Chemical and genetic rescue of in vivo progranulin-deficient lysosomal and autophagic defects.

In 2006, GRN mutations were first linked to frontotemporal dementia (FTD), the leading cause of non-Alzheimer dementias. While much research has been dedicated to understanding the genetic causes of the disease, our understanding of the mechanistic impacts of GRN deficiency has only recently begun to take shape. With no known cure or treatment available for GRN-related FTD, there is a growing need to rapidly advance genetic and/or small-molecule therapeutics for this disease. This issue is complicated by the fact that, while lysosomal dysfunction seems to be a key driver of pathology, the mechanisms linking a loss of GRN to a pathogenic state remain unclear. In our attempt to address these key issues, we have turned to the nematode, Caenorhabditis elegans, to model, study, and find potential therapies for GRN-deficient FTD. First, we show that the loss of the nematode GRN ortholog, pgrn-1, results in several behavioral and molecular defects, including lysosomal dysfunction and defects in autophagic flux. Our investigations implicate the sphingolipid metabolic pathway in the regulation of many of the in vivo defects associated with pgrn-1 loss. Finally, we utilized these nematodes as an in vivo tool for high-throughput drug screening and identified two small molecules with potential therapeutic applications against GRN/pgrn-1 deficiency. These compounds reverse the biochemical, cellular, and functional phenotypes of GRN deficiency. Together, our results open avenues for mechanistic and therapeutic research into the outcomes of GRN-related neurodegeneration, both genetic and molecular.

Detection of Caenorhabditis elegans Germ Cell Apoptosis Following Exposure to Environmental Contaminant Mixtures: A Crude Oil-Dispersant Mixture Example.

Crude oil disasters, such as the Deepwater Horizon accident, have caused severe environmental contamination and damage, affecting the health of marine and terrestrial organisms. Some previous studies have demonstrated cleanup efforts using chemical dispersant induced more potent toxicities than oil alone due to an increase in bioavailability of crude oil components, such as PAHs. However, there still lacks a systematic procedure that provides methods to determine genotypic and phenotypic changes following exposure to environmental toxicants or toxicant mixture, such as dispersed crude oil. Here, we describe methods for identifying a mechanism of dispersed crude oil-induced reproductive toxicity in the model organisms, Caenorhabditis elegans (C. elegans). Due to the genetic malleability of C. elegans, two mutant strains outlined in this chapter were used to identify a pathway responsible for inducing apoptosis: MD701 bcIs39 [lim-7p::ced-1::GFP + lin-15(+)], a mutant strain that allows visualization of apoptotic bodies via a green fluorescent protein fused to CED-1; and TJ1 (cep-1(gk138) I.), a p53/CEP-1 defective strain that is unable to activate apoptosis via the p53/CEP-1 pathway. In addition, qRT-PCR was utilized to demonstrate the aberrant expression of apoptosis (ced-13, ced-3, ced-4, ced-9, cep-1, dpl-1, efl-1, efl-2, egl-1, egl-38, lin-35, pax-2, and sir-2.1) and cytochrome P450 (cyp14a3, cyp35a1, cyp35a2, cyp35a5, and cyp35c1) protein-coding genes following exposure to dispersed crude oil. The procedure outlined here can be applicable to determine whether environmental contaminants, most of time contaminant mixture, cause reproductive toxicity by activation of the proapoptotic, p53/CEP-1 pathway.

3D mechanical characterization of single cells and small organisms using acoustic manipulation and force microscopy.

Quantitative micromechanical characterization of single cells and multicellular tissues or organisms is of fundamental importance to the study of cellular growth, morphogenesis, and cell-cell interactions. However, due to limited manipulation capabilities at the microscale, systems used for mechanical characterizations struggle to provide complete three-dimensional coverage of individual specimens. Here, we combine an acoustically driven manipulation device with a micro-force sensor to freely rotate biological samples and quantify mechanical properties at multiple regions of interest within a specimen. The versatility of this tool is demonstrated through the analysis of single Lilium longiflorum pollen grains, in combination with numerical simulations, and individual Caenorhabditis elegans nematodes. It reveals local variations in apparent stiffness for single specimens, providing previously inaccessible information and datasets on mechanical properties that serve as the basis for biophysical modelling and allow deeper insights into the biomechanics of these living systems.

