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Rec-8 dimorphism affects longevity, stress resistance and X-chromosome nondisjunction in C. elegans, and replicative lifespan in S. cerevisiae.
Front Genet
PUBLISHED: 08-04-2014
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A quantitative trait locus (QTL) in the nematode C. elegans, "lsq4," was recently implicated by mapping longevity genes. QTLs for lifespan and three stress-resistance traits coincided within a span of <300 kbp, later narrowed to <200 kbp. A single gene in this interval is now shown to modulate all lsq4-associated traits. Full-genome analysis of transcript levels indicates that lsq4 contains a dimorphic gene governing the expression of many sperm-specific genes, suggesting an effect on spermatogenesis. Quantitative analysis of allele-specific transcripts encoded within the lsq4 interval revealed significant, 2- to 15-fold expression differences for 10 of 33 genes. Fourteen "dual-candidate" genes, implicated by both position and expression, were tested for RNA-interference effects on QTL-linked traits. In a strain carrying the shorter-lived allele, knockdown of rec-8 (encoding a meiotic cohesin) reduced its transcripts 4-fold, to a level similar to the longer-lived strain, while extending lifespan 25-26%, whether begun before fertilization or at maturity. The short-lived lsq4 allele also conferred sensitivity to oxidative and thermal stresses, and lower male frequency (reflecting X-chromosome non-disjunction), traits reversed uniquely by rec-8 knockdown. A strain bearing the longer-lived lsq4 allele, differing from the short-lived strain at <0.3% of its genome, derived no lifespan or stress-survival benefit from rec-8 knockdown. We consider two possible explanations: high rec-8 expression may include increased "leaky" expression in mitotic cells, leading to deleterious destabilization of somatic genomes; or REC-8 may act entirely in germ-line meiotic cells to reduce aberrations such as non-disjunction, thereby blunting a stress-resistance response mediated by innate immunity. Replicative lifespan was extended 20% in haploid S. cerevisiae (BY4741) by deletion of REC8, orthologous to nematode rec-8, implying that REC8 disruption of mitotic-cell survival is widespread, exemplifying antagonistic pleiotropy (opposing effects on lifespan vs. reproduction), and/or balancing selection wherein genomic disruption increases genetic variation under harsh conditions.
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A Small-Molecule Inhibitor of RAD51 Reduces Homologous Recombination and Sensitizes Multiple Myeloma Cells to Doxorubicin.
Front Oncol
PUBLISHED: 01-01-2014
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We previously reported high expression of RAD51 and increased homologous recombination (HR) rates in multiple myeloma (MM) cells, and showed that genomic instability and disease progression are commensurate with HR levels. Moreover, high RAD51 expression in vivo is associated with chemoresistance and poor patient survival. Doxorubicin (DOX) is one of the most widely used drug treatments in MM chemotherapy. DOX is cytotoxic because it induces DNA double-strand breaks, which can be repaired by RAD51-mediated HR; activation of this pathway thus contributes to resistance. To investigate the role of RAD51 in MM drug resistance, we assessed the ability of B02, a small-molecule inhibitor of RAD51, to enhance DOX sensitivity of MM cells. Combining low-toxicity doses of DOX and B02 resulted in significant synthetic lethality, observed as increased apoptosis and reduced viability compared to either agent alone, or to the product of their individual effects. In contrast, the combination did not produce significant synergy against normal human CD19(+) B cells from peripheral blood. DOX induced RAD51 at both mRNA and protein levels, while arresting cells in S and G2. DOX treatment also increased the number of RAD51 foci, a marker of HR repair, so that the fraction of cells with ?5 foci rose fourfold, whereas ?H2AX foci rose far less, implying that most new breaks are repaired. When B02 treatment preceded DOX exposure, the induction of RAD51 foci was severely blunted, whereas, ?H2AX foci rose significantly relative to basal levels or either agent alone. In MM cells carrying a chromosomally integrated reporter of HR repair, DOX increased HR events while B02 inhibition of RAD51 blocked the HR response. These studies demonstrate the crucial role of RAD51 in protecting MM cells from genotoxic agents such as DOX, and suggest that specific inhibition of RAD51 may be an effective means to block DNA repair in MM cells and thus to enhance the efficacy of chemotherapy.
