A Real-Time PCR-Based Diagnostic Test for Organisms in Respiratory Tract Infection

Respiratory tract infection, especially pneumonia, is a significant cause of morbidity and mortality worldwide. Although rapid and accurate identification of the pathogens and the corresponding treatment, which is based on the microbiological results, is required in the healthcare setting, the current clinical tests lack high sensitivity and flexibility. As of yet, a comprehensive approach has not been able to work these issues out. Meanwhile, the development of molecular techniques enables the detection of organisms from respiratory specimens speedily as well as precisely and aids the settlement of such issues. With our novel approach that employs relative quantification, we successfully set the cutoff value to discriminate the causative pathogen from colonizing commensal organisms by real-time PCR. In this way, a diagnostic system for respiratory pathogens was devised and validated through clinical sample testing. In this chapter, a real-time PCR-based test capable of differentiating causative pathogens in respiratory specimens is described, and also its principle and the utility of this approach are illustrated

Synthesis of 13C-labelled, bicyclic mimetics of natural enediynes

Using a versatile synthesis with 13CH3PPh3I and CH313CO2Et as 13C sources, the first examples of nine membered chromophores, which have been differentially labelled with 13C in their carbocyclic enediyne cores, are described

Phosphatidylserine dictates the assembly and dynamics of caveolae in the plasma membrane

Caveolae are bulb-shaped nanodomains of the plasma membrane that are enriched in cholesterol and sphingolipids. They have many physiological functions, including endocytic transport, mechanosensing, and regulation of membrane and lipid transport. Caveola formation relies on integral membrane proteins termed caveolins (Cavs) and the cavin family of peripheral proteins. Both protein families bind anionic phospholipids, but the precise roles of these lipids are unknown. Here, we studied the effects of phosphatidylserine (PtdSer), phosphatidylinositol 4-phosphate (PtdIns4P), and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) on caveolar formation and dynamics. Using live-cell, single-particle tracking of GFP-labeled Cav1 and ultrastructural analyses, we compared the effect of PtdSer disruption or phosphoinositide depletion with caveola disassembly caused by cavin1 loss. We found that PtdSer plays a crucial role in both caveola formation and stability. Sequestration or depletion of PtdSer decreased the number of detectable Cav1-GFP puncta and the number of caveolae visualized by electron microscopy. Under PtdSer-limiting conditions, the co-localization of Cav1 and cavin1 was diminished, and cavin1 degradation was increased. Using rapamycin-recruitable phosphatases, we also found that the acute depletion of PtdIns4P and PtdIns(4,5)P2 has minimal impact on caveola assembly but results in decreased lateral confinement. Finally, we show in a model of phospholipid scrambling, a feature of apoptotic cells, that caveola stability is acutely affected by the scrambling. We conclude that the predominant plasmalemmal anionic lipid PtdSer is essential for proper Cav clustering, caveola formation, and caveola dynamics and that membrane scrambling can perturb caveolar stability

Induction of spontaneous curvature and endocytosis: Unwanted consequences of cholesterol extraction using methyl-β-Cyclodextrin

Membrane curvature is a property of biological membranes essential for organelle morphology and the formation of tubulovesicular carriers. Curvature generation is influenced by the lipid composition of the membrane and protein-mediated processes. Lipids with small headgroups, such as phosphatidic acid, are conical and impose negative curvature on a monolayer. Conversely, lipids with large headgroups relative to the hydrophobic tail(s), such as lysophosphatidylcholine, have an inverted conical shape and impose positive curvature. Due to its abundance and high rates of spontaneous flip-flop between membrane leaflets cholesterol is proposed to buffer the formation of membrane curvature. Recently, we demonstrated that cholesterol is also crucial for maintaining the proper spacing of anionic phospholipids. Upon extraction of cholesterol with cyclodextrin there is a sharp increase in the negative surface charge density of the plasma membrane, which promotes electrostatic repulsion between anionic headgroups, the generation of spontaneous positive curvature and rapid membrane internalization

Radiologic types of Mycobacterium xenopi pulmonary disease:different patients with similar short-term outcomes

AbstractMycobacterium xenopi pulmonary disease (Mxe-PD) is common among nontuberculous mycobacterial infections in Europe andCanada. Associations between radiological pattern and clinical features and outcomes are inadequately studied in Mxe-PD. Wesought to investigate clinical characteristics and outcomes according to the dominant radiological pattern among patients withMxe-PD. We retrospectively studied patients with Mxe-PD seen in our clinic, categorizing their predominant CT pattern asnodular bronchiectasis, fibrocavitary, or unclassifiable, and compared clinical characteristics, treatment, and outcomes betweenradiologic groups. Of 94 patients with Mxe-PD, CT patterns comprised nodular bronchiectasis (40/94, 42.6%), fibrocavitary (37/94, 39.4%), and unclassifiable (17/94, 18.1%). Compared with fibrocavitation, patients with nodular bronchiectasis were femaledominant, less often had COPD, less often had AFB smear-positive sputum, and more frequently had co-isolation ofPseudomonas. Patients with nodular bronchiectasis were less often treated (65% versus 91.9%) and when treated, they receivedfewer anti-mycobacterial drugs (on average 3 versus 4). Outcomes did not differ significantly by radiological pattern. Nodularbronchiectasis was common among Mxe-PD patients in our clinic. Compared with fibrocavitary disease, patients with nodularbronchiectasis had features suggestive of milder disease and were less often treated. Among treated patients, outcomes did notdiffer by radiologic pattern.Keywords Mycobacterium xenopi . Nontuberculous mycobacteria . Chest CT . Nodular bronchiectasi

Diastereoselective additions of ethynyl Grignard reagent to erythrulose derivatives

Through systematic changes in reaction conditions and with the use of Ti(OiPr)4, the typical stereochemical outcome of ethynylmagnesium bromide on α,β-O-isopropylidene-erythrulose derivatives has been reversed with exceptional levels of control

Spin trapping of 13C-labeled p-benzynes generated by Masamune–Bergman cyclization of bicyclic nine-membered enediynes

Our understanding of the chemistry and biology of the enediyne class of antitumor antibiotics is still developing and faces further challenges. The cleavage of double-stranded DNA through hydrogen abstraction by enediyne-derived radicals is a generally well-accepted mechanism. However, the mechanisms of several unexpected and complex cytotoxic effects of enediynes,such as RNA and protein damage or the generation of p-quinone species, have yet to be fully elucidated. Despite several carefully crafted experimental and theoretical studies on simplified systems,the behavior of the major chemical perpetrators of such cytotoxicity, namely the biradical intermediates formed by natural enediynes, remains more a matter of conjecture than a point of fact