The Caenorhabditis elegans homolog of human copper chaperone Atox1, CUC-1, aids in distal tip cell migration.

Cell migration is a fundamental biological process involved in for example embryonic development, immune system and wound healing. Cell migration is also a key step in cancer metastasis and the human copper chaperone Atox1 was recently found to facilitate this process in breast cancer cells. To explore the role of the copper chaperone in other cell migration processes, we here investigated the putative involvement of an Atox1 homolog in Caenorhabditis elegans, CUC-1, in distal tip cell migration, which is a key process during the development of the C. elegans gonad. Using knock-out worms, in which the cuc-1 gene was removed by CRISPR-Cas9 technology, we probed life span, brood size, as well as distal tip cell migration in the absence or presence of supplemented copper. Upon scoring of gonads, we found that cuc-1 knock-out, but not wild-type, worms exhibited distal tip cell migration defects in approximately 10-15% of animals and, had a significantly reduced brood size. Importantly, the distal tip cell migration defect was rescued by a wild-type cuc-1 transgene provided to cuc-1 knock-out worms. The results obtained here for C. elegans CUC-1 imply that Atox1 homologs, in addition to their well-known cytoplasmic copper transport, may contribute to developmental cell migration processes.

Sensory cilia as the Achilles heel of nematodes when attacked by carnivorous mushrooms.

Fungal predatory behavior on nematodes has evolved independently in all major fungal lineages. The basidiomycete oyster mushroom Pleurotus ostreatus is a carnivorous fungus that preys on nematodes to supplement its nitrogen intake under nutrient-limiting conditions. Its hyphae can paralyze nematodes within a few minutes of contact, but the mechanism had remained unclear. We demonstrate that the predator-prey relationship is highly conserved between multiple Pleurotus species and a diversity of nematodes. To further investigate the cellular and molecular mechanisms underlying rapid nematode paralysis, we conducted genetic screens in Caenorhabditis elegans and isolated mutants that became resistant to P. ostreatus We found that paralysis-resistant mutants all harbored loss-of-function mutations in genes required for ciliogenesis, demonstrating that the fungus induced paralysis via the cilia of nematode sensory neurons. Furthermore, we observed that P. ostreatus caused excess calcium influx and hypercontraction of the head and pharyngeal muscle cells, ultimately resulting in rapid necrosis of the entire nervous system and muscle cells throughout the entire organism. This cilia-dependent predatory mechanism is evolutionarily conserved in Pristionchus pacificus, a nematode species estimated to have diverged from C. elegans 280 to 430 million y ago. Thus, P. ostreatus exploits a nematode-killing mechanism that is distinct from widely used anthelmintic drugs such as ivermectin, levamisole, and aldicarb, representing a potential route for targeting parasitic nematodes in plants, animals, and humans.

Silica nanoparticles disrupt OPT-2/PEP-2-dependent trafficking of nutrient peptides in the intestinal epithelium.

Despite of the increasing application of silica nanoparticles and identification of oral exposure as a major entry portal, we lack understanding of nanosilica effects in the gut. Thus, we investigated biointeractions of nanosilica with single intestinal cells. The invertebrate nematode Caenorhabditis elegans was chosen as model organism with a tractable intestine and realistic target organism of nanomaterials in the environment. We found that nanosilica impairs the intestinal uptake of oligopeptides. Downstream to absorption by the apical OPT-2/PEP-2 transporter dipeptides were trapped in aberrant vesicles that grow over time and reach diameters of ≥6 µm. The peptide vesicles do not correspond to known organelles such as gut granules and form independently of related gene products GLO-1 or GLO-3. Formation of aberrant peptide vesicles also occurred independently of insulin/IGF-I receptor (DAF-2) signaling and daf-2 loss of function mutants showed specific vesicle patterns including distinct localization along the apical membrane of single intestinal cells. As malnutrition of exposed C. elegans manifested in reduced growth and a petite phenotype similar to OPT-2/PEP-2 transporter deficient mutants, we conclude that nanosilica-induced peptide vesicles represent a new compartment of di- and tripeptide trapping which disrupts hydrolysis of nutrient peptides and metabolism.

