Amyotrophic lateral sclerosis is a fatal paralytic disease that targets motor neurons, leading to motor neuron death and widespread denervation atrophy of muscle. Previous electrophysiological data have shown that some motor axon branches attempt to compensate for loss of innervation, resulting in enlarged axonal arbors. Recent histological assays have shown that during the course of the disease some axonal branches die back. We thus asked whether the two types of behavior, die-back and compensatory growth, occur in different branches of single neurons or, alternatively, whether entire motor units are of one type or the other. We used high-resolution in vivo imaging in the G93A SOD1 mouse model, bred to express transgenic yellow fluorescent protein in all or subsets of motor neurons. Time-lapse imaging showed that degenerative axon branches are easily distinguished from those undergoing compensatory reinnervation, showing fragmentation of terminal branches but sparing of the more proximal axon. Reconstruction of entire motor units showed that some were abnormally large. Surprisingly, these large motor units contained few if any degenerating synapses. Some small motor units, however, no longer possessed any neuromuscular contacts at all, giving the appearance of "winter trees." Thus, degenerative versus regenerative changes are largely confined to distinct populations of neurons within the same motor pool. Identification of factors that protect "compensatory" motor neurons from degenerative changes may provide new targets for therapeutic intervention.

Low back pain (LBP) is defined as pain localised between the 12th rib and the inferior gluteal folds, with or without leg pain. Most cases are non-specific, but in about 10% of cases a specific cause is identified. Red flags are typical signs or symptoms that are frequently associated with specific LBP. Yellow flags are prognostic factors associated with a more unfavourable and often chronic disabling course of the disease. LBP has a lifetime prevalence of 60-85%. At any one time, about 15% of adults have LBP. LBP poses an economic burden to society, mainly in terms of the large number of work days lost (indirect costs) and less so by direct treatment costs. A substantial proportion of individuals with chronic LBP has been found to have chronic widespread pain. LBP is often associated with other pain manifestations such as headache, abdominal pain and pain in different locations of the extremities. Widespread pain is associated with a worse prognosis compared to localised LBP. Treatment targets are reduction of pain and better activity/participation, including prevention of disability as well as maintainance of work capacity. The evidence from selected and appraised guidelines, systematic reviews and major clinical studies was classified into four levels, level Ia being the best level with evidence from meta-analysis of randomised controlled trials. Key recommendations (level Ia): fitness programmes and advice to stay active can reduce pain, improve function and can prevent LBP becoming chronic. Simple analgesics, NSAIDs and muscle relaxants can reduce pain and can improve and maintain function. Maintaining physical activity, avoiding rest and manual therapy can reduce pain and maintain and restore function in acute LBP. Behavioural treatment can prevent LBP becoming chronic. Aerobic fitness and endurance training, behavioural treatment and multi-disciplinary treatment programmes can reduce pain and can improve/maintain function in chronic LBP.

High performance liquid chromatography (HPCL) has been employed to study the distribution throughout the human retina of zeaxanthin and lutein, the two major components of the macular pigment. Differences between individuals have also been studied with a view to uncovering possible age-related effects. Both pigments were detected in prenatal eyes (approximately 20 weeks gestation) but did not form a visible yellow spot. Generally they were not easily discernible until about 6 months after birth. For 87 donors between the ages of 3 and 95, no dependence on age was observed in the quantity of either pigment. For approximately 90% of these, zeaxanthin was dominant. For the remaining 10%, as well as for the seven youngest donors, all below the age of 2, and in prenatal eyes, lutein was the major pigment. In individual retinas, the lutein:zeaxanthin ratio increased from an average of approximately 1:2.4 in the central 0-0.25 mm to over 2:1 in the periphery (8.7-12.2 mm). The variation in this ratio with eccentricity was linearly correlated with the corresponding rod:cone ratio. A selective mechanism of uptake, which results in cones and rods preferentially acquiring zeaxanthin and lutein, respectively, could explain this correlation.

