Super-resolution microscopy reveals cell wall dynamics and peptidoglycan architecture in ovococcal bacteria

Cell morphology and viability in Eubacteria is dictated by the architecture of peptidoglycan, the major and essential structural component of the cell wall. Although the biochemical composition of peptidoglycan is well understood, how the peptidoglycan architecture can accommodate the dynamics of growth and division while maintaining cell shape remains largely unknown. Here, we elucidate the peptidoglycan architecture and dynamics of bacteria with ovoid cell shape (ovococci), which includes a number of important pathogens, by combining biochemical analyses with atomic force and super-resolution microscopies. Atomic force microscopy analysis showed preferential orientation of the peptidoglycan network parallel to the short axis of the cell, with distinct architectural features associated with septal and peripheral wall synthesis. Super-resolution three-dimensional structured illumination fluorescence microscopy was applied for the first time in bacteria to unravel the dynamics of peptidoglycan assembly in ovococci. The ovococci have a unique peptidoglycan architecture and growth mode not observed in other model organisms.     

Characterization of the elongasome core PBP2 : MreC complex of Helicobacter pylori

The definition of bacterial cell shape is a complex process requiring the participation of multiple components of an intricate macromolecular machinery. We aimed at characterizing the determinants involved in cell shape of the helical bacterium Helicobacter pylori. Using a yeast two-hybrid screen with the key cell elongation protein PBP2 as bait, we identified an interaction between PBP2 and MreC. The minimal region of MreC required for this interaction ranges from amino acids 116 to 226. Using recombinant proteins, we showed by affinity and size exclusion chromatographies and surface plasmon resonance that PBP2 and MreC form a stable complex. In vivo, the two proteins display a similar spatial localization and their complex has an apparent 1:1 stoichiometry; these results were confirmed in vitro by analytical ultracentrifugation and chemical cross-linking. Small angle X-ray scattering analyses of the PBP2 : MreC complex suggest that MreC interacts directly with the C-terminal region of PBP2. Depletion of either PBP2 or MreC leads to transition into spherical cells that lose viability. Finally, the specific expression in trans of the minimal interacting domain of MreC with PBP2 in the periplasmic space leads to cell rounding, suggesting that the PBP2/MreC complex formation in vivo is essential for cell morphology.     

Salivary melatonin rhythm as a marker of the circadian system in healthy children and those with attention-deficit/hyperactivity disorder

Attention-deficit/hyperactivity disorder (ADHD) is the most common neurobehavioral disorder of childhood. Problems with sleep structure, efficiency, and timing have been reported in some, but not all, studies on ADHD children. As the sleep-wake cycle belongs to circadian rhythms, the timekeeping circadian system might be involved in ADHD. To assess whether the circadian system of ADHD children differs from that of controls, the rhythm of the pineal hormone melatonin was used as a reliable marker of the system. Saliva from 34 ADHD and 43 control 6- to 12-yr-old children was sampled at 2-h intervals throughout the entire 24-h cycle, and the melatonin profiles of the ADHD and control children were compared. The nocturnal melatonin peaks of the ADHD and control group did not differ significantly. The high nocturnal interindividual variability of the peaks seen in adulthood was present already in the studied children. The 24-h melatonin profiles of all the ADHD subjects did not differ significantly from those of the control subjects. Categorization of subjects according to age, into groups of 6- to 7-yr-old (9 ADHD, 5 control), 8- to 9-yr-old (16 ADHD, 26 control), and 10- to 12-yr-old (9 ADHD, 12 control) children, revealed significant differences between the ADHD and control group in the melatonin rhythm waveform, but not in nocturnal melatonin peaks; the peaks were about the same in both groups and did not change significantly with increasing age. In the oldest, but not in the younger, children, the melatonin signal duration in the ADHD group was shorter than in the control group. The difference might be due to the fact that whereas in the control group both the evening melatonin onset and the morning offset phase delayed in the oldest children relative to those in the youngest children, in the ADHD group only the onset, but not the offset, phase delayed with increasing age. The data may indicate subtle differences between the circadian system of ADHD and control children during development.     

Atomic model of CPV reveals the mechanism used by this single-shelled virus to economically carry out functions conserved in multishelled reoviruses

Unlike the multishelled viruses in the Reoviridae, cytoplasmic polyhedrosis virus (CPV) is single shelled, yet stable and fully capable of carrying out functions conserved within Reoviridae. Here, we report a 3.1 ? resolution cryo electron microscopy structure of CPV and derive its atomic model, consisting of 60 turret proteins (TPs), 120 each of capsid shell proteins (CSPs) and large protrusion proteins (LPPs). Two unique segments of CSP contribute to CPV's stability: an inserted protrusion domain interacting with neighboring proteins, and an N-anchor tying up CSPs together through strong interactions such as β sheet augmentation. Without the need to interact with outer shell proteins, LPP retains only the N-terminal two-third region containing a conserved helix-barrel core and interacts exclusively with CSP. TP is also simplified, containing only domains involved in RNA capping. Our results illustrate how CPV proteins have evolved in a coordinative manner to economically carry out their conserved functions.     

