Specificity and Strain-Typing Capabilities of Nanorod Array-Surface Enhanced Raman Spectroscopy for Mycoplasma pneumoniae Detection

Mycoplasma pneumoniae is a cell wall-less bacterial pathogen of the human respiratory tract that accounts for > 20% of all community-acquired pneumonia (CAP). At present the most effective means for detection and strain-typing is quantitative polymerase chain reaction (qPCR), which can exhibit excellent sensitivity and specificity but requires separate tests for detection and genotyping, lacks standardization between available tests and between labs, and has limited practicality for widespread, point-of-care use. We have developed and previously described a silver nanorod array-surface enhanced Raman Spectroscopy (NA-SERS) biosensing platform capable of detecting M. pneumoniae with statistically significant specificity and sensitivity in simulated and true clinical throat swab samples, and the ability to distinguish between reference strains of the two main genotypes of M. pneumoniae. Furthermore, we have established a qualitative lower endpoint of detection for NA-SERS of < 1 genome equivalent (cell/μl) and a quantitative multivariate detection limit of 5.3 ± 1 cells/μl. Here we demonstrate using partial least squares- discriminatory analysis (PLS-DA) of sample spectra that NA-SERS correctly identified M. pneumoniae clinical isolates from globally diverse origins and distinguished these from a panel of 12 other human commensal and pathogenic mycoplasma species with 100% cross-validated statistical accuracy. Furthermore, PLS-DA correctly classified by strain type all 30 clinical isolates with 96% cross-validated accuracy for type 1 strains, 98% cross-validated accuracy for type 2 strains, and 90% cross-validated accuracy for type 2V strains.     

Innate Immune Response to Streptococcus pyogenes Depends on the Combined Activation of TLR13 and TLR2

Innate immune recognition of the major human-specific Gram-positive pathogen Streptococcus pyogenes is not understood. Here we show that mice employ Toll-like receptor (TLR) 2- and TLR13-mediated recognition of S. pyogenes. These TLR pathways are non-redundant in the in vivo context of animal infection, but are largely redundant in vitro, as only inactivation of both of them abolishes inflammatory cytokine production by macrophages and dendritic cells infected with S. pyogenes. Mechanistically, S. pyogenes is initially recognized in a phagocytosis-independent manner by TLR2 and subsequently by TLR13 upon internalization. We show that the TLR13 response is specifically triggered by S. pyogenes rRNA and that Tlr13 ^−/− cells respond to S. pyogenes infection solely by engagement of TLR2. TLR13 is absent from humans and, remarkably, we find no equivalent route for S. pyogenes RNA recognition in human macrophages. Phylogenetic analysis reveals that TLR13 occurs in all kingdoms but only in few mammals, including mice and rats, which are naturally resistant against S. pyogenes. Our study establishes that the dissimilar expression of TLR13 in mice and humans has functional consequences for recognition of S. pyogenes in these organisms.     

On the minimum value of the Colless index and the bifurcating trees that achieve it

Journal of Mathematical Biology - Measures of tree balance play an important role in the analysis of phylogenetic trees. One of the oldest and most popular indices in this regard is the Colless...     

Drosophila metalloproteases in development and differentiation: the role of ADAM proteins and their relatives

ADAM metalloproteases are membrane bound glycoproteins that control many biological processes during development and differentiation, mainly by acting as ectodomain sheddases. The Drosophila genome contains five genes that code for classical ADAM proteins which are characterized by a highly conserved domain structure with the respective catalytic domains facing the extracellular space. More than 50 genes encode related proteins such as those that have lost their primary enzymatic activity while retaining, e.g., their adhesive properties. The physiological relevance of many Drosophila ADAMs and their relatives is still unknown, however for others, a striking role during organogenesis and tissue maintenance has been demonstrated during the last few years. We have carried out genetic screenings combined with candidate approaches, aiming to identify new components involved in cardiogenesis and muscle differentiation. Herein we summarize our results with a particular focus on metalloproteases with known or potential roles in tissue differentiation.     