The thymus provides an essential environment for the development of T cells from haemopoietic progenitors. This environment is separated into cortical and medullary regions, each containing functionally distinct epithelial populations that are important at successive stages of T-cell development and selection. However, the developmental origin and lineage relationships between cortical and medullary epithelial cell types remain controversial. Here we describe a clonal assay to investigate the developmental potential of single, individually selected, thymic epithelial progenitors (marked with enhanced yellow fluorescent protein) developing within the normal architecture of the thymus. Using this approach, we show that cortical and medullary epithelial cells share a common origin in bipotent precursors, providing definitive evidence that they have a single rather than dual germ layer origin during embryogenesis. Our findings resolve a long-standing issue in thymus development, and are important in relation to the development of cell-based strategies for thymus disorders and the possibility of restoring function of the atrophied adult thymus.

We recently identified multivesicular bodies (MVBs) as prevacuolar compartments (PVCs) in the secretory and endocytic pathways to the lytic vacuole in tobacco (Nicotiana tabacum) BY-2 cells. Secretory carrier membrane proteins (SCAMPs) are post-Golgi, integral membrane proteins mediating endocytosis in animal cells. To define the endocytic pathway in plants, we cloned the rice (Oryza sativa) homolog of animal SCAMP1 and generated transgenic tobacco BY-2 cells expressing yellow fluorescent protein (YFP)-SCAMP1 or SCAMP1-YFP fusions. Confocal immunofluorescence and immunogold electron microscopy studies demonstrated that YFP-SCAMP1 fusions and native SCAMP1 localize to the plasma membrane and mobile structures in the cytoplasm of transgenic BY-2 cells. Drug treatments and confocal immunofluorescence studies demonstrated that the punctate cytosolic organelles labeled by YFP-SCAMP1 or SCAMP1 were distinct from the Golgi apparatus and PVCs. SCAMP1-labeled organelles may represent an early endosome because the internalized endocytic markers FM4-64 and AM4-64 reached these organelles before PVCs. In addition, wortmannin caused the redistribution of SCAMP1 from the early endosomes to PVCs, probably as a result of fusions between the two compartments. Immunogold electron microscopy with high-pressure frozen/freeze-substituted samples identified the SCAMP1-positive organelles as tubular-vesicular structures at the trans-Golgi with clathrin coats. These early endosomal compartments resemble the previously described partially coated reticulum and trans-Golgi network in plant cells.

A Kunjin (KUN) virus cDNA sequence of 10664 nucleotides was obtained and it encoded a single open reading frame for 3433 amino acids. Partial N-terminal amino acid analyses of KUN virus-specified proteins identified the polyprotein cleavage sites and the definitive gene order. The gene order relative to that proposed for yellow fever (YF) virus is as follows: KUN 5'-C.GP20.E.GP44.P19.P10.P71.(?).P21.P98-3' YF 5'-C.prM.E.NS1.ns2a.ns2b.NS3.ns4a.ns4b. NS5-3'. The order of putative signal sequences and stop transfer sequences indicated that KUN NS1, NS2A and NS4B are probably cleaved in the lumen of the endoplasmic reticulum, at a consensus site Val-X-Ala decreases where X is an uncharged residue, and NS2B, NS3 and NS5 are cleaved in the cytosol at the site Lys-Arg decreases Gly. Comparisons with the complete amino acid sequences of YF and West Nile (WN) viruses showed that KUN virus shared 93% homology with WN virus, but only 46% homology with YF virus. Comparisons among individual gene products of six flaviviruses showed that E, NS1, NS3 and NS5 tended to be the most highly conserved, and C among the least conserved. Homologous cleavage sites were evident, and six domains in NS5, a total of over 170 residues, shared at least 85% homology. Comparisons with the KUN C to NS2B sequence defined a gradient of relationships of all gene products in decreasing order WN greater than Murray Valley greater than Japanese encephalitis greater than St Louis encephalitis viruses within this closely related serological complex. A non-coding 5' sequence (75 nucleotides) of KUN virus shared 95% homology with WN virus and a shorter imperfect match with Murray Valley encephalitis virus (15 of 18 nucleotides). The KUN non-coding 3' sequence of 290 nucleotides contained several short and imperfectly matched sequences, and shared 87% homology over the distal region of 191 nucleotides with the corresponding region of WN virus RNA.

