In-hospital blood collection increases the rate of indeterminate results in interferon-gamma release assays.

BACKGROUND: The interferon (IFN)-γ release assay (IGRA) has recently been established as a method to evaluate the infection status of tuberculosis instead of the tuberculin skin test. However, indeterminate results can create challenges to interpretation. The IGRA has been available in Japan since 2005, including the recently launched QuantiFERON-TB Gold Plus (QFT-plus) assay. OBJECTIVES: The aim of this study was to investigate the clinical features and predictors of indeterminate results by the QFT-plus test in routine practice. METHODS: This was a cross-sectional study of 1258 patients. Multivariate logistic regression models were employed to investigate the clinical factors related to indeterminate results by the QFT-plus. RESULTS: Overall, 91.8% of results were found to be conclusive and 8.2% were indeterminate. The QFT-plus indeterminate results were predominantly due to a low level of IFN-γ production by mitogens. Multivariate analysis indicated that an indeterminate result was significantly associated with age, sex, corticosteroid use, autoimmune disease, and inpatient setting. CONCLUSION: Certain types of individuals are at higher risk of an indeterminate IGRA result. The QFT-plus test for hospitalized patients should be avoided as much as possible, and it is better to perform the test for those patients in outpatient settings.

Evidence for intermolecular forces involved in ladybird beetle tarsal setae adhesion.

Why can beetles such as the ladybird beetle Coccinella septempunctata walk vertically or upside-down on a smooth glass plane? Intermolecular and/or capillary forces mediated by a secretion fluid on the hairy footpads have commonly been considered the predominant adhesion mechanism. However, the main contribution of physical phenomena to the resulting overall adhesive force has yet to be experimentally proved, because it is difficult to quantitatively analyse the pad secretion which directly affects the adhesion mechanism. We observed beetle secretion fluid by using inverted optical microscopy and cryo-scanning electron microscopy, which showed the fluid secretion layer and revealed that the contact fluid layer between the footpad and substrate was less than 10-20 nm thick, thus indicating the possibility of contribution of intermolecular forces. If intermolecular force is the main physical phenomenon of adhesion, the force will be proportional to the work of adhesion, which can be described by the sum of the square roots of dispersive and polar parts of surface free energy. We measured adhesion forces of ladybird beetle footpads to flat, smooth substrates with known surface free energies. The adhesive force was proportional to the square-root of the dispersive component of the substrate surface free energy and was not affected by the polar component. Therefore, intermolecular forces are the main adhesive component of the overall adhesion force of the ladybird beetle. The footpads adhere more strongly to surfaces with higher dispersive components, such as wax-covered plant leaves found in the natural habitat of ladybird beetles. Based on the present findings, we assume ladybird beetles have developed this improved performance as an adaptation to the variety of plant species in its habitat.

Nascent peptide-mediated translation elongation arrest of Arabidopsis thaliana CGS1 mRNA occurs autonomously.

The Arabidopsis thaliana CGS1 gene encodes cystathionine gamma-synthase, the first committed enzyme of methionine biosynthesis in higher plants. Expression of CGS1 is feedback-regulated at the step of mRNA degradation in response to S-adenosyl-L-methionine (AdoMet). A short stretch of amino acid sequence, termed the MTO1 region, encoded within the first exon of CGS1 itself acts in cis in the regulation. In vitro analyses using wheat germ extract (WGE) revealed that AdoMet induces temporal translation arrest of CGS1 mRNA prior to mRNA degradation. This translational pausing occurs immediately downstream of the MTO1 region and is mediated by the nascent MTO1 peptide. In order to elucidate further the nature of this unique regulatory mechanism, we have examined whether a non-plant system also contains the post-transcriptional regulation activity. Despite the fact that mammals do not carry cystathionine gamma-synthase, AdoMet was able to induce the MTO1 sequence-dependent translation elongation arrest in rabbit reticulocyte lysate (RRL) in a similar manner to that observed in WGE. This result suggests that MTO1 peptide-mediated translation arrest does not require a plant-specific factor and rather most probably occurs via a direct interaction between the nascent MTO1 peptide and the ribosome that has translated it. In contrast, decay intermediates of CGS1 mRNA normally observed upon induction of CGS1 mRNA decay in plant systems were not detected in RRL, raising the possibility that CGS1 mRNA degradation involves a plant-specific mechanism.

Ribosome stacking defines CGS1 mRNA degradation sites during nascent peptide-mediated translation arrest.

Expression of the Arabidopsis CGS1 gene that codes for cystathionine gamma-synthase is feedback-regulated at the step of mRNA degradation in response to S-adenosyl-L-methionine (AdoMet). This regulation occurs during translation and involves AdoMet-induced temporal translation arrest prior to the mRNA degradation. Here, we have identified multiple intermediates of CGS1 mRNA degradation with different 5' ends that are separated by approximately 30 nucleotides. Longer intermediates were found to be produced as the number of ribosomes loaded on mRNA was increased. Sucrose density gradient centrifugation experiments showed that the shortest mRNA degradation intermediate was associated with monosomes, whereas longer degradation intermediates were associated with multiple ribosomes. Immunoblot analyses revealed a ladder of premature polypeptides whose molecular weights corresponded to products of ribosomes in a stalled stack. An increase in smaller premature polypeptides was observed as the number of ribosomes loaded on mRNA increased. These results show that AdoMet induces the stacking of ribosomes on CGS1 mRNA and that multiple mRNA degradation sites probably correspond to each stacked ribosome.

Nascent peptide-mediated translation elongation arrest coupled with mRNA degradation in the CGS1 gene of Arabidopsis.

Expression of the Arabidopsis CGS1 gene that codes for cystathionine gamma-synthase is feedback regulated at the step of mRNA stability in response to S-adenosyl-L-methionine (AdoMet). A short stretch of amino acid sequence, called the MTO1 region, encoded by the first exon of CGS1 itself is involved in this regulation. Here, we demonstrate, using a cell-free system, that AdoMet induces temporal translation elongation arrest at the Ser-94 codon located immediately downstream of the MTO1 region, by analyzing a translation intermediate and performing primer extension inhibition (toeprint) analysis. This translation arrest precedes the formation of a degradation intermediate of CGS1 mRNA, which has its 5' end points near the 5' edge of the stalled ribosome. The position of ribosome stalling also suggests that the MTO1 region in nascent peptide resides in the ribosomal exit tunnel when translation elongation is temporarily arrested. In addition to the MTO1 region amino acid sequence, downstream Trp-93 is also important for the AdoMet-induced translation arrest. This is the first example of nascent peptide-mediated translation elongation arrest coupled with mRNA degradation in eukaryotes. Furthermore, our data suggest that the ribosome stalls at the step of translocation rather than at the step of peptidyl transfer.