Steroid-induced psychosis.

Steroid-induced psychosis is a well-documented phenomenon. It usually occurs with oral systemic steroid treatment and is more common at higher doses, although there are case reports of occurrence with local steroid injections. We report a case of a 35-year-old man with no previous history of psychosis who was seen for follow-up after a brief psychotic episode following an injection of 5 mg of dexamethasone into his scrotum the previous day. The injection was given to treat chronic pain from a combat injury.

Spatial overlap links seemingly unconnected genotype-matched TB cases in rural Uganda.

INTRODUCTION: Incomplete understanding of TB transmission dynamics in high HIV prevalence settings remains an obstacle for prevention. Understanding where transmission occurs could provide a platform for case finding and interrupting transmission. METHODS: From 2012-2015, we sought to recruit all adults starting TB treatment in a Ugandan community. Participants underwent household (HH) contact investigation, and provided names of social contacts, sites of work, healthcare and socializing, and two sputum samples. Mycobacterium tuberculosis culture-positive specimens underwent 24-loci MIRU-VNTR and spoligotyping. We sought to identify epidemiologic links between genotype-matched cases by analyzing social networks and mapping locations where cases reported spending ≥12 hours over the one-month pre-treatment. Sites of spatial overlap (≤100m) between genotype-matched cases were considered potential transmission sites. We analyzed social networks stratified by genotype clustering status, with cases linked by shared locations, and compared network density by location type between clustered vs. non-clustered cases. RESULTS: Of 173 adults with TB, 131 (76%) were enrolled, 108 provided sputum, and 84/131 (78%) were MTB culture-positive: 52% (66/131) tested HIV-positive. Of 118 adult HH contacts, 105 (89%) were screened and 3 (2.5%) diagnosed with active TB. Overall, 33 TB cases (39%) belonged to 15 distinct MTB genotype-matched clusters. Within each cluster, no cases shared a HH or reported shared non-HH contacts. In 6/15 (40%) clusters, potential epidemiologic links were identified by spatial overlap at specific locations: 5/6 involved health care settings. Genotype-clustered TB social networks had significantly greater network density based on shared clinics (p<0.001) and decreased density based on shared marketplaces (p<0.001), compared to non-clustered networks. CONCLUSIONS: In this molecular epidemiologic study, links between MTB genotype-matched cases were only identifiable via shared locations, healthcare locations in particular, rather than named contacts. This suggests most transmission is occurring between casual contacts, and emphasizes the need for improved infection control in healthcare settings in rural Africa.

General Staining and Segmentation Procedures for High Content Imaging and Analysis.

Automated quantitative fluorescence microscopy, also known as high content imaging (HCI), is a rapidly growing analytical approach in cell biology. Because automated image analysis relies heavily on robust demarcation of cells and subcellular regions, reliable methods for labeling cells is a critical component of the HCI workflow. Labeling of cells for image segmentation is typically performed with fluorescent probes that bind DNA for nuclear-based cell demarcation or with those which react with proteins for image analysis based on whole cell staining. These reagents, along with instrument and software settings, play an important role in the successful segmentation of cells in a population for automated and quantitative image analysis. In this chapter, we describe standard procedures for labeling and image segmentation in both live and fixed cell samples. The chapter will also provide troubleshooting guidelines for some of the common problems associated with these aspects of HCI.

Tools to Measure Autophagy Using High Content Imaging and Analysis.

Macroautophagy, hereafter referred to as autophagy, is a predominately pro-survival catabolic process responsible for the degradation of long-lived or aggregated proteins, invading microorganisms and damaged or redundant intracellular organelles. Removal of these entities is achieved through encompassment of the target by the autophagosome and subsequent delivery to the lysosome. The use of fluorescence microscopy is a common method to investigate autophagy through monitoring the spatial and temporal recruitment both of autophagosomal markers and cargo to the autophagosome. In this section, we will discuss the use of high content imaging (HCI) and analysis in the study of autophagy with reference to commonly used markers of autophagosomal formation.

Nitration of Hsp90 on Tyrosine 33 Regulates Mitochondrial Metabolism.