ES cells are defined as self-renewing, pluripotent cell lines derived from early embryos. Cultures of ES cells are also characterized by the expression of certain markers thought to represent the pluripotent state. However, despite the widespread expression of key markers such as Oct4 and the appearance of a characteristic undifferentiated morphology, functional ES cells may represent only a small fraction of the cultures grown under self-renewing conditions. Thus phenotypically "undifferentiated" cells may consist of a heterogeneous population of functionally distinct cell types. Here we use a transgenic allele designed to detect low level transcription in the primitive endoderm lineage as a tool to identify an immediate early endoderm-like ES cell state. This reporter employs a tandem array of internal ribosomal entry sites to drive translation of an enhanced Yellow Fluorescent Protein (Venus) from the transcript that normally encodes for the early endodermal marker Hex. Expression of this Venus transgene reports on single cells with low Hex transcript levels and reveals the existence of distinct populations of Oct4 positive undifferentiated ES cells. One of these cells types, characterized by both the expression of the Venus transgene and the ES cells marker SSEA-1 (V(+)S(+)), appears to represent an early step in primitive endoderm specification. We show that the fraction of cells present within this state is influenced by factors that both promote and suppress primitive endoderm differentiation, but conditions that support ES cell self-renewal prevent their progression into differentiation and support an equilibrium between this state and at least one other that resembles the Nanog positive inner cell mass of the mammalian blastocysts. Interestingly, while these subpopulations are equivalently and clonally interconvertible under self-renewing conditions, when induced to differentiate both in vivo and in vitro they exhibit different behaviours. Most strikingly when introduced back into morulae or blastocysts, the V(+)S(+) population is not effective at contributing to the epiblast and can contribute to the extra-embryonic visceral and parietal endoderm, while the V(-)S(+) population generates high contribution chimeras. Taken together our data support a model in which ES cell culture has trapped a set of interconvertible cell states reminiscent of the early stages in blastocyst differentiation that may exist only transiently in the early embryo.

A high-throughput growth assay for the protozoan parasite Toxoplasma gondii was developed based on a highly fluorescent transgenic parasite line. These parasites are stably transfected with a tandem yellow fluorescent protein (YFP) and are 1,000 times more fluorescent than the wild type. Parasites were inoculated in optical-bottom 384-well culture plates containing a confluent monolayer of host cells, and growth was monitored by using a fluorescence plate reader. The signal was linearly correlated with parasite numbers over a wide array. Direct comparison of the YFP growth assay with the beta-galactosidase growth assay by using parasites expressing both reporters demonstrated that the assays' sensitivities were comparable but that the accuracy of the YFP assay was higher, especially at higher numbers of parasites per well. Determination of the 50%-inhibitory concentrations of three known growth-inhibiting drugs (cytochalasin D, pyrimethamine, and clindamycin) resulted in values comparable to published data. The delayed parasite death kinetics of clindamycin could be measured without modification of the assay, making this assay very versatile. Additionally, the temperature-dependent effect of pyrimethamine was assayed in both wild-type and engineered drug-resistant parasites. Lastly, the development of mycophenolic acid resistance after transfection of a resistance gene in T. gondii was followed. In conclusion, the YFP growth assay limits pipetting steps to a minimum, is highly versatile and amendable to automation, and should enable rapid screening of compounds to fulfill the need for more efficient and less toxic antiparasitic drugs.

The dye Procion Yellow M4RS crosses junctional membranes from cytoplasm to cytoplasm at electrotonic synapses between segments of the crayfish septate axon. The dye does not enter the cells from extracellular space. Thus permeability of junctional membranes is qualitatively different from that of nonjunctional membranes. Electron microscopy after fixation in the presence of lanthanum hydroxide indicates that these synapses are "gap junctions" and that there is a network of channels continuous with extracellular space between apposed junctional membranes. These channels must be interlaced with intercytoplasmic channels that are not open to extracellular space.