The role of the heat shock proteins (HSP70 and sHSP) in the thermotolerance of freshwater amphipods from contrasting habitats

The aim of this study was to clarify the significance of HSP70 and sHSP for thermotolerance in freshwater amphipods. We compared four amphipod species from different freshwater habitats and biogeographical regions (Central Europe vs. Lake Baikal). Test individuals were exposed to thermal stress generated by a water temperature of 25 °C. The thermotolerance of the species, determined by median lethal time (LT50), followed in decreasing order by Gmelinoides fasciatus, Echinogammarus berilloni, Gammarus pulex, Eulimnogammarus verrucosus. HSP70 and sHSP base level concentrations for the species were determined at control (i.e. non-stress) conditions. For HSP70, the base levels were positively correlated to the species' thermotolerances. For sHSP, however, only thermotolerant G. fasciatus showed a high level. Thermal stress at 25 °C water temperature caused a deferred onset of HSP70 and sHSP expression followed by a subsequent offset, delineating a unimodal response curve. The time lag to the expression onset of HSP70 was shorter in the thermosensitive species, compared to thermotolerant ones. Conversely, the time span until the maximum level of HSP70 was variable, not showing a dependence on the thermotolerance properties of the species. The peak concentration in G. pulex was distinctly higher than in the other species, whereas E. verrucosus did not develop a well-defined response maximum at all. In sHSP, the temporal pattern of expression was even more variable than in HSP70. However, the thermosensitive species E. verrucosus showed a time lag of expression onset significantly shorter than the other species and thermotolerant G. fasciatus developed the most pronounced response maximum. Basing on these results, the cellular response to thermal stress in amphipods is more consistently reflected by HSP70, compared to sHSP.     

Extraskeletal osteosarcoma of the thorax in a goat: case report

Background

This case report describes the clinical and ultrasonographic findings in a Swiss Braunvieh cow with lymphosarcoma of the abomasum.

Case Presentation

The main clinical findings were vomiting in response to eating and melena. The results of serum biochemistry and rumen fluid analysis were indicative of abomasal reflux syndrome. The main ultrasonographic findings were two enlarged lymph nodes caudal to the reticulum and a severely enlarged abomasum with thickening of the abomasal wall and folds. Based on all the findings, pyloric stenosis caused by lymphosarcoma was tentatively diagnosed and later confirmed at postmortem examination.

Conclusions

This is an interesting case, which broadens the spectrum of abomasal reflux syndrome.     

The amino-terminus of nitric oxide sensitive guanylyl cyclase α₁ does not affect dimerization but influences subcellular localization

Background

Nitric oxide sensitive guanylyl cyclase (NOsGC) is a heterodimeric enzyme formed by an α- and a β₁-subunit. A splice variant (C-α₁) of the α₁-subunit, lacking at least the first 236 amino acids has been described by Sharina et al. 2008 and has been shown to be expressed in differentiating human embryonic cells. Wagner et al. 2005 have shown that the amino acids 61–128 of the α₁-subunit are mandatory for quantitative heterodimerization implying that the C-α₁-splice variant should lose its capacity to dimerize quantitatively.

Methodology/Principal Findings

In the current study we demonstrate preserved quantitative dimerization of the C-α₁-splice by co-purification with the β₁-subunit. In addition we used fluorescence resonance energy transfer (FRET) based on fluorescence lifetime imaging (FLIM) using fusion proteins of the β₁-subunit and the α₁-subunit or the C-α₁ variant with ECFP or EYFP. Analysis of the respective combinations in HEK-293 cells showed that the fluorescence lifetime was significantly shorter (≈0.3 ns) for α₁/β₁ and C-α₁/β₁ than the negative control. In addition we show that lack of the amino-terminus in the α₁ splice variant directs it to a more oxidized subcellular compartment.

Conclusions/Significance

We conclude that the amino-terminus of the α₁-subunit is dispensable for dimerization in-vivo and ex-vivo, but influences the subcellular trafficking.