Peroxynitrite production and tyrosine nitration are present in several pathological conditions, including neurodegeneration, stroke, aging, and cancer. Nitration of the pro-survival chaperone heat shock protein 90 (Hsp90) in position 33 and 56 induces motor neuron death through a toxic gain-of-function. Here we show that nitrated Hsp90 regulates mitochondrial metabolism independently of the induction of cell death. In PC12 cells, a small fraction of nitrated Hsp90 was located on the mitochondrial outer membrane and down-regulated mitochondrial membrane potential, oxygen consumption, and ATP production. Neither endogenous Hsp90 present in the homogenate nor unmodified and fully active recombinant Hsp90 was able to compete with the nitrated protein for the binding to mitochondria. Moreover, endogenous or recombinant Hsp90 did not prevent the decrease in mitochondrial activity but supported nitrated Hsp90 mitochondrial gain-of-function. Nitrotyrosine in position 33, but not in any of the other four tyrosine residues prone to nitration in Hsp90, was sufficient to down-regulate mitochondrial activity. Thus, in addition to induction of cell death, nitrated Hsp90 can also regulate mitochondrial metabolism, suggesting that depending on the cell type, distinct Hsp90 nitration states regulate different aspects of cellular metabolism. This regulation of mitochondrial homeostasis by nitrated Hsp90 could be of particular relevance in cancer cells.

Next day determination of ejaculatory sperm motility after overnight shipment of semen to remote locations.

PURPOSE: To develop a method for delayed assessment of sperm motility, after shipment of semen to a remote laboratory. Sperm in semen were labeled with the MitoTracker(®) Red CM-H(2)XRos reagent, and fixed with 3.7 % formaldehyde by the laboratory technicians at the origin of the semen. This treatment reflected well sperm mitochondrial activity, and the MitoTracker(®) signal was related to sperm motility and velocity for 2-3 days following ejaculation. METHODS: Sperm motility and velocity were evaluated manually and by computer assisted semen analysis (CASA), respectively. Fluorescence assessment of individual sperm was carried out with the computer assisted Metamorph v4.6.9 program. Emission levels of MitoTracker(®) spermatozoa were studied in room temperature and cooled semen, or in the respective room temperature swim-up sperm fractions following ejaculation, and on the second day (N = 103 samples, 89 men) and third day (N = 10 samples, 8 men). RESULTS: Sperm with optical density (O.D.) ≥0.7 showed close correlations with ejaculatory sperm motility and velocity even after second day (r = 0.92, p < 0.001, N = 103 samples). Further, the multiple of sperm motility and velocity was also related to the proportion of high MitoTracker(®) reagent emission sperm (r = 0.83, p < 0.001, N = 103 samples). MitoTracker(®) dye fluorescence on the second day accurately reflected the ejaculatory sperm motility (r = 0.90, p < 0.001). Thus, a shipping delay would not adversely affect the results. CONCLUSIONS: The delayed assessment of sperm motility in samples treated with MitoTracker(®) Red CM-H(2)XRos reagent and shipped to remote laboratory truly reflects the level of sperm motility at the time of the ejaculation.

A new species of Ovabunda (Octocorallia, Xeniidae) from the Andaman Sea, Thailand with notes on the biogeography of this genus.

A survey of xeniid octocorals was carried out in the waters off Southwestern Thailand in September, 2007. Microscopic investigation of the colonies revealed that three specimens belonged to the genus Ovabunda. Gross morphological examination is presented here accompanied by scanning electron micrographs of the sclerites. Molecular phylogenetic analysis showed identical genotypes at mtMutS, COI, and 28S rDNA for all three specimens and supports their generic assignment. Colony size and shape, sclerite size, and pinnule arrangement differ from nominal species of Ovabunda and thus a new species, O. andamanensis is introduced here. This work also presents a new eastern geographical record for the genus Ovabunda.

On the extent and role of the small proteome in the parasitic eukaryote Trypanosoma brucei.

BACKGROUND: Although technical advances in genomics and proteomics research have yielded a better understanding of the coding capacity of a genome, one major challenge remaining is the identification of all expressed proteins, especially those less than 100 amino acids in length. Such information can be particularly relevant to human pathogens, such as Trypanosoma brucei, the causative agent of African trypanosomiasis, since it will provide further insight into the parasite biology and life cycle. RESULTS: Starting with 993 T. brucei transcripts, previously shown by RNA-Sequencing not to coincide with annotated coding sequences (CDS), homology searches revealed that 173 predicted short open reading frames in these transcripts are conserved across kinetoplastids with 13 also conserved in representative eukaryotes. Mining mass spectrometry data sets revealed 42 transcripts encoding at least one matching peptide. RNAi-induced down-regulation of these 42 transcripts revealed seven to be essential in insect-form trypanosomes with two also required for the bloodstream life cycle stage. To validate the specificity of the RNAi results, each lethal phenotype was rescued by co-expressing an RNAi-resistant construct of each corresponding CDS. These previously non-annotated essential small proteins localized to a variety of cell compartments, including the cell surface, mitochondria, nucleus and cytoplasm, inferring the diverse biological roles they are likely to play in T. brucei. We also provide evidence that one of these small proteins is required for replicating the kinetoplast (mitochondrial) DNA. CONCLUSIONS: Our studies highlight the presence and significance of small proteins in a protist and expose potential new targets to block the survival of trypanosomes in the insect vector and/or the mammalian host.