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Extreme Depletion of PIP3 Accompanies the Increased Life Span and Stress Tolerance of PI3K-null C. elegans Mutants.
Front Genet
PUBLISHED: 01-05-2013
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The regulation of animal longevity shows remarkable plasticity, in that a variety of genetic lesions are able to extend lifespan by as much as 10-fold. Such studies have implicated several key signaling pathways that must normally limit longevity, since their disruption prolongs life. Little is known, however, about the proximal effectors of aging on which these pathways are presumed to converge, and to date, no pharmacologic agents even approach the life-extending effects of genetic mutation. In the present study, we have sought to define the downstream consequences of age-1 nonsense mutations, which confer 10-fold life extension to the nematode Caenorhabditis elegans - the largest effect documented for any single mutation. Such mutations insert a premature stop codon upstream of the catalytic domain of the AGE-1/p110? subunit of class-I PI3K. As expected, we do not detect class-I PI3K (and based on our sensitivity, it constitutes <14% of wild-type levels), nor do we find any PI3K activity as judged by immunodetection of phosphorylated AKT, which strongly requires PIP3 for activation by upstream kinases, or immunodetection of its product, PIP3. In the latter case, the upper 95%-confidence limit for PIP3 is 1.4% of the wild-type level. We tested a variety of commercially available PI3K inhibitors, as well as three phosphatidylinositol analogs (PIAs) that are most active in inhibiting AKT activation, for effects on longevity and survival of oxidative stress. Of these, GDC-0941, PIA6, and PIA24 (each at 1 or 10??M) extended lifespan by 7-14%, while PIAs 6, 12, and 24 (at 1 or 10??M) increased survival time in 5?mM peroxide by 12-52%. These effects may have been conferred by insulinlike signaling, since a reporter regulated by the DAF-16/FOXO transcription factor, SOD-3::GFP, was stimulated by these PIAs in the same rank order (PIA24?>?PIA6?>?PIA12) as lifespan. A second reporter, PEPCK::GFP, was equally activated (?40%) by all three.
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Gene categories differentially expressed in C. elegans age-1 mutants of extraordinary longevity: new insights from novel data-mining procedures.
J. Gerontol. A Biol. Sci. Med. Sci.
PUBLISHED: 10-21-2011
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Two nonsense mutants of age-1, the Caenorhabditis elegans gene encoding phosphoinositide 3-kinase, live nearly 10-fold longer than wild-type controls and are exceptionally resistant to several stresses. Genome-wide expression analyses implicated downregulation of many more genes than were upregulated in second-generation age-1 homozygotes. Functional-annotation analysis, based on Gene Ontology terms, suggested that novel mechanisms may mediate the stronger phenotypes observed for these worms than with milder age-1 disruption. For the current study, the same microarray data were reanalyzed using novel meta-analytic procedures that we developed recently. First, gene p values were corrected for systematic biases based on the observed distribution for nonexpressed genes; these values were then combined to derive an aggregate p value for each functional-annotation term while adjusting for intergene covariance. This resulted in much better coverage of relevant gene categories, including many that were independently supported by other data. The number of nonredundant GO categories significantly distinguishing age-1 alleles of exceptional longevity increased from sevenfold to greater than ninefold, improving both sensitivity and specificity of selection for altered pathways and implicating previously unsuspected longevity mechanisms. Of 150 genes whose differential expression underlay significant GO terms in both comparisons, over half were up- or down-regulated in accord with longevity, whereas one third showed altered expression uniquely in the longest-lived age-1-null strains, consistent with the activation or suppression of pathways peculiar to strong age-1 mutants.
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A narrow quantitative trait locus in C. elegans coordinately affects longevity, thermotolerance, and resistance to paraquat.
