Structural implications of lipoarabinomannan glycans from global clinical isolates in diagnosis of Mycobacterium tuberculosis infection.

In Mycobacterium tuberculosis (Mtb), surface-exposed Lipoarabinomannan (LAM) is a key determinant of immunogenicity, yet its intrinsic heterogeneity confounds typical structure-function analysis. Recently, LAM gained a strong foothold as a validated marker for active tuberculosis (TB) infection and has shown great potential in new diagnostic efforts. However, no efforts have yet been made to model or evaluate the impact of mixed polyclonal Mtb infections (infection with multiple strains) on TB diagnostic procedures other than antibiotic susceptibility testing. Here, we selected three TB clinical isolates (HN878, EAI, and IO) and purified LAM from these strains to present an integrated analytical approach of one-dimensional and two-dimensional Nuclear Magnetic Resonance (NMR) spectroscopy, as well as enzymatic digestion and site-specific mass spectrometry (MS) to probe LAM structure and behavior at multiple levels. Overall, we found that the glycan was similar in all LAM preparations, albeit with subtle variations. Succinates, lactates, hydroxybutyrate, acetate, and the hallmark of Mtb LAM-methylthioxylose (MTX), adorned the nonreducing terminal arabinan of these LAM species. Newly identified acetoxy/hydroxybutyrate was present only in LAM from EAI and IO Mtb strains. Notably, detailed LC/MS-MS unambiguously showed that all acyl modifications and the lactyl ether in LAM are at the 3-OH position of the 2-linked arabinofuranose adjacent to the terminal ß-arabinofuranose. Finally, after sequential enzymatic deglycosylation of LAM, the residual glycan that has ∼50% of α-arabinofuranose -(1→5) linked did not bind to monoclonal antibody CS35. These data clearly indicate the importance of the arabinan termini arrangements for the antigenicity of LAM.

Culturing Mycobacteria.

Building upon the foundational research of Robert Koch, who demonstrated the ability to grow Mycobacterium tuberculosis for the first time in 1882 using media made of coagulated bovine serum, microbiologists have continued to develop new and more efficient ways to grow mycobacteria. Presently, all known mycobacterial species can be grown in the laboratory using either axenic culture techniques or in vivo passage in laboratory animals. This chapter provides conventional protocols to grow mycobacteria for diagnostic purposes directly from clinical specimens, as well as in research laboratories for scientific purposes. Detailed protocols used for production of M. tuberculosis in large scale (under normoxic and hypoxic conditions) in bioreactors and for production of obligate intracellular pathogens such as Mycobacterium leprae and "Mycobacterium lepromatosis" using athymic nude mice and armadillos are provided.

Do Image-Assisted Mobile Applications Improve Dietary Habits, Knowledge, and Behaviours in Elite Athletes? A Pilot Study.

To date, there has been a paucity of research on optimal ways to educate and promote dietary behavioural change within athletes. Optimising athlete nutrition is fundamental to reaching peak performance and maintaining athlete wellbeing. MealLogger® is a smartphone application that incorporates the use of an image-based food record and social-media functionality to provide in-application personalised feedback to individuals or groups, peer-support, and a platform to deliver nutrition education material. This study measured the feasibility of MealLogger® within New Zealand elite male field hockey players (n = 17) aged 18⁻20 to increase athlete knowledge and nutrition promoting behaviours. During a six-week intervention, participants were instructed to log images of their meals three days per week and they received individualised dietetic feedback on logged meals. Weekly nutrition-education fact-sheets and videos were delivered through the application. Nutrition knowledge increased moderately from baseline (%Pre 54.7 ± 14.3; %Post 61.1 ± 11.45, p = 0.01). Participants report a highly positive experience of application use (8/10) with 82.3% attempting to make positive changes in dietary behaviours based on in-app education. All participants preferred this method to traditional methods of dietary analysis. Using image-based applications such as MealLogger® is an effective approach to monitor dietary intake and deliver education to optimise the nutritional behaviours of elite athletes.

Light-induced Fos expression is attenuated in the suprachiasmatic nucleus of serotonin 1B receptor knockout mice.