The CD11c enhanced yellow fluorescent protein (EYFP) transgenic mouse was constructed to identify dendritic cells in the periphery (Lindquist et al. [2004] Nat. Immunol. 5:1243-1250). In this study, we used this mouse to characterize dendritic cells within the CNS. Our anatomic results showed discrete populations of EYFP(+) brain dendritic cells (EYFP(+) bDC) that colocalized with a small fraction of microglia immunoreactive for Mac-1, Iba-1, CD45, and F4/80 but not for NeuN, Dcx, NG2 proteoglycan, or GFAP. EYFP(+) bDC, isolated by fluorescent activated cell sorting (FACS), expressed mRNA for the Itgax (CD11c) gene, whereas FACS anlaysis of EYFP(+) bDC cultures revealed the presence of CD11c protein. Light microscopy studies revealed that EYFP(+) bDC were present in the embryonic CNS when the blood-brain barrier is formed and postnatally when brain cells are amenable to culturing. In adult male mice, EYFP(+) bDC distribution was prominent within regions of the CNS that 1) are subject to structural plasticity and neurogenesis, 2) receive sensory and humoral input from the external environment, and 3) lack a blood-brain barrier. Ultrastructural analysis of EYFP(+) bDC in adult neurogenic niches showed their proximity to developing neurons and a morphology characteristic of immune/microglia cells. Kainic acid-induced seizures revealed that EYFP(+) bDC responded to damage of the hippocampus and displayed morphologies similar to those described for seizure-activated EGFP(+) microglia in the hippocampus of cfms (CSF-1R) EGFP mice. Collectively, these findings suggest a new member of the dendritic cell family residing among the heterogeneous microglia population.

The transcription factor FNR (fumarate nitrate reduction) requires the presence of an iron-sulfur (Fe-S) cluster for its function as a global transcription regulator in Escherichia coli when oxygen becomes scarce. To define the oxidation state and type of Fe-S cluster present in the active form of FNR, we have studied anaerobically purified FNR with Mössbauer spectroscopy. Our data showed that this form of FNR contained a [4Fe-4S]2+ cluster (delta = 0.45 mm/s; DeltaEQ = 1.22 mm/s) and that the [4Fe-4S]2+ cluster was rapidly destroyed on exposure of FNR to air. Under these conditions, the yellow-green active form of FNR turned deep red; analysis of sulfide indicated that 70% of the labile sulfide was still present, suggesting that the Fe-S cluster had been converted into a different form. Little [3Fe-4S] cluster was, however, detected by EPR. According to Mössbauer spectroscopy, the [4Fe-4S]2+ cluster was converted in about 60% yield to a [2Fe-2S]2+ cluster (delta = 0.28 mm/s; DeltaEQ = 0.58 mm/s) following 17 min of exposure to air. The [2Fe-2S]2+ cluster form of FNR was much more stable to oxygen, but was unable to sustain biological activity (e.g., DNA binding). However, DNA binding and the absorption spectrum characteristic of the [4Fe-4S]2+ cluster could be largely restored from the [2Fe-2S]2+ form when Cys, Fe, DTT, and the NifS protein were added. It has yet to be determined whether the form of FNR containing the [2Fe-2S]2+ cluster has any biological significance, e.g., as an in vivo intermediate that is more rapidly converted to the active form than the apoprotein.

The complement system plays an essential protective role in the initial defense against many microorganisms. Flavivirus NS1 is a secreted nonstructural glycoprotein that accumulates in blood, is displayed on the surface of infected cells, and has been hypothesized to have immune evasion functions. Herein, we demonstrate that dengue virus (DENV), West Nile virus (WNV), and yellow fever virus (YFV) NS1 attenuate classical and lectin pathway activation by directly interacting with C4. Binding of NS1 to C4 reduced C4b deposition and C3 convertase (C4b2a) activity. Although NS1 bound C4b, it lacked intrinsic cofactor activity to degrade C4b, and did not block C3 convertase formation or accelerate decay of the C3 and C5 convertases. Instead, NS1 enhanced C4 cleavage by recruiting and activating the complement-specific protease C1s. By binding C1s and C4 in a complex, NS1 promotes efficient degradation of C4 to C4b. Through this mechanism, NS1 protects DENV from complement-dependent neutralization in solution. These studies define a novel immune evasion mechanism for restricting complement control of microbial infection.