Tricarboxylic acid cycle activity suppresses acetylation of mitochondrial proteins during early embryonic development in Caenorhabditis elegans.

The tricarboxylic acid (TCA) cycle (or citric acid cycle) is responsible for the complete oxidation of acetyl-CoA and formation of intermediates required for ATP production and other anabolic pathways, such as amino acid synthesis. Here, we uncovered an additional mechanism that may help explain the essential role of the TCA cycle in the early embryogenesis of Caenorhabditis elegans. We found that knockdown of citrate synthase (cts-1), the initial and rate-limiting enzyme of the TCA cycle, results in early embryonic arrest, but that this phenotype is not because of ATP and amino acid depletions. As a possible alternative mechanism explaining this developmental deficiency, we observed that cts-1 RNAi embryos had elevated levels of intracellular acetyl-CoA, the starting metabolite of the TCA cycle. Of note, we further discovered that these embryos exhibit hyperacetylation of mitochondrial proteins. We found that supplementation with acetylase-inhibiting polyamines, including spermidine and putrescine, counteracted the protein hyperacetylation and developmental arrest in the cts-1 RNAi embryos. Contrary to the hypothesis that spermidine acts as an acetyl sink for elevated acetyl-CoA, the levels of three forms of acetylspermidine, N1-acetylspermidine, N8-acetylspermidine, and N1,N8-diacetylspermidine, were not significantly increased in embryos treated with exogenous spermidine. Instead, we demonstrated that the mitochondrial deacetylase sirtuin 4 (encoded by the sir-2.2 gene) is required for spermidine's suppression of protein hyperacetylation and developmental arrest in the cts-1 RNAi embryos. Taken together, these results suggest the possibility that during early embryogenesis, acetyl-CoA consumption by the TCA cycle in C. elegans prevents protein hyperacetylation and thereby protects mitochondrial function.

Reflective imaging improves spatiotemporal resolution and collection efficiency in light sheet microscopy.

Light-sheet fluorescence microscopy (LSFM) enables high-speed, high-resolution, and gentle imaging of live specimens over extended periods. Here we describe a technique that improves the spatiotemporal resolution and collection efficiency of LSFM without modifying the underlying microscope. By imaging samples on reflective coverslips, we enable simultaneous collection of four complementary views in 250 ms, doubling speed and improving information content relative to symmetric dual-view LSFM. We also report a modified deconvolution algorithm that removes associated epifluorescence contamination and fuses all views for resolution recovery. Furthermore, we enhance spatial resolution (to <300 nm in all three dimensions) by applying our method to single-view LSFM, permitting simultaneous acquisition of two high-resolution views otherwise difficult to obtain due to steric constraints at high numerical aperture. We demonstrate the broad applicability of our method in a variety of samples, studying mitochondrial, membrane, Golgi, and microtubule dynamics in cells and calcium activity in nematode embryos.

Limits and possibilities experienced by nurses in the treatment of women with chronic venous ulcers / Límites y posibilidades vividas por enfermeras en el tratamiento de mujeres con úlcera venosa crónica / Limites e possibilidades vivenciados por enfermeiras no tratamento de mulheres com úlcera venosa crônica

Objective To understand the experiences and expectations of nurses in the treatment of women with chronic venous ulcers. Method Phenomenological research was based on Alfred Schütz, whose statements were obtained in January, 2012, through semi-structured interviews with seven nurses. Results The nurse reveals the difficulties presented by the woman in performing self-care, the perceived limitations in the treatment anchored in motivation, and the values and beliefs of women. It showed professional frustration because venous leg ulcer recurrence, lack of inputs, interdisciplinary work and training of nursing staff. There was an expected adherence to the treatment of women, and it emphasized the need for ongoing care, supported self-care and standard practices in treatment. Conclusion That treatment of chronic venous leg ulcers constitutes a challenge that requires collective investment, involving women, professionals, managers and health institutions. .

