Spectral Phasor Analysis of Nile Red Identifies Membrane Microenvironment Changes in the Presence of Amyloid Peptides.

The interaction of protein and peptide amyloid oligomers with membranes is thought to be one of the mechanisms contributing to cellular toxicity. However, techniques to study these interactions in the complex membrane environment of live cells are lacking. Spectral phasor analysis is a recently developed biophysical technique that can enable visualisation and analysis of membrane-associated fluorescent dyes. When the spectral profile of these dyes changes as a result of changes to the membrane microenvironment, spectral phasor analysis can localise those changes to discrete membrane regions. In this study, we investigated whether spectral phasor analysis could detect changes in the membrane microenvironment of live cells in the presence of fibrillar aggregates of the disease-related Aß42 peptide or the functional amyloid neurokinin B. Our results show that the fibrils cause distinct changes to the microenvironment of nile red associated with both the plasma and the nuclear membrane. We attribute these shifts in nile red spectral properties to changes in membrane fluidity. Results from this work suggest that cells have mechanisms to avoid or control membrane interactions arising from functional amyloids which have implications for how these peptides are stored in dense core vesicles. Furthermore, the work highlights the utility of spectral phasor analysis to monitor microenvironment changes to fluorescent probes in live cells.

Endocytic recycling prevents copper accumulation in astrocytoma cells stimulated with copper-bound neurokinin B.

Neurokinin B (NKB) is a key neuropeptide in reproductive endocrinology where it contributes to the generation of pulses of gonadotropin-releasing hormone. NKB is a copper-binding peptide; in the absence of metal NKB rapidly adopts an amyloid structure, but copper binding inhibits amyloid formation and generates a structure that can activate the neurokinin 3 receptor. The fate of copper once it binds NKB and activates the neurokinin 3 receptor is not understood, but endocytosis of NKB occurs even when the peptide is coordinated to copper. Using astrocytoma cells that express endogenous neurokinin 3 receptor, this work shows that endocytosis of apo- and copper-bound NKB occurs in concert with the receptor via a trafficking pathway that includes the early endosome. When cells are stimulated with copper-bound NKB the cellular copper concentration does not significantly increase, however when the cells are pre-treated with the recycling inhibitor, brefeldin A, they are capable of accumulating copper. This data shows that copper-bound NKB can activate the neurokinin 3 receptor then endocytosis abstracts metal, peptide and receptor from the cell surface. The cell does not accumulate the copper but instead it enters recycling pathways that ultimately leads to metal release from the cell. The work reveals a novel receptor-mediated copper trafficking pathway that retains metal in membrane bound organelles until it is exported from the cell.

Endocytosis of G Protein-Coupled Receptors and Their Ligands: Is There a Role in Metal Trafficking?

The prevalence of metal dysregulation in many neurodegenerative and neurocognitive disorders has compelled many studying such diseases to investigate the mechanisms underlying metal regulation in the central nervous system. Metal homoeostasis is often complex, with sophisticated, multilayered pathways in operation. G protein-coupled receptors are omnipresent on cell membranes and have intriguing mechanisms of endocytosis and trafficking that may be useful in metal homoeostasis. Indeed, many receptors and/or their cognate ligands are able to bind metals, and in many cases metals are considered to have neuromodulatory roles as a result of receptor binding. In this mini-review, we outline the structural and functional aspects of G protein-coupled receptors with a focus on the mechanisms leading to endocytosis and cellular trafficking. We further highlight how this may help in the trafficking of metal ions, notably copper.

Etiology and Anti-microbial Sensitivity of Organisms Causing Community Acquired Pneumonia: A Single Hospital Study.

OBJECTIVE: The objective of this study was to identify the common etiological pathogens causing community acquired pneumonia (CAP) in our hospital and sensitivity patterns to the common antibiotics used. MATERIALS AND METHODS: This study was undertaken in a 750 bedded multi-specialty referral hospital in Kerala catering to both urban and semi-urban populations. It is a prospective study of patients who attended the medical out-patient department and those admitted with a clinical diagnosis of CAP, during the year 2009. Data were collected based on detailed patient interview, clinical examination and laboratory investigations. The latter included sputum culture and sensitivity pattern. These were tabulated and percentage incidence of etiological pathogens calculated. The antimicrobial sensitivity pattern was also classified by percentage and expressed as bar diagram. RESULTS: The study showed Streptococcus pneumoniae to be the most common etiological agent for CAP, in our hospital setting. The other organisms isolated in order of frequency were Klebsiella pneumoniae, Pseudomonas aeruginosa, Alpha hemolytic streptococci, Escherichia coli, Beta hemolytic streptococci and atypical coli. S. pneumoniae was most sensitive to linezolid, followed by amoxicillin-clavulanate (augmentin), cloxacillin and ceftriaxone. Overall, the common pathogens causing CAP showed highest sensitivity to amikacin, followed by ofloxacin, gentamycin, amoxicillin-clavulanate (augmentin), ceftriaxone and linezolid. The least sensitivity rates were shown to amoxicillin and cefoperazone. CONCLUSION: In a hospital setting, empirical management for cases of CAP is not advisable. The present study has shown S. pneumoniae as the most likely pathogen and either linezolid or amikacin as the most likely effective antimicrobial in cases of CAP, in our setting.