We have developed a set of plasmids containing fluorescent protein cassettes for use in PCR-mediated gene tagging in Candida albicans. We engineered YFP and CFP variants of the GFP sequence optimized for C. albicans codon usage. The fluorescent protein sequences, linked to C. albicans auxotrophic marker sequences, were amplified by PCR and transformed directly into yeast. Gene-specific sequence was incorporated into the PCR primers, such that the tag-cassette integrates by homologous recombination at the 3'-end of the gene of interest. This technique was used to tag Cdc3 and Tub1 with GFP, YFP and CFP, which were readily visualized by fluorescence microscopy and localized as expected. In addition, Tub1-YFP and Cdc3-CFP were visualized in the same cells. Thus, this technique directs one-step construction of multiple fluorescent protein fusions, facilitating the study of protein co-expression and co-localization in C. albicans cells in vivo.

Notch1 regulates neural stem cell (NSC) number during development, but its role in adult neurogenesis is unclear. We generated nestin-CreER(T2)/R26R-YFP/Notch1(loxP/loxP) [Notch1inducible knock-out (iKO)] mice to allow tamoxifen (TAM)-inducible elimination of Notch1 and concomitant expression of yellow fluorescent protein (YFP) in nestin-expressing Type-1 NSCs and their progeny in the adult hippocampal subgranular zone (SGZ). Consistent with previous research, YFP+ cells in all stages of neurogenesis were evident in the subgranular zone (SGZ) of wild-type (WT) mice (nestin-CreER(T2)/R26R-YFP/Notch1(w/w)) after tamoxifen (post-TAM), producing adult-generated YFP+ dentate gyrus neurons. Compared with WT littermates, Notch1 iKO mice had similar numbers of total SGZ YFP+ cells 13 and 30 d post-TAM but had significantly fewer SGZ YFP+ cells 60 and 90 d post-TAM. Significantly fewer YFP+ Type-1 NSCs and transiently amplifying progenitors (TAPs) resulted in generation of fewer YFP+ granule neurons in Notch1 iKO mice. Strikingly, 30 d of running rescued this deficit, as the total YFP+ cell number in Notch iKO mice was equivalent to WT levels. This was even more notable given the persistent deficits in the Type-1 NSC and TAP reservoirs. Our data show that Notch1 signaling is required to maintain a reservoir of undifferentiated cells and ensure continuity of adult hippocampal neurogenesis, but that alternative Notch- and Type-1 NSC-independent pathways compensate in response to physical activity. These data shed light on the complex relationship between Type-1 NSCs, adult neurogenesis, the neurogenic niche, and environmental stimuli.

The y2 mutation resulted from the insertion of the gypsy element into the X-linked yellow locus of Drosophila melanogaster. As a consequence of this insertion, transcriptional enhancers that control the expression of the yellow gene in the wings and body cuticle of adult flies are unable to act on the yellow promoter, resulting in a tissue-specific phenotype characterized by mutant coloration in these structures. Some yellow null alleles (yn) are able to complement the y2 phenotype giving rise to near wild type y2/yn females. The molecular structure of the yellow locus in complementing and noncomplementing mutations was determined by cloning and sequencing the various alleles examined. From the information obtained in these studies, we propose a model suggesting that the complementing wild type phenotype of y2/yn flies might be due to the ability of functional wing and body cuticle transcriptional enhancers located in the yn locus to act in trans on the promoter of the yellow gene found in the y2-containing chromosome. Furthermore, this transactivation is abolished by the presence of an intact promoter in cis, suggesting that promoter competition between the yellow genes located on each homolog precludes the activation in trans by transcriptional enhancers in favour of cis effects on their own promoter.