Objetivo Comprender las experiencias y expectativas de enfermeras en el tratamiento de mujeres con úlcera venosa crónica. Método Investigación fenomenológica fundamentada en Alfred Schutz, que buscó Se realizó entrevista semiestructurada con siete enfermeras, en enero del 2012. Resultados La enfermera revela dificultades presentadas por la mujer para realizar el autocuidado, percibe limitaciones en el tratamiento relacionadas con la desmotivación, los valores y las creencias de las mujeres. Refiere frustración profesional debido a la recidiva de la lesión, a la falta de insumos, al deficiente trabajo interdisciplinar y a la limitada capacitación del equipo de enfermeras. Espera la adhesión de la mujer al tratamiento y resalta la necesidad del cuidado continuo, del autocuidado apoyado y de estandarizar conductas de tratamiento. Conclusión El tratamiento de la úlcera venosa crónica es un desafío que requiere contribución colectiva, involucrando a las mujeres, a los profesionales, a los gestores y a las instituciones de salud. .

Objetivo Compreender as experiências e expectativas de enfermeiras no tratamento de mulheres com úlcera venosa crônica na Atenção Primária à Saúde. Método Pesquisa fundamentada na fenomenologia social de Alfred Schütz, com depoimentos obtidos em janeiro de 2012, por meio de entrevista semiestruturada com sete enfermeiras. Resultados As enfermeiras revelam dificuldades apresentadas pelas mulheres com úlcera venosa crônica para realizar o autocuidado, percebem limitações na terapêutica ancoradas na desmotivação e nos valores e crenças das mulheres. Referem frustração profissional em razão da recidiva da lesão, falta de insumos e tecnologia, de trabalho interdisciplinar e da capacitação da equipe de enfermagem. Esperam a adesão das mulheres ao tratamento e ressaltam a necessidade do cuidado contínuo, do autocuidado apoiado e da padronização de condutas no tratamento. Conclusão O tratamento da úlcera venosa crônica constitui-se em um desafio que requer investimento coletivo, envolvendo a mulher, os profissionais, os gestores e as instituições de saúde. .

Caffeic acid phenethylester increases stress resistance and enhances lifespan in Caenorhabditis elegans by modulation of the insulin-like DAF-16 signalling pathway.

UNLABELLED: CAPE is an active constituent of propolis which is widely used in traditional medicine. This hydroxycinnamic acid derivate is a known activator of the redox-active Nrf2 signalling pathway in mammalian cells. We used C. elegans to investigate the effects of this compound on accumulation of reactive oxygen species and the modulation of the pivotal redox-active pathways SKN-1 and DAF-16 (homologues of Nrf2 and FoxO, respectively) in this model organism; these results were compared to the effects in Hct116 human colon carcinoma cells. CAPE exerts a strong antioxidative effect in C. elegans: The increase of reactive oxygen species induced by thermal stress was diminished by about 50%. CAPE caused a nuclear translocation of DAF-16, but not SKN-1. CAPE increased stress resistance of the nematode against thermal stress and finally a prolongation of the median and maximum lifespan by 9 and 17%, respectively. This increase in stress resistance and lifespan was dependent on DAF-16 as shown in experiments using a DAF-16 loss of function mutant strain. Life prolongation was retained under SKN-1 RNAi conditions showing that the effect is SKN-1 independent. The results of CAPE obtained in C. elegans differed from the results obtained in Hct116 colon carcinoma cells: CAPE also caused strong antioxidative effects in the mammalian cells, but no activation of the FoxO4 signalling pathway was detectable. Instead, an activation of the Nrf2 signalling pathway was shown by luciferase assay and western blots. CONCLUSION: CAPE activates the insulin-like DAF-16, but not the SKN-1 signalling pathway in C. elegans and therefore enhances the stress resistance and lifespan of this organism. Since modulation of the DAF-16 pathway was found to be a pivotal effect of CAPE in C. elegans, this has to be taken into account for the investigation of the molecular mechanisms of the traditional use of propolis.

A method to identify and validate mitochondrial modulators using mammalian cells and the worm C. elegans.

