytes (ML ratio) in peripheral blood is associated with tuberculosis and malaria disease risk and cancer and cardiovascular disease outcomes. We studied anti-mycobacterial function and the transcriptome of monocytes in relation to the ML ratio. Mycobacterial growth inhibition assays of whole or sorted blood were performed and mycobacteria were enumerated by liquid culture. Transcriptomes of unstimulated CD14 + monocytes isolated by magnetic bead sorting were characterised by microarray. Transcript expression was tested for association with ML ratio calculated from leucocyte differential counts by linear regression. The ML ratio was associated with mycobacterial growth in vitro (β = 2.23, SE 0.91, p = 0.02). Using sorted monocytes and lymphocytes, in vivo ML ratio (% variance explained R(2) = 11%, p = 0.02) dominated over in vitro ratios (R(2) = 5%, p = 0.10) in explaining mycobacterial growth. Expression of 906 genes was associated with the ML ratio and 53 with monocyte count alone. ML-ratio associated genes were enriched for type-I and -II interferon signalling (p = 1.2 × 10(− 8)), and for genes under transcriptional control of IRF1, IRF2, RUNX1, RELA and ESRRB. The ML-ratio-associated gene set was enriched in TB disease (3.11-fold, 95% CI: 2.28-4.19, p = 5.7 × 10(− 12)) and other inflammatory diseases including atopy, HIV, IBD and SLE. The ML ratio is associated with distinct transcriptional and anti-mycobacterial profiles of monocytes that may explain the disease associations of the ML ratio

TRAPPC9 Mediates the Interaction between p150Glued and COPII Vesicles at the Target Membrane

Background: The transport of endoplasmic reticulum (ER)-derived COPII vesicles toward the ER-Golgi intermediate compartment (ERGIC) requires cytoplasmic dynein and is dependent on microtubules. p150Glued, a subunit of dynactin, has been implicated in the transport of COPII vesicles via its interaction with COPII coat components Sec23 and Sec24. However, whether and how COPII vesicle tether, TRAPP (Transport protein particle), plays a role in the interaction between COPII vesicles and microtubules is currently unknown. Principle Findings: We address the functional relationship between COPII tether TRAPP and dynactin. Overexpressed TRAPP subunits interfered with microtubule architecture by competing p150Glued away from the MTOC. TRAPP subunit TRAPPC9 bound directly to p150Glued via the same carboxyl terminal domain of p150Glued that binds Sec23 and Sec24. TRAPPC9 also inhibited the interaction between p150Glued and Sec23/Sec24 both in vitro and in vivo, suggesting that TRAPPC9 serves to uncouple p150Glued from the COPII coat, and to relay the vesicle-dynactin interaction at the target membrane. Conclusions: These findings provide a new perspective on the function of TRAPP as an adaptor between the ERGIC membrane and dynactin. By preserving the connection between dynactin and the tethered and/or fused vesicles, TRAPP allows nascent ERGIC to continue the movement along the microtubules as they mature into the cis-Golgi

A DNA aptamer recognising a malaria protein biomarker can function as part of a DNA origami assembly

DNA aptamers have potential for disease diagnosis and as therapeutics, particularly when interfaced with programmable molecular technology. Here we have combined DNA aptamers specific for the malaria biomarker Plasmodium falciparum lactate dehydrogenase (PfLDH) with a DNA origami scaffold. Twelve aptamers that recognise PfLDH were integrated into a rectangular DNA origami and atomic force microscopy demonstrated that the incorporated aptamers preserve their ability to specifically bind target protein. Captured PfLDH retained enzymatic activity and protein-aptamer binding was observed dynamically using high-speed AFM. This work demonstrates the ability of DNA aptamers to recognise a malaria biomarker whilst being integrated within a supramolecular DNA scaffold, opening new possibilities for malaria diagnostic approaches based on DNA nanotechnology

DNA Extraction Method Development for Solid Tissues

Although germline variation testing is traditionally performed using DNA obtained from blood or other liquid samples, determining somatic variation in cancer samples requires DNA extraction directly from tissues. Additionally, epigenetic markers, such as 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are tissue-specific and change in selected disease states. However, several substances present in tissues are known to inhibit downstream reactions, including polymerase chain reaction PCR). For this project, we are assessing the quantity and quality of DNA obtained from extractions of various vital organs using 30 different commercially available DNA extraction kits to determine optimal kits for each tissue