Chikungunya virus (CHIKV), a member of the alphavirus genus, is of considerable public health concern in Southeast Asian and African countries. However, despite serological evidence, the diagnosis of this arthropod-borne human disease is confirmed infrequently and needs to be improved. In fact, illness caused by CHIKV can be confused with diseases such as dengue or yellow fever, based on the similarity of the symptoms, and laboratory confirmation of suspected cases is required to launch control measures during an epidemic. Moreover, no quantitative molecular tool is described to study CHIKV replication or detection in clinical samples and cell culture supernatants. In this study, a specific and sensitive CHIKV one-step TaqMan RT-PCR assay was developed as a tool for the diagnosis of African CHIKV as well as a rapid indicator of active infection by quantifying viral load. This study also showed that a simple heat viral RNA release during the reverse transcription step constituted an alternative to the conventional RNA extraction method.

The rostral part of the agranular frontal cortex (area 6) can be subdivided on the basis of its cytoarchitecture, enzymatic properties, and connections into two large sectors: a superior region, lying medial to the spur of the arcuate sulcus, and an inferior region, lying lateral to it. In this study we traced the afferent and efferent connections of the inferior region of area 6 by injecting small amounts of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) and fluorescent tracers (fast blue and diamidino yellow) into restricted parts of inferior area 6 and in physiologically determined fields of area 4. There is an ordered topographic pattern of connections between inferior area 6 and area 4. The region near the spur of the arcuate sulcus (hand field) projects to the area 4 hand field while the lateral part of inferior area 6 (mouth field) is connected with the corresponding field in area 4. The organization of the connections between the two fields is, however, different. The hand fields in area 6 and 4 have direct reciprocal projections, whereas the mouth field in the postarcuate cortex relays information to area 4 via a zone intermediate between the arcuate and the central sulcus. This zone corresponds to the cytochrome oxidase area F4 (Matelli, Luppino, and Rizzolatti: Behav. Brain Res. 18: 125-137, '85). The inferior area 6 also has topographically organized connections with the supplementary motor area. The inferior area 6 receives and sends fibers to a series of discrete cortical areas located in the lower cortical moiety (Sanides: The Structure and Function of the Nervous Tissue, Vol. 5. New York: Academic Press, pp 329-453, '72). These areas that form a broad ring around the central sulcus are the ventral bank of the principal sulcus and the adjacent area 46, the precentral operculum (PrOC), area SII (Jones and Burton: J. Comp. Neurol. 168:197-248, '76), the parietal operculum, and the rostral part of the inferior parietal lobule including the lower bank of the intraparietal sulcus. Finally, the inferior area 6 has sparse but consistent connections with insular and cingulate cortices. The functional significance of this complex pattern of connections is discussed.

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the X-linked gene MECP2. Girls with RTT show dramatic changes in brain function, but relatively few studies have explored the structure of neural circuits. Examining two mouse models of RTT (Mecp2B and Mecp2J), we previously documented changes in brain anatomy. Herein, we use confocal microscopy to study the effects of MeCP2 deficiency on the morphology of dendrites and axons in the fascia dentata (FD), CA1 area of hippocampus, and motor cortex following Lucifer yellow microinjection or carbocyanine dye tracing. At 3 weeks of age, most (33 of 41) morphological parameters were significantly altered in Mecp2B mice; fewer (23 of 39) were abnormal in Mecp2J mice. There were striking changes in the density and size of the dendritic spines and density and orientation of axons. In Mecp2B mice, dendritic spine density was decreased in the FD (approximately 11%), CA1 (14-22%), and motor cortex (approximately 16%). A decreased spine head size (approximately 9%) and an increased spine neck length (approximately 12%) were found in Mecp2B FD. In addition, axons in the motor cortex were disorganized. In Mecp2J mice, spine density was significantly decreased in CA1 (14-26%). In both models, dendritic swelling and elongated spine necks were seen in all areas studied. Marked variation in the type and extent of changes was noted in dendrites of adjacent neurons. Electron microscopy confirmed abnormalities in dendrites and axons and showed abnormal mitochondria. Our findings document widespread abnormalities of dendrites and axons that recapitulate those seen in RTT.