Front Genet
PUBLISHED: 06-28-2011
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By linkage mapping of quantitative trait loci, we previously identified at least 11 natural genetic variants that significantly modulate Caenorhabditis elegans life-span (LS), many of which would have eluded discovery by knock-down or mutation screens. A region on chromosome IV between markers stP13 and stP35 had striking effects on longevity in three inter-strain crosses (each P?
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Prior exposure to oxidized low-density lipoprotein limits apoptosis in subsequent generations of endothelial cells by altering promoter methylation.
Am. J. Physiol. Heart Circ. Physiol.
PUBLISHED: 05-20-2011
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Oxidized LDL (ox-LDL) plays a critical role in atherogenesis, including apoptosis. As hypercholesterolemia causes epigenetic changes resulting in long-term phenotypic consequences, we hypothesized that repeated and continuous exposure to ox-LDL may alter the pattern of apoptosis in human umbilical vein endothelial cells (HUVECs). We also analyzed global and promoter-specific methylation of apoptosis-related genes. As expected, ox-LDL evoked a dose-dependent increase in apoptosis in the first passage HUVECs that was completely abrogated by lectin-like ox-LDL receptor (LOX-1)-neutralizing antibody. Ox-LDL-induced apoptosis was associated with upregulation of proapoptotic LOX-1, ANXA5, BAX, and CASP3 and inhibition of antiapoptotic BCL2 and cIAP-1 genes accompanied with reciprocal changes in the methylation of promoter regions of these genes. Subsequent passages of cells displayed attenuated apoptotic response to repeat ox-LDL challenge with blunted gene expression and exaggerated methylation of LOX-1, BAX, ANXA5, and CASP3 genes (all P < 0.05 vs. first exposure to ox-LDL). Treatment of cells with LOX-1 antibody before initial ox-LDL treatment prevented both gene-specific promoter methylation and expression changes and reduction of apoptotic response to repeat ox-LDL challenge. Based on these data, we conclude that exposure of HUVECs to ox-LDL induces epigenetic changes leading to resistance to apoptosis in subsequent generations and that this effect may be related to the LOX-1-mediated increase in DNA methylation.
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Modulation of lipid biosynthesis contributes to stress resistance and longevity of C. elegans mutants.
Aging (Albany NY)
PUBLISHED: 03-10-2011
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Many lifespan-modulating genes are involved in either generation of oxidative substrates and end-products, or their detoxification and removal. Among such metabolites, only lipoperoxides have the ability to produce free-radical chain reactions. For this study, fatty-acid profiles were compared across a panel of C. elegans mutants that span a tenfold range of longevities in a uniform genetic background. Two lipid structural properties correlated extremely well with lifespan in these worms: fatty-acid chain length and susceptibility to oxidation both decreased sharply in the longest-lived mutants (affecting the insulinlike-signaling pathway). This suggested a functional model in which longevity benefits from a reduction in lipid peroxidation substrates, offset by a coordinate decline in fatty-acid chain length to maintain membrane fluidity. This model was tested by disrupting the underlying steps in lipid biosynthesis, using RNAi knockdown to deplete transcripts of genes involved in fatty-acid metabolism. These interventions produced effects on longevity that were fully consistent with the functions and abundances of their products. Most knockdowns also produced concordant effects on survival of hydrogen peroxide stress, which can trigger lipoperoxide chain reactions.
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Antiangiogenic and antimitotic effects of aspirin in hypoxia–reoxygenation modulation of the LOX-1-NADPH oxidase axis as a potential mechanism.