Rapid validation of cancer genes in chimeras derived from established genetically engineered mouse models

Recent technological advances have opened the door for the fast and cost-effective generation of genetically engineered mouse models (GEMMs) to study cancer. We describe here a conceptually novel approach for the generation of chimeric GEMMs based on the controlled introduction of various genetic elements in embryonic stem cells (ESCs) that are derived from existing mouse strains with a predisposition for cancer. The isolation of GEMM-derived ESC lines is greatly facilitated by the availability of the newly defined culture media containing inhibitors that effectively preserve ESC pluripotency. The feasibility of the GEMM-ESC approach is discussed in light of current literature and placed into the context of existing models. This approach will allow for fast and flexible validation of candidate cancer genes and drug targets and will result in a repository of GEMM-ESC lines and corresponding vector collections that enable easy distribution and use of preclinical models to the wider scientific community.     

Soil and Plant Contamination with <Emphasis Type="Italic">Mycobacterium Avium</Emphasis> subsp. <Emphasis Type="Italic">Paratuberculosis</Emphasis> After Exposure to Naturally Contaminated Mouflon Feces

The aim of this study was to demonstrate the persistence of Mycobacterium avium subsp. paratuberculosis (MAP) in soil and colonization of different plant parts after deliberate exposure to mouflon feces naturally contaminated with different amounts of MAP. Samples of aerial parts of plants, their roots, and the soil below the roots were collected after 15 weeks and examined using IS900 real-time quantitative PCR (qPCR) and cultivation. Although the presence of viable MAP cells was not demonstrated, almost all samples were found to be positive using qPCR. MAP IS900 was not only found in the upper green parts, but also in the roots and soil samples (from 1.00 × 10⁰ to 6.43 × 10³). The level of soil and plant contamination was influenced mainly by moisture, clay content, and the depth from which the samples were collected, rather than by the initial concentration of MAP in the feces at the beginning of the experiment.     

Psychometric validation of the Croatian version of the Quality of Life in Epilepsy Inventory (QOLIE-31)

The primary goals of this study were to adapt the Quality of Life in Epilepsy Inventory-31 items (QOLIE-31) questionnaire to the Croatian language and to assess the translated questionnaire's psychometric properties. Translation/retranslation of the English version of the QOLIE-31 was done, and all steps for cross-cultural adaptation process were performed properly by an expert committee. Later, QOLIE-31 questionnaires and previously validated Short Form-36 (SF-36) outcome instruments were given to 200 patients with epilepsy. 172 patients (86%) responded to the first set of questionnaires, and 114 of the first time respondents (66%) returned their second survey. The two measures of reliability as internal consistency and reproducibility were determined by Cronbach alpha statistics and intraclass correlation coefficient, respectively. Concurrent validity was measured by comparing with a SF-36 questionnaire, and measurement was made using the Pearson correlation coefficient (r). The study demonstrated satisfactory internal consistency with high Cronbach a values for all of the corresponding domains (seizure worry 0.84, medication effects 0.80, emotional well-being 0.73, energy/fatigue 0.76, cognitive functioning 0.71, social functioning 0.77, overall quality of life 0.65). The intraclass correlation coefficient for six domains of QOLIE-31 questionnaire demonstrated excellent test/retest reproducibility (ICC > or = 0.75), and good test/retest reproducibility (ICC 0.71) in one domain (cognitive functioning). Considering concurrent validity, three domains had excellent correlation (r = 0.75-1), while 11 had good correlation (r = 0.50 to 0.75), and 3 had moderate correlation (r = 0.25-0.50). This study demonstrated that, if measures are to be used across cultures, the items must not only be translated well linguistically but also must be culturally adapted to maintain the content validity of the instrument at a conceptual level across different cultures. Croatian version of QOLIE-31 will be a valuable contribution to outcome measurement in epilepsy patients, particularly in the context of treatment trials, but als in a wider research context.     

Active site residues and mechanism of UDP-glucose dehydrogenase.

UDP-glucose dehydrogenase catalyzes the NAD+-dependent twofold oxidation of UDP-glucose to give UDP-glucuronic acid. A sequestered aldehyde intermediate is produced in the first oxidation step and a covalently bound thioester is produced in the second oxidation step. This work demonstrates that the Streptococcus pyogenes enzyme incorporates a single solvent-derived oxygen atom during catalysis and probably does not generate an imine intermediate. The reaction of UDP-[6",6"-di-2H]-d-glucose is not accompanied by a primary kinetic isotope effect, indicating that hydride transfer is not rate determining in this reaction. Studies with a mutant of the key active site nucleophile, Cys260Ala, show that it is capable of both reducing the aldehyde intermediate, and oxidizing the hydrated form of the aldehyde intermediate but is incapable of oxidizing UDP-glucose to UDP-glucuronic acid. In the latter case, a ternary Cys260Ala/aldehyde intermediate/NADH complex is presumably formed, but it does not proceed to product as both release and hydration of the bound aldehyde occur slowly. A washout experiment demonstrates that the NADH in this ternary complex is not exchangeable with external NADH, indicating that dissociation only occurs after the addition of a nucleophile to the aldehyde carbonyl. Studies on Thr118Ala show that the value of kcat is reduced 160-fold by this mutation, and that the reaction of UDP-D-[6",6"-di-2H]-glucose is now accompanied by a primary kinetic isotope effect. This indicates that the barriers for the hydride transfer steps have been selectively increased and supports a mechanism in which an ordered water molecule (H-bonded to Thr118) serves as the catalytic base in these steps.