We demonstrate far-field optical imaging with subdiffraction resolution of the endoplasmic reticulum (ER) in the interior of a living mammalian cell. The diffraction barrier is overcome by applying stimulated emission depletion (STED) on a yellow fluorescent protein tag. Imaging individual structural elements of the ER revealed a focal plane (x, y) resolution of <50 nm inside the living cell, corresponding to a 4-fold improvement over that of a confocal microscope and a 16-fold reduction in the focal-spot cross-sectional area. A similar gain in resolution is realized with both pulsed- and continuous-wave laser illumination. Images of highly convoluted parts of the ER reveal a similar resolution improvement in 3D optical sectioning by a factor of 3 along the optic axis (z). Time-lapse STED recordings document morphological changes of the ER over time. Thus, nanoscale 3D imaging of organelles in the interior of living cells greatly expands the scope of light microscopy in cell biology.

Hypocretin (orexin) is synthesized by neurons in the lateral hypothalamus and has been reported to increase food intake and regulate the neuroendocrine system. In the present paper, long descending axonal projections that contain hypocretin were found that innervate all levels of the spinal cord from cervical to sacral segments, as studied in mouse, rat, and human spinal cord and not previously described. High densities of axonal innervation are found in regions of the spinal cord related to modulation of sensation and pain, notably in the marginal zone (lamina 1). Innervation of the intermediolateral column and lamina 10 as well as strong innervation of the caudal region of the sacral cord suggest that hypocretin may participate in the regulation of both the sympathetic and parasympathetic parts of the autonomic nervous system. Double-labeling experiments in mice combining retrograde transport of diamidino yellow after spinal cord injections and immunocytochemistry support the concept that hypocretin-immunoreactive fibers in the cord originate from the neurons in the lateral hypothalamus. Digital-imaging physiological studies with fura-2 detected a rise in intracellular calcium in response to hypocretin in cultured rat spinal cord neurons, indicating that spinal cord neurons express hypocretin-responsive receptors. A greater number of cervical cord neurons responded to hypocretin than another hypothalamo-spinal neuropeptide, oxytocin. These data suggest that in addition to possible roles in feeding and endocrine regulation, the descending hypocretin fiber system may play a role in modulation of sensory input, particularly in regions of the cord related to pain perception and autonomic tone.

Curcumin, a yellow pigment in the Indian spice Turmeric (Curcuma longa), which is chemically known as diferuloylmethane, was first isolated exactly two centuries ago in 1815 by two German Scientists, Vogel and Pelletier. However, according to the pubmed database, the first study on its biological activity as an antibacterial agent was published in 1949 in Nature and the first clinical trial was reported in The Lancet in 1937. Although the current database indicates almost 9000 publications on curcumin, until 1990 there were less than 100 papers published on this nutraceutical. At the molecular level, this multitargeted agent has been shown to exhibit anti-inflammatory activity through the suppression of numerous cell signalling pathways including NF-κB, STAT3, Nrf2, ROS and COX-2. Numerous studies have indicated that curcumin is a highly potent antimicrobial agent and has been shown to be active against various chronic diseases including various types of cancers, diabetes, obesity, cardiovascular, pulmonary, neurological and autoimmune diseases. Furthermore, this compound has also been shown to be synergistic with other nutraceuticals such as resveratrol, piperine, catechins, quercetin and genistein. To date, over 100 different clinical trials have been completed with curcumin, which clearly show its safety, tolerability and its effectiveness against various chronic diseases in humans. However, more clinical trials in different populations are necessary to prove its potential against different chronic diseases in humans. This review's primary focus is on lessons learnt about curcumin from clinical trials.

This article is part of a themed section on Principles of Pharmacological Research of Nutraceuticals. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.11/issuetoc.

