The Influence of Substitution on Thiol-Induced Oxanorbornadiene Fragmentation

Oxanorbornadienes (ONDs) undergo facile Michael addition with thiols and then fragment by a retro-Diels-Alder (rDA) reaction, a unique two-step sequence among electrophilic cleavable linkages. The rDA reaction rate was explored as a function of the furan structure, with substituents at the 2- and 5-positions found to be the most influential and the fragmentation rate to be inversely correlated with electron-withdrawing ability. Density functional theory calculations provided an excellent correlation with the experimentally measured OND rDA rates

Advantages and consecvences of using puppet in drama education of mentaly disabled people

Objective of this thesis is to get closer and clarify advantages of drama educational influence by working with mental disabled people. It refers to the advantages of working with puppet within educational process and it shows possibilities in progress of single character elements of people with mental disablement. Thesis is divided into three parts. First part is focused to theory of drama-education and dramatherapy. First part thinks also of historical context of origin of alternative pedagogical trends and describes the influence of drama education on single character elements. Second part dedicates deeply to benefits of puppet in connection with general personal character progress. Third one, practical part, describes drama-educational lessons and theatre project named "Where the stars fall". The benefit of both styles by working on progress of single character elements of mental disabled people, is pointed at the close

Full color palette of fluorescent d-amino acids for in situ labeling of bacterial cell walls† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c7sc01800b Click here for additional data file.

Fluorescent d-amino acids (FDAAs) enable efficient in situ labeling of peptidoglycan in diverse bacterial species. Conducted by enzymes involved in peptidoglycan biosynthesis, FDAA labeling allows specific probing of cell wall formation/remodeling activity, bacterial growth and cell morphology. Their broad application and high biocompatibility have made FDAAs an important and effective tool for studies of peptidoglycan synthesis and dynamics, which, in turn, has created a demand for the development of new FDAA probes. Here, we report the synthesis of new FDAAs, with emission wavelengths that span the entire visible spectrum. We also provide data to characterize their photochemical and physical properties, and we demonstrate their utility for visualizing peptidoglycan synthesis in Gram-negative and Gram-positive bacterial species. Finally, we show the permeability of FDAAs toward the outer-membrane of Gram-negative organisms, pinpointing the probes available for effective labeling in these species. This improved FDAA toolkit will enable numerous applications for the study of peptidoglycan biosynthesis and dynamics

Structured illumination microscopy of DAAD PG labeling in pathogenic <i>Chlamydia</i>.

<p>Structured illumination microscopy (SIM) was conducted on four pathogenic <i>Chlamydia</i> species. EDA-DA was added 2 hours post infection (hpi) and coverslips were fixed at 18 hpi. PG labeling (represented by EDA—DA) is shown in green, the major <i>C</i>. <i>trachomatis</i> outer membrane protein (MOMP) is shown in red, and cell nuclei are in blue (this labeling scheme is maintained in all subsequent figures, unless otherwise stated). MOMP staining is not shown for the three other species. Images are representative of ~20 inclusions viewed per strain. Scale bar = 1 μm.</p

3D SIM visualization of labeled chlamydial PG.

<p><b>(a-e)</b> Maximum intensity projection (7 μm thick Z stack) of <i>C</i>. <i>trachomatis</i> incubated with 4 mM EDA-DA at 2 hpi and fixed at 18 hpi. Panels (<b>b-e)</b> are maximum intensity projections of 0.5 μm thick planes of interest selected from panel <b>a</b>. Arrowheads indicate areas of punctate PG staining (as opposed to PG rings). <b>(f)</b> The PG ring dimensions: diameter x, width y, and thickness z. <b>(g-k)</b> 3D-SIM of cases of asymmetric cell division are outlined and marked with arrowheads for clarity. Panels <b>(h)</b> and <b>(i)</b> are magnifications of the image in panel <b>(g).</b> Images in panels <b>(j)</b> and <b>(k)</b> are independent cases. MOMP and PG labeling is the same as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005590#ppat.1005590.g002" target="_blank">Fig 2</a>. Image is representative of ~ 20 inclusions analyzed. Scale bar = 1 μm.</p

Relevant steps of PG biosynthesis and D-amino acid dipeptide (DAAD) probes used in this study.

<p>(<b>a</b>) DAADs are taken up by bacteria where they compete with endogenous D-Ala-D-Ala (DA—DA) for incorporation into PG. De novo synthesis of DA—DA is inhibited by D-cyloserine. The pentapeptide PG subunit is then flipped across the inner membrane into the periplasm where it is transglycosylated to form glycan polymers (nascent PG) and crosslinked by penicillin binding proteins (PBPs). Transpeptidation causes cleavage of the terminal D-Ala at position 5. Because the N-terminally labeled portion of DAAD becomes the amino acid at position 4 of the pentapeptide, the label is resistant to this processing and remains on the stem peptide. (<b>b</b>) PG-labeling reagents used in this study. Clickable DAADs EDA—DA and ADA—DA. Star represents the clickable amino acid.</p

Chlamydial EBs do not retain PG labeling.

<p><b>(a)</b> SIM of EDA—DA labeled <i>C</i>. <i>trachomatis</i> inclusions at 22 hpi. Arrowheads indicate locations of EBs. Tissue culture cells were infected with <i>C</i>. <i>trachomatis</i> and placed on a rocker for 2 hours, ensuring asynchronous infection of the cell monolayer. Bacteria were then incubated with 4 mM EDA-DA for 22 hours. <b>(b)</b> Confocal maximum intensity projections of a mature, <i>Chlamydia</i> inclusion (40 hpi) in which bacteria were grown in the presence of 4 mM EDA—DA for the entire developmental cycle. The arrowheads point to EBs (distinguished by their MOMP labeling and smaller size). <b>(c)</b> Confocal maximum intensity projections of <i>Chlamydia</i>-infected cells 4 hpi. EBs used for infection were harvested from cells that had been incubated with EDA—DA for 18 hours. Exogenous EDA—DA (4 mM) was present throughout the EB harvest as well as the subsequent reinfection. MOMP and PG labeling is the same as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005590#ppat.1005590.g002" target="_blank">Fig 2</a>. Images are all representative of at least three separate experiments. Scale bar = 1 μm.</p

Proposed model for PG biosynthesis and maintenance in pathogenic <i>Chlamydia</i>.

<p><b>(a)</b> Upon invading a host cell, MreB facilitates mid-cell PG (green) ring formation 8 hpi and the first cell division occurs. Inhibition of MreB (red) prior to EB-RB transition prevents this first division and results in accumulation of intracellular ‘EB-like’ particles in the host cytosol. Once RBs form, PG is constantly synthesized and incorporated into the ring non-uniformly by dynamic MreB patches. If MreB polymerization is inhibited, the PG ring dissociates, resulting in slightly enlarged, static, PG-less aberrant bodies. Inhibition of PG crosslinking by β-lactams does not affect transglycosylase activity and MreB localization, but instead results in unchecked enlargement of the RB. Bottom, upon cell division, the new division planes form immediately and perpendicular to the previous division plane. During transition to non-replicative EBs, MreB and PG biosynthesis enzymes are downregulated [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005590#ppat.1005590.ref058" target="_blank">58</a>], resulting in the complete disassembly of the PG ring. <b>(b)</b> Chlamydial PG biosynthesis and degradation mechanisms overlap and are active throughout the cell cycle. MreB (red) moves along the ring plane (black arrows), initiating the non-uniform synthesis of new PG (green) and influencing the action of an unknown degradation mechanism that continuously removes older material (white arrows). PG degradation enzymes (amidases and lytic transglycosylases) are visualized as blue pacmen.</p