Mitochondria are semi-autonomous organelles regulated by a complex network of proteins that are vital for many cellular functions. Because mitochondrial modulators can impact many aspects of cellular homeostasis, their identification and validation has proven challenging. It requires the measurement of multiple parameters in parallel to understand the exact nature of the changes induced by such compounds. We developed a platform of assays scoring for mitochondrial function in two complementary models systems, mammalian cells and C. elegans. We first optimized cell culture conditions and established the mitochondrial signature of 1,200 FDA-approved drugs in liver cells. Using cell-based and C. elegans assays, we further defined the metabolic effects of two pharmacological classes that emerged from our hit list, i.e. imidazoles and statins. We found that these two drug classes affect respiration through different and cholesterol-independent mechanisms in both models. Our screening strategy enabled us to unequivocally identify compounds that have toxic or beneficial effects on mitochondrial activity. Furthermore, the cross-species approach provided novel mechanistic insight and allowed early validation of hits that act on mitochondrial function.

Dispersed crude oil amplifies germ cell apoptosis in Caenorhabditis elegans, followed a CEP-1-dependent pathway.

The Deepwater Horizon oil spill is among the most severe environmental disasters in US history. The extent of crude oil released and the subsequent dispersant used for cleanup was unprecedented. The dispersed crude oil represents a unique form of environmental contaminant that warrants investigations of its environmental and human health impacts. Lines of evidence have demonstrated that dispersed oil affects reproduction in various organisms, in a more potent manner than oil- and dispersant-only exposures. However, the action mechanism of dispersed oil remains largely unknown. In this study, we utilized the model organism Caenorhabditis elegans to investigate impacts of dispersed oil exposure on sex cell apoptosis and related gene expressions. Worms were exposed to different diluted levels of crude oil-dispersant (oil-dis) mixtures (20:1, v/v; at 500×, 2,000×, and 5,000× dilutions). The dispersed crude oil significantly increases the number of apoptotic germ cells in treated worms when compared with control at all exposure levels (p < 0.05). Genes involved in the apoptosis pathway were dysregulated, which include ced-13, ced-3, ced-4, ced-9, cep-1, dpl-1, efl-1, efl-2, egl-1, egl-38, lin-35, pax-2, and sir-2.1. Many aberrant expressed genes encoding for core components in apoptosis machinery (cep-1/p53, ced-13/BH3, ced-9/Bcl-2, ced-4/Apaf-1, and ced-3/caspase) displayed consistent expression patterns across all exposure levels. Significantly ced-3/caspase was upregulated at all dispersed oil-treated groups, consistent with the observed apoptosis phenotype. Given cep-1/p53 was activated at all dispersed oil treatments and the germ cell apoptosis was suppressed in the CEP-1 loss of function mutant, the increased apoptosis is likely CEP-1 dependent. In addition, the anti-apoptotic ced-9/Bcl-2 was activated in response to the increase in cell death. This study provides a mechanism understanding of dispersed crude oil-induced reproductive toxicity.

Microscopic analysis of chromatin localization and dynamics in C. elegans.

During development, the genome undergoes drastic reorganization within the nuclear space. To determine tridimensional genome folding, genome-wide techniques (damID/Hi-C) can be applied using cell populations, but these have to be calibrated using microscopy and single-cell analysis of gene positioning. Moreover, the dynamic behavior of chromatin has to be assessed on living samples. Combining fast stereotypic development with easy genetics and microscopy, the nematode C. elegans has become a model of choice in recent years to study changes in nuclear organization during cell fate acquisition. Here we present two complementary techniques to evaluate nuclear positioning of genes either by fluorescence in situ hybridization in fixed samples or in living worm embryos using the GFP-lacI/lacO chromatin-tagging system.

Hydrogen sulfide in cell signaling, signal transduction, cellular bioenergetics and physiology in C. elegans.

Hydrogen sulfide (H2S), long viewed as a toxic gas and environmental hazard, is emerging as a biological mediator with remarkable physiological and pathophysiological relevance. H2S is now viewed as the third main gasotransmitter in the mammalian body. Its pharmacological characteristic possesses similarities to the other two gasotransmitters - nitric oxide (NO) and carbon monoxide (CO). Many of the biological effects of H2S follow a bell-shaped concentration-response; at low concentration or at lower release rates it has beneficial and cytoprotective effects, while at higher concentrations or fast release rates toxicity becomes apparent. Cellular bioenergetics is a prime example for this bell-shaped dose-response, where H2S, at lower concentrations/rates serves as an inorganic substrate and electron donor for mitochondrial ATP generation, while at high concentration it inhibits mitochondrial respiration by blocking the Complex IV in the mitochondrial electron transport chain. The current review is aimed to focus on the following aspects of H2S biology: 1) a general overview of the general pharmacological characteristics of H2S, 2) a summary of the key H2S-mediated signal transduction pathways, 3) an overview of role of H2S in regulation of cellular bioenergetics, 4) key aspects of H2S physiology in C. elegans (a model system) and, finally 5) the therapeutic potential of H2S donating molecules in various disease states.