The hypothalamic suprachiasmatic nucleus (SCN) is a circadian oscillator that receives a dense serotonergic innervation from the median raphe nucleus. Serotonin (5-HT) modulates the effects of light on circadian behavior by acting on 5-HT1B receptors on retinohypothalamic (RHT) terminals in the SCN. Activation of 5-HT1B presynaptic receptors on RHT terminals inhibits glutamate release. However, 5-HT1B receptor knockout (5-HT1B KO) mice have attenuated behavioral responses to light [P.J. Sollars, M.D. Ogilvie, A.M. Simpson, G.E. Pickard, Photic entrainment is altered in the 5-HT1B receptor knockout mouse, J. Biol. Rhythms 21 (2006) 21-32]. To assess the cellular response of the 5-HT1B KO SCN to light, light-induced Fos expression was analyzed in 5-HT1B KO and wild-type (WT) mice. In addition, the distribution of melanopsin containing retinal ganglion cells that contribute the majority of axons to the RHT was examined in 5-HT1B KO mice and compared to that of WT mice. Light-induced Fos expression in the SCN was reduced in 5-HT1B KO mice compared to WT mice at circadian time (CT) 16 and CT 23 in a manner similar to the reduction previously described in light-induced behavioral phase shifts. The number of melanopsin retinal ganglion cells was similar in WT and 5-HT1B KO mice. These data taken together with previous data suggest that functional removal of the 5-HT1B receptor results in reduced functional light input to the SCN.

Photic entrainment is altered in the 5-HT1B receptor knockout mouse.

The hypothalamic suprachiasmatic nucleus (SCN) is a circadian oscillator that receives glutamatergic afferents from the retina and serotonergic afferents from the midbrain. Activation of presynaptic serotonin 1B (5-HT1B) receptors on retinal terminals in the SCN inhibits retinohypothalamic neurotransmission and light-induced behavioral phase shifts. To assess the role of 5-HT1B receptors in photic entrainment, 5-HT1B receptor knockout (5-HT1B KO) and wild-type (WT) mice were maintained in non-24 h L:D cycles (T cycles). WT mice entrained to T = 21 h and T = 22 h cycles, whereas 5-HT1B KO animals did not. 5-HT1B KO animals did entrain to T = 23 h and T = 26 h cycles, although their phase angle of entrainment was altered compared to WT animals. 5-HT1B KO mice were significantly more phase delayed under T = 23 h conditions and significantly more phase advanced under T = 26 h conditions compared to WT mice. When 5-HT1B KO mice were housed in a T = 23 h short-day photoperiod (9.5L:13.5D), the delayed phase angle of entrainment was more pronounced. Light-induced phase shifts were reduced in 5-HT1B KO mice, consistent with their behavior in T cycles, suggesting an attenuated response to light. Based on previous work, this attenuated response to light might not have been predicted but can be explained by consideration of GABAergic mechanisms within the SCN. Phase-delayed circadian rhythms during the short days of winter are characteristic of patients suffering from seasonal affective disorder, and 5-HT has been implicated in its pathophysiology. The 5-HT1B KO mouse may be useful for investigating the altered entrainment evident during this serious mood disorder.

Cultural issues and elder mistreatment.

The article is a brief discussion of a few of the complex issues that may represent a cultural component of elder mistreatment. Reference is made to historical perspectives and subcultures. The goals of the article are to broaden the discussion around the issues of elder abuse in various population groups and to consider some methods of intervention.

Cryo-sectioning and chemical-fixing ultramicrotomy techniques for imaging rubber latex particle morphology.

Two methods adapted from biological microscopy are described for a new application in imaging the morphology of rubbery latex particles. In the first method, a drop of latex is frozen in liquid nitrogen, sectioned with a diamond knife and vapour-stained with osmium tetroxide, then viewed by transmission electron microscopy. When applied to latexes made by emulsion polymerization of methyl methacrylate in a natural rubber latex seed, inclusions are clearly visible. A chemical fixation method is then described for imaging the morphology of such rubbery latex particles. Glutaraldehyde is added to the latex, followed by osmium tetroxide. The sample is then dehydrated in ethanol, epoxy resin added, and the sample cured, ultramicrotomed, and imaged with transmission electron microscopy. An inclusion morphology is again clearly seen.