Tools and techniques to measure mitophagy using fluorescence microscopy.

Mitophagy is a specialized form of autophagy that removes damaged mitochondria, thereby maintaining efficient cellular metabolism and reducing cellular stress caused by aberrant oxidative bursts. Deficits in mitophagy underlie several diseases, and a substantial body of research has elucidated key steps in the pathways that lead to and execute autophagic clearance of mitochondria. Many of these studies employ fluorescence microscopy to visualize mitochondrial morphology, mass, and functional state. Studies in this area also examine colocalization/recruitment of accessory factors, components of the autophagic machinery and signaling molecules to mitochondria. In this review, we provide a brief summary of the current understanding about the processes involved in mitophagy followed by a discussion of probes commonly employed and important considerations of the methodologies to study and analyze mitophagy using fluorescence microscopy. Representative data, where appropriate, are provided to highlight the use of key probes to monitor mitophagy. The review will conclude with a consideration of new possibilities for mitophagy research and a discussion of recently developed technologies for this emerging area of cell biology.

Development of a functional schwann cell phenotype from autologous porcine bone marrow mononuclear cells for nerve repair.

Adult bone marrow mononuclear cells (BM-MNCs) are a potential resource for making Schwann cells to repair damaged peripheral nerves. However, many methods of producing Schwann-like cells can be laborious with the cells lacking a functional phenotype. The objective of this study was to develop a simple and rapid method using autologous BM-MNCs to produce a phenotypic and functional Schwann-like cell. Adult porcine bone marrow was collected and enriched for BM-MNCs using a SEPAX device, then cells cultured in Neurobasal media, 4 mM L-glutamine and 20% serum. After 6-8 days, the cultures expressed Schwann cell markers, S-100, O4, GFAP, were FluoroMyelin positive, but had low p75(NGF) expression. Addition of neuregulin (1-25 nM) increased p75(NGF) levels at 24-48 hrs. We found ATP dose-dependently increased intracellular calcium [Ca(2+)](i), with nucleotide potency being UTP = ATP > ADP > AMP > adenosine. Suramin blocked the ATP-induced [Ca(2+)](i) but α, ß,-methylene-ATP had little effect suggesting an ATP purinergic P2Y2 G-protein-coupled receptor is present. Both the Schwann cell markers and ATP-induced [Ca(2+)](i) sensitivity decreased in cells passaged >20 times. Our studies indicate that autologous BM-MNCs can be induced to form a phenotypic and functional Schwann-like cell which could be used for peripheral nerve repair.

Detection of intracellular glutathione using ThiolTracker violet stain and fluorescence microscopy.

Glutathione plays an important role in protecting mammalian cells from oxidative stress and cell death. Because reduced glutathione (GSH) represents the large majority of intracellular free thiols, cell-permeant, thiol-reactive fluorescent probes represent potentially useful indicators of intracellular GSH. The ThiolTracker Violet stain (a registered trademark of Invitrogen) is a bright fluorescent probe that is highly reactive to thiols and can be used as a convenient and effective indicator of intracellular GSH and general redox status by a variety of detection modalities. While this probe has been validated in flow cytometry and microplate fluorimetry assays, the following method will describe details on the use of the ThiolTracker Violet dye in traditional fluorescence microscopy, as well as high-content imaging and analysis.

Yeast artificial chromosomes employed for random assembly of biosynthetic pathways and production of diverse compounds in Saccharomyces cerevisiae.

BACKGROUND: Natural products are an important source of drugs and other commercially interesting compounds, however their isolation and production is often difficult. Metabolic engineering, mainly in bacteria and yeast, has sought to circumvent some of the associated problems but also this approach is impeded by technical limitations. Here we describe a novel strategy for production of diverse natural products, comprising the expression of an unprecedented large number of biosynthetic genes in a heterologous host. RESULTS: As an example, genes from different sources, representing enzymes of a seven step flavonoid pathway, were individually cloned into yeast expression cassettes, which were then randomly combined on Yeast Artificial Chromosomes and used, in a single transformation of yeast, to create a variety of flavonoid producing pathways. Randomly picked clones were analysed, and approximately half of them showed production of the flavanone naringenin, and a third of them produced the flavonol kaempferol in various amounts. This reflected the assembly of 5-7 step multi-species pathways converting the yeast metabolites phenylalanine and/or tyrosine into flavonoids, normally only produced by plants. Other flavonoids were also produced that were either direct intermediates or derivatives thereof. Feeding natural and unnatural, halogenated precursors to these recombinant clones demonstrated the potential to further diversify the type of molecules that can be produced with this technology. CONCLUSION: The technology has many potential uses but is particularly suited for generating high numbers of structurally diverse compounds, some of which may not be amenable to chemical synthesis, thus greatly facilitating access to a huge chemical space in the search for new commercially interesting compounds.