Selective fluorescent detection of RNA in living cells by using imidazolium-based cyclophane.

A water-soluble imidazolium-based fluorescent chemosensor senses RNA selectively through fluorescence enhancement over other biologically relevant biomolecules in aqueous solution at physiological pH 7.4. Fluorescence image detection of RNA in living cells such as onion cells, HeLa cells, and animal model cells was successfully demonstrated which displays a chelation-enhanced fluorescence effect. These affinities can be attributed to the strong electrostatic (C-H)(+)···A(-) ionic H-bonding and the aromatic moiety driven π-stacking of imidazolium-based cyclophane with the size-complementary major groove of RNA.

Elemental mapping by electron energy loss spectroscopy in biology.

Over the past decades there have been significant advances in transmission electron microscopy for biological applications, including in energy filtering and spectrum imaging, which are techniques based on the principles of electron energy loss spectroscopy. These imaging modalities allow quantitative mapping of specific chemical elements with high sensitivity and spatial resolution. This chapter describes the experimental and computational procedures for elemental mapping in two dimensions as well as a more recent extension to three dimensions, which can reveal quantitative distributions of elements in cells on a macromolecular scale.

Laser microsurgery in Caenorhabditis elegans.

Laser killing of cell nuclei has long been a powerful means of examining the roles of individual cells in C. elegans. Advances in genetics, laser technology, and imaging have further expanded the capabilities and usefulness of laser surgery. Here, we review the implementation and application of currently used methods for target edoptical disruption in C. elegans.

Medicinal plants and antioxidants: what do we learn from cell culture and Caenorhabditis elegans studies?

Traditional medicinal plants have a long history of therapeutic use. The beneficial health effects of medicinal plants rich in polyphenols are often attributed to their potent antioxidant activities, as established in vitro, since diets rich in polyphenols are epidemiologically associated with a decreased incidence of age-related diseases in humans. However, medicinal plants may also exert pro-oxidant effects that up-regulate endogenous protective enzymes. Care is needed when studying the biological effects of medicinal plants in cell culture because some polyphenols oxidize readily in culture media. This review summarizes the data we have obtained from in vitro and in vivo (Caenorhabditis elegans) studies examining the diverse effects of traditional medicinal plants and their modes of action.

Nuclear hormone receptor regulation of microRNAs controls developmental progression.

In response to small-molecule signals such as retinoids or steroids, nuclear receptors activate gene expression to regulate development in different tissues. MicroRNAs turn off target gene expression within cells by binding complementary regions in messenger RNA transcripts, and they have been broadly implicated in development and disease. Here we show that the Caenorhabditis elegans nuclear receptor DAF-12 and its steroidal ligand directly activate promoters of let-7 microRNA family members to down-regulate the microRNA target hbl-1, which drives progression of epidermal stem cells from second to third larval stage patterns of cell division. Conversely, the receptor without the ligand represses microRNA expression during developmental arrest. These findings identify microRNAs as components of a hormone-coupled molecular switch that shuts off earlier developmental programs to allow for later ones.

Effects of sterols on the development and aging of Caenorhabditis elegans.

Although Caenorhabditis elegans lacks several components of the de novo sterol biosynthetic pathway, it requires sterols as essential nutrients. Supplemental cholesterol undergoes extensive enzymatic modification in C. elegans to form certain sterols of unknown function. Since sterol metabolism in C. elegans differs from that in other species, such as mammals and yeast, it is important to examine how sterols regulate worm physiology. To examine the functions of sterols in C. elegans, a sterol-feeding experiment was carried out and several critical parameters, such as brood size, growth rate, and life span, were measured. In addition, the change in lipid distribution in C. elegans can be both qualitatively and quantitatively determined by various methods, including staining and chromatographic techniques. Taken together, the effects of sterols on C. elegans are very prominent and can be easily assessed using the techniques described here.