Evaluation of the current knowledge limitations in breast cancer research: a gap analysis

BACKGROUND A gap analysis was conducted to determine which areas of breast cancer research, if targeted by researchers and funding bodies, could produce the greatest impact on patients. METHODS Fifty-six Breast Cancer Campaign grant holders and prominent UK breast cancer researchers participated in a gap analysis of current breast cancer research. Before, during and following the meeting, groups in seven key research areas participated in cycles of presentation, literature review and discussion. Summary papers were prepared by each group and collated into this position paper highlighting the research gaps, with recommendations for action. RESULTS Gaps were identified in all seven themes. General barriers to progress were lack of financial and practical resources, and poor collaboration between disciplines. Critical gaps in each theme included: (1) genetics (knowledge of genetic changes, their effects and interactions); (2) initiation of breast cancer (how developmental signalling pathways cause ductal elongation and branching at the cellular level and influence stem cell dynamics, and how their disruption initiates tumour formation); (3) progression of breast cancer (deciphering the intracellular and extracellular regulators of early progression, tumour growth, angiogenesis and metastasis); (4) therapies and targets (understanding who develops advanced disease); (5) disease markers (incorporating intelligent trial design into all studies to ensure new treatments are tested in patient groups stratified using biomarkers); (6) prevention (strategies to prevent oestrogen-receptor negative tumours and the long-term effects of chemoprevention for oestrogen-receptor positive tumours); (7) psychosocial aspects of cancer (the use of appropriate psychosocial interventions, and the personal impact of all stages of the disease among patients from a range of ethnic and demographic backgrounds). CONCLUSION Through recommendations to address these gaps with future research, the long-term benefits to patients will include: better estimation of risk in families with breast cancer and strategies to reduce risk; better prediction of drug response and patient prognosis; improved tailoring of treatments to patient subgroups and development of new therapeutic approaches; earlier initiation of treatment; more effective use of resources for screening populations; and an enhanced experience for people with or at risk of breast cancer and their families. The challenge to funding bodies and researchers in all disciplines is to focus on these gaps and to drive advances in knowledge into improvements in patient care

Measuring luteinising hormone pulsatility with a robotic aptamer-enabled electrochemical reader

Assessment of luteinising hormone pulsatility is important in the diagnosis of reproductive disorders. Here the authors develop a DNA aptamer-based electrochemical analysis integrated into a robotic platform for high-throughput and sensitive analysis

Interactions between Connected Half-Sarcomeres Produce Emergent Mechanical Behavior in a Mathematical Model of Muscle

Most reductionist theories of muscle attribute a fiber's mechanical properties to the scaled behavior of a single half-sarcomere. Mathematical models of this type can explain many of the known mechanical properties of muscle but have to incorporate a passive mechanical component that becomes ∼300% stiffer in activating conditions to reproduce the force response elicited by stretching a fast mammalian muscle fiber. The available experimental data suggests that titin filaments, which are the mostly likely source of the passive component, become at most ∼30% stiffer in saturating Ca2+ solutions. The work described in this manuscript used computer modeling to test an alternative systems theory that attributes the stretch response of a mammalian fiber to the composite behavior of a collection of half-sarcomeres. The principal finding was that the stretch response of a chemically permeabilized rabbit psoas fiber could be reproduced with a framework consisting of 300 half-sarcomeres arranged in 6 parallel myofibrils without requiring titin filaments to stiffen in activating solutions. Ablation of inter-myofibrillar links in the computer simulations lowered isometric force values and lowered energy absorption during a stretch. This computed behavior mimics effects previously observed in experiments using muscles from desmin-deficient mice in which the connections between Z-disks in adjacent myofibrils are presumably compromised. The current simulations suggest that muscle fibers exhibit emergent properties that reflect interactions between half-sarcomeres and are not properties of a single half-sarcomere in isolation. It is therefore likely that full quantitative understanding of a fiber's mechanical properties requires detailed analysis of a complete fiber system and cannot be achieved by focusing solely on the properties of a single half-sarcomere