The selective detection of mitochondrial superoxide by live cell imaging.

A general protocol is described to improve the specificity for imaging superoxide formation in live cells via fluorescence microscopy with either hydroethidine (HE) or its mitochondrially targeted derivative Mito-HE (MitoSOX Red). Two different excitation wavelengths are used to distinguish the superoxide-dependent hydroxylation of Mito-HE (385-405 nm) from the nonspecific formation of ethidium (480-520 nm). Furthermore, the dual wavelength imaging in live cells can be combined with immunocolocalization, which allows superoxide formation to be compared simultaneously in cocultures of two types of genetically manipulated cells in the same microscopic field. The combination of these approaches can greatly improve the specificity for imaging superoxide formation in cultured cells and tissues.

Selective fluorescent imaging of superoxide in vivo using ethidium-based probes.

The putative oxidation of hydroethidine (HE) has become a widely used fluorescent assay for the detection of superoxide in cultured cells. By covalently joining HE to a hexyl triphenylphosphonium cation (Mito-HE), the HE moiety can be targeted to mitochondria. However, the specificity of HE and Mito-HE for superoxide in vivo is limited by autooxidation as well as by nonsuperoxide-dependent cellular processes that can oxidize HE probes to ethidium (Etd). Recently, superoxide was shown to react with HE to generate 2-hydroxyethidium [Zhao, H., Kalivendi, S., Zhang, H., Joseph, J., Nithipatikom, K., Vasquez-Vivar, J. & Kalyanaraman, B. (2003) Free Radic. Biol. Med. 34, 1359-1368]. However, 2-hydroxyethidium is difficult to distinguish from Etd by conventional fluorescence techniques exciting at 510 nm. While investigating the oxidation of Mito-HE by superoxide, we found that the superoxide product of both HE and Mito-HE could be selectively excited at 396 nm with minimal interference from other nonspecific oxidation products. The oxidation of Mito-HE monitored at 396 nm by antimycin-stimulated mitochondria was 30% slower than at 510 nm, indicating that superoxide production may be overestimated at 510 nm by even a traditional superoxide-stimulating mitochondrial inhibitor. The rate-limiting step for oxidation by superoxide was 4x10(6) M-1.s-1, which is proposed to involve the formation of a radical from Mito-HE. The rapid reaction with a second superoxide anion through radical-radical coupling may explain how Mito-HE and HE can compete for superoxide in vivo with intracellular superoxide dismutases. Monitoring oxidation at both 396 and 510 nm of excitation wavelengths can facilitate the more selective detection of superoxide in vivo.

Rapid analysis of mitochondrial DNA depletion by fluorescence in situ hybridization and immunocytochemistry: potential strategies for HIV therapeutic monitoring.

Nucleoside reverse transcriptase inhibitors (NRTIs) have been a mainstay in the treatment of human immunodeficiency virus since the introduction of azidothymidine (AZT) in 1987. However, none of the current therapies can completely eradicate the virus, necessitating long-term use of anti-retroviral drugs to prevent viral re-growth. One of the side effects associated with long-term use of NRTIs is mitochondrial toxicity stemming from inhibition of the mitochondrial DNA (mtDNA) polymerase gamma, which leads to mtDNA depletion and consequently to mitochondrial dysfunction. Here we report the use of fluorescence in situ hybridization (FISH) and immunocytochemistry (ICC) to monitor mtDNA depletion in cultured fibroblasts treated with the NRTI 2',3'-dideoxycytidine (ddC). These techniques are amenable to both microscopy and flow cytometry, allowing analysis of populations of cells on a single-cell basis. We show that, as mtDNA depletion progresses, a mosaic population develops, with some cells being depleted of and others retaining mtDNA. These techniques could be useful as potential therapeutic monitors to indicate when NRTI therapy should be interrupted to prevent mitochondrial toxicity and could aid in the development of less toxic NRTIs by providing an assay suitable for pharmacodynamic evaluation of candidate molecules.

Immunological approaches to the characterization and diagnosis of mitochondrial disease.

Monoclonal antibodies (mAbs) are important tools in the diagnosis and characterization of mitochondrial diseases. They can be used in immunohistochemical and/or Western blotting approaches to identify misassembled OXPHOS complexes or pyruvate dehydrogenase deficiencies where the intact complex is not formed which is the great majority of cases. The advantage of antibody based approaches is that they can be quantitative, require very small amounts of tissue sample and are fast, simple and relatively cheap to perform. Here we provide details of the mAbs currently available and describe optimized protocols for both immunohistochemistry using patient fibroblasts as well as Western blotting using either cell culture or biopsy material.