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1.  Tissue Expression Pattern of PMK-2 p38 MAPK Is Established by the miR-58 Family in C. elegans 
PLoS Genetics  2015;11(2):e1004997.
Analyses of gene expression profiles in evolutionarily diverse organisms have revealed a role for microRNAs in tuning tissue-specific gene expression. Here, we show that the relatively abundant and constitutively expressed miR-58 family of microRNAs sharply defines the tissue-specific expression of the broadly transcribed gene encoding PMK-2 p38 MAPK in Caenorhabditis elegans. Whereas PMK-2 functions redundantly with PMK-1 in the nervous system to regulate neuronal development and behavioral responses to pathogenic bacteria, the miR-58, miR-80, miR-81, and miR-82 microRNAs function redundantly to destabilize pmk-2 mRNA in non-neuronal cells with switch-like potency. Our data suggest a role for the miR-58 family in the establishment of neuronal-specific gene expression in C. elegans, and support a more general role for microRNAs in the establishment of tissue-specific gene expression.
Author Summary
MicroRNAs are small, noncoding RNAs that act post-transcriptionally to inhibit expression of their target mRNAs. Gene expression studies of microRNAs and their target transcripts in diverse organisms have suggested that microRNAs may function to shape patterns of tissue expression. In this paper, we show that the miR-58/80-82 family of microRNAs, which accounts for roughly half of all C. elegans microRNAs at all developmental stages, defines the spatial expression pattern of PMK-2 p38 MAPK. While the pmk-2 gene is broadly transcribed, its tissue-specific expression is established by the redundant activities of miR-58, miR-80, miR-81, and miR-82, which switch off expression of PMK-2 through destabilization of pmk-2 mRNA in non-neuronal tissues. Our data suggest a housekeeping role for the miR-58/80-82 family in establishing and maintaining neuronal patterns of gene expression in C. elegans, and supports a more general role for microRNAs in establishing patterns of tissue expression.
PMCID: PMC4335502  PMID: 25671546
2.  The Unfolded Protein Response in a Pair of Sensory Neurons Promotes Entry of C. elegans into Dauer Diapause 
Current biology : CB  2013;23(24):10.1016/j.cub.2013.10.058.
In response to unfavorable environmental conditions such as starvation, crowding and elevated temperature, Caenorhabditis elegans larvae enter an alternative developmental stage known as dauer [1], which exhibit dramatic remodeling of stress resistance and metabolism [2–3]. The genetic dissection of the molecular mechanisms of the C. elegans dauer developmental decision has defined evolutionarily conserved signaling pathways of organismal neuroendocrine physiology [2–4]. Here, we have identified a mechanism by which a dominant mutation in a neuronal insulin gene, daf-28(sa