J. Cardiovasc. Pharmacol.
PUBLISHED: 10-01-2010
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Hypoxia–reoxygenation (HR) is a primary driver of angiogenesis in both atherogenesis and tumorigenesis. The main target of hypoxia-driven proangiogenic signaling is adherens junctions responsible for contact inhibition of endothelial cells. We analyzed the effects of hypoxia (8–12 hours) followed by a brief period of reoxygenation (2 hours) (HR) on angiogenesis and integrity of adherens junction in cultured human umbilical vein endothelial cells as well as the effects of aspirin on modulation of human umbilical vein endothelial cells response to HR. Cells exposed to HR displayed considerable enhancement of tube formation (angiogenesis) on matrigel. Immunocytostaining of near-confluent cells revealed that HR caused disruption of adherens junctions and internalization of their components VE-cadherin, p120 catenin, and b-catenin. Additionally, HR resulted in the appearance of binucleated cells, and VE-cadherin in colocalization with b-catenin was found to be positioned between the separating nuclei. Presence of aspirin (acetylsalicylic acid, 1 mM) resulted in preservation of adherens junctions on the cellular membrane and prevented angiogenesis as well as mitosis. HR caused upregulation LOX-1, the p47(phox) subunit of NADPH, while reducing transcription of endothelial nitric oxide synthase. Aspirin had no effect on endothelial nitric oxide synthase and canceled the transcriptional activation of the LOX-1 and p47(phox) subunit of NADPH oxidase. Based on these data, we hypothesize that aspirin preserves the integrity of adherens junctions and thus blunts angiogenic response to HR through downregulation of LOX-1 and the LOX-1-mediated p47(phox) component of NADPH oxidase transcription, thus preventing NADPH oxidase assembly and function.
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Histoplasma capsulatum and Caenorhabditis elegans: a simple nematode model for an innate immune response to fungal infection.
Med. Mycol.
PUBLISHED: 12-24-2009
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Histoplasma capsulatum is a primary fungal pathogen of mammals responsible for histoplasmosis. During pathogenesis H. capsulatum yeast proliferate in phagosomes of macrophages. This extensive host/pathogen interaction involves a complex cascade of responses in both organisms. In the mammalian host, infection results in complex branched immunity that is initiated with an innate response and later induces an adaptive response but each response is difficult to resolve during fungal infection. Therefore, in an effort to identify less complex systems and to gain understanding of the host innate response to H. capsulatum, we constructed a mini-host survival assay. With this assay, we found ingestion of virulent Histoplasma capsulatum NAm 1 strain yeasts to be lethal to a Bristol-N2 Caenorhabditis elegans host. The virulent H. capsulatum NAm1 strain shows differential lethality under live/heat-killed infective conditions. Specifically, after ingestion of live yeast lethality is > or = 90% within 48 to 72 h, whereas worms ingesting heat-killed yeast reach equivalent mortality only after 10-14 days. On the other hand, ingestion of live H. capsulatum yeast of the nonvirulent NAm 1 (ura(-)) strain is no more lethal to the nematode than heat-killed yeast. Therefore, C. elegans provides an attractive model for further investigations of the ancient innate immune response during early host/pathogen (H. capsulatum/worm) interaction and pathogenesis.
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Caenorhabditis elegans PI3K mutants reveal novel genes underlying exceptional stress resistance and lifespan.
Aging Cell
PUBLISHED: 09-17-2009
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Two age-1 nonsense mutants, truncating the class-I phosphatidylinositol 3-kinase catalytic subunit (PI3K(CS)) before its kinase domain, confer extraordinary longevity and stress-resistance to Caenorhabditis elegans. These traits, unique to second-generation homozygotes, are blunted at the first generation and are largely reversed by additional mutations to DAF-16/FOXO, a transcription factor downstream of AGE-1 in insulin-like signaling. The strong age-1 alleles (mg44, m333) were compared with the weaker hx546 allele on expression microarrays, testing four independent cohorts of each allele. Among 276 genes with significantly differential expression, 92% showed fewer transcripts in adults carrying strong age-1 alleles rather than hx546. This proportion is significantly greater than the slight bias observed when contrasting age-1 alleles to wild-type worms. Thus, transcriptional changes peculiar to nonsense alleles primarily involve either gene silencing or failure of transcriptional activation. A subset of genes responding preferentially to age-1-nonsense alleles was reassessed by real-time polymerase chain reaction, in worms bearing strong or weak age-1 alleles; nearly all of these were significantly more responsive to the age-1(mg44) allele than to age-1(hx546). Additional mutation of daf-16 reverted the majority of altered mg44-F2 expression levels to approximately wild-type values, although a substantial number of genes remained significantly distinct from wild-type, implying that age-1(mg44) modulates transcription through both DAF-16/FOXO-dependent and -independent channels. When age-1-inhibited genes were targeted by RNA interference (RNAi) in wild-type or age-1(hx546) adults, most conferred significant oxidative-stress protection. RNAi constructs targeting two of those genes were shown previously to extend life, and RNAis targeting five novel genes were found here to increase lifespan. PI3K-null mutants may thus implicate novel mechanisms of life extension.