Little is known about the signalling processes involved in the response of roots to abiotic stresses. The Arabidopsis root is a model system of root anatomy with a simple architecture and is amenable to genetic manipulation. Although it is known that the root responds to cold, drought and salt stress with increases in cytoplasmic free calcium, there is currently no information about the role(s) of the functionally diverse cell types that comprise the root. Transgenic Arabidopsis with enhancer-trapped GAL4 expression in specific cell types was used to target the calcium reporting protein, aequorin, fused to a modified yellow fluorescent protein (YFP). The luminescence output of targeted aequorin enabled in vivo measurement of changes in cytosolic free calcium concentrations ([Ca2+]cyt) in specific cell types during acute cold, osmotic and salt stresses. In response to an acute cold stress, all cell types tested as well as plants constitutively expressing aequorin displayed rapid [Ca2+]cyt peaks. However, there were significant quantitative differences between different cell types in terms of their response to cold stress, osmotic stress (440 mM mannitol) and salt stress (220 mM NaCl), implying specific roles for certain cell types in the detection and/or response to these stimuli. In response to osmotic and salt stress, the endodermis and pericycle displayed prolonged oscillations in cytosolic calcium that were distinct from the responses of the other cell types tested. Targeted expression of aequorin circumvented the technical difficulties involved in fluorescent dye injection as well as the lack of cell specificity of constitutively expressed aequorin, and revealed a new level of complexity in root calcium signalling.

A study of Zika virus infections was carried out in four communities in Oyo State, Nigeria. Virus isolation studies between 1971 and 1975 yielded two virus isolations from human cases of mild febrile illness. Haemagglutination-inhibition tests revealed a high prevalence of antibodies to Zika and three other flaviviruses used. The percentages of positive sera were as follows: Zika (31%), Yellow fever (50%), West Nile (46%), and Wesselsbron (59%). Neutralization tests showed that 40% of Nigerians had Zika virus neutralizing antibody. Fifty per cent of zika virus immune persons had neutralizing antibody to Zika alone or to Zika and one other flavivirus. A total of 121 sera had antibody to Zika virus; of these 48 (40%) also showed antibody to two other flaviviruses, and 12 (10%) had antibodies to three or more other viruses. The percentage of neutralizing antibodies to other flaviviruses in Zika virus immune sera was 81% to Dengue type 1, 58% to Yellow fever, 7% to Wesselsbron, 6% to West Nile and 3% to Uganda S.

Bipolar cell and amacrine cell synaptic inputs to alpha ganglion cells (alphaGCs) in dark-adapted mouse retinas were studied by recording the light-evoked excitatory cation current (DeltaIC) and inhibitory chloride current (DeltaICl) under voltage-clamp conditions, and the cell morphology was revealed by Lucifer yellow fluorescence with a confocal microscope. Three types of alphaGCs were identified. (1) ONalphaGCs exhibits no spike activity in darkness, increased spikes in light, sustained inward DeltaIC, sustained outward DeltaICl of varying amplitude, and large soma (20-25 microm in diameter) with alpha-cell-like dendritic field approximately 180-350 microm stratifying near 70% of the inner plexiform layer (IPL) depth. (2) Transient OFFalphaGCs (tOFFalphaGCs) exhibit no spike activity in darkness, transient increased spikes at light offset, small sustained outward DeltaIC in light, a large transient inward DeltaIC at light offset, a sustained outward DeltaICl, and a morphology similar to the ONalphaGCs except for that their dendrites stratified near 30% of the IPL depth. (3) Sustained OFFalphaGCs exhibit maintained spike activity of 5-10 Hz in darkness, sustained decrease of spikes in light, sustained outward DeltaIC, sustained outward DeltaICl, and a morphology similar to the tOFFalphaGCs. By comparing the response thresholds and dynamic ranges of alphaGCs with those of the preganglion cells, our data suggest that the light responses of each type of alphaGCs are mediated by different sets of bipolar cells and amacrine cells. This detailed physiological analysis complements the existing anatomical results and provides new insights on the functional roles of individual synapses in the inner mammalian retina.