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Extreme-longevity mutations orchestrate silencing of multiple signaling pathways.
Biochim. Biophys. Acta
PUBLISHED: 03-23-2009
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Long-lived mutants provide unique insights into the genetic factors that limit lifespan in wild-type animals. Most mutants and RNA interference targets found to extend life, typically by 1.5- to 2.5-fold, were discovered in C. elegans. Several longevity-assurance pathways are conserved across widely divergent taxa, indicating that mechanisms of lifespan regulation evolved several hundred million years ago. Strong mutations to the C. elegans gene encoding AGE-1/PI3KCS achieve unprecedented longevity by orchestrating the modulation (predominantly silencing) of multiple signaling pathways. This is evident in a profound attenuation of total kinase activity, leading to reduced phosphoprotein content. Mutations to the gene encoding the catalytic subunit of PI3K (phosphatidylinositol 3-kinase) have the potential to modulate all enzymes that depend on its product, PIP3, for membrane tethering or activation by other kinases. Remarkably, strong mutants inactivating PI3K also silence multiple signaling pathways at the transcript level, partially but not entirely mediated by the DAF-16/FOXO transcription factor. Mammals have a relatively large proportion of somatic cells, and survival depends on their replication, whereas somatic cell divisions in nematodes are limited to development and reproductive tissues. Thus, translation of longevity gains from nematodes to mammals requires disentangling the downstream consequences of signaling mutations, to avoid their deleterious consequences.
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Positive feedback between transcriptional and kinase suppression in nematodes with extraordinary longevity and stress resistance.
PLoS Genet.
PUBLISHED: 03-11-2009
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Insulin/IGF-1 signaling (IIS) regulates development and metabolism, and modulates aging, of Caenorhabditis elegans. In nematodes, as in mammals, IIS is understood to operate through a kinase-phosphorylation cascade that inactivates the DAF-16/FOXO transcription factor. Situated at the center of this pathway, phosphatidylinositol 3-kinase (PI3K) phosphorylates PIP(2) to form PIP(3), a phospholipid required for membrane tethering and activation of many signaling molecules. Nonsense mutants of age-1, the nematode gene encoding the class-I catalytic subunit of PI3K, produce only a truncated protein lacking the kinase domain, and yet confer 10-fold greater longevity on second-generation (F2) homozygotes, and comparable gains in stress resistance. Their F1 parents, like weaker age-1 mutants, are far less robust-implying that maternally contributed trace amounts of PI3K activity or of PIP(3) block the extreme age-1 phenotypes. We find that F2-mutant adults have <10% of wild-type kinase activity in vitro and <60% of normal phosphoprotein levels in vivo. Inactivation of PI3K not only disrupts PIP(3)-dependent kinase signaling, but surprisingly also attenuates transcripts of numerous IIS components, even upstream of PI3K, and those of signaling molecules that cross-talk with IIS. The age-1(mg44) nonsense mutation results, in F2 adults, in changes to kinase profiles and to expression levels of multiple transcripts that distinguish this mutant from F1 age-1 homozygotes, a weaker age-1 mutant, or wild-type adults. Most but not all of those changes are reversed by a second mutation to daf-16, implicating both DAF-16/ FOXO-dependent and -independent mechanisms. RNAi, silencing genes that are downregulated in long-lived worms, improves oxidative-stress resistance of wild-type adults. It is therefore plausible that attenuation of those genes in age-1(mg44)-F2 adults contributes to their exceptional survival. IIS in nematodes (and presumably in other species) thus involves transcriptional as well as kinase regulation in a positive-feedback circuit, favoring either survival or reproduction. Hyperlongevity of strong age-1(mg44) mutants may result from their inability to reset this molecular switch to the reproductive mode.
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Aspirin inhibits oxidant stress, reduces age-associated functional declines, and extends lifespan of Caenorhabditis elegans.
Antioxid. Redox Signal.
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Oxidative stress and inflammation are leading risk factors for age-associated functional declines. We assessed aspirin effects on endogenous oxidative-stress levels, lifespan, and age-related functional declines, in the nematode Caenorhabditis elegans.
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Reduced kidney lipoprotein lipase and renal tubule triglyceride accumulation in cisplatin-mediated acute kidney injury.
Am. J. Physiol. Renal Physiol.
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Peroxisome proliferator-activated receptor-? (PPAR?) activation attenuates cisplatin (CP)-mediated acute kidney injury by increasing fatty acid oxidation, but mechanisms leading to reduced renal triglyceride (TG) accumulation could also contribute. Here, we investigated the effects of PPAR? and CP on expression and enzyme activity of kidney lipoprotein lipase (LPL) as well as on expression of angiopoietin protein-like 4 (Angptl4), glycosylphosphatidylinositol-anchored-HDL-binding protein (GPIHBP1), and lipase maturation factor 1 (Lmf1), which are recognized as important proteins that modulate LPL activity. CP caused a 40% reduction in epididymal white adipose tissue (WAT) mass, with a reduction of LPL expression and activity. CP also reduced kidney LPL expression and activity. Angptl4 mRNA levels were increased by ninefold in liver and kidney tissue and by twofold in adipose tissue of CP-treated mice. Western blots of two-dimensional gel electrophoresis identified increased expression of a neutral pI Angptl4 protein in kidney tissue of CP-treated mice. Immunolocalization studies showed reduced staining of LPL and increased staining of Angptl4 primarily in proximal tubules of CP-treated mice. CP also increased TG accumulation in kidney tissue, which was ameliorated by PPAR? ligand. In summary, a PPAR? ligand ameliorates CP-mediated nephrotoxicity by increasing LPL activity via increased expression of GPHBP1 and Lmf1 and by reducing expression of Angptl4 protein in the proximal tubule.
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Photothermal confocal spectromicroscopy of multiple cellular chromophores and fluorophores.
Biophys. J.
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Confocal fluorescence microscopy is a powerful biological tool providing high-resolution, three-dimensional (3D) imaging of fluorescent molecules. Many cellular components are weakly fluorescent, however, and thus their imaging requires additional labeling. As an alternative, label-free imaging can be performed by photothermal (PT) microscopy (PTM), based on nonradiative relaxation of absorbed energy into heat. Previously, little progress has been made in PT spectral identification of cellular chromophores at the 3D microscopic scale. Here, we introduce PTM integrating confocal thermal-lens scanning schematic, time-resolved detection, PT spectral identification, and nonlinear nanobubble-induced signal amplification with a tunable pulsed nanosecond laser. The capabilities of this confocal PTM were demonstrated for high-resolution 3D imaging and spectral identification of up to four chromophores and fluorophores in live cells and Caenorhabditis elegans. Examples include cytochrome c, green fluorescent protein, Mito-Tracker Red, Alexa-488, and natural drug-enhanced or genetically engineered melanin as a PT contrast agent. PTM was able to guide spectral burning of strong absorption background, which masked weakly absorbing chromophores (e.g., cytochromes in the melanin background). PTM provided label-free monitoring of stress-related changes to cytochrome c distribution, in C. elegans at the single-cell level. In nonlinear mode ultrasharp PT spectra from cyt c and the lateral resolution of 120 nm during calibration with 10-nm gold film were observed, suggesting a potential of PTM to break through the spectral and diffraction limits, respectively. Confocal PT spectromicroscopy could provide a valuable alternative or supplement to fluorescence microscopy for imaging of nonfluorescent chromophores and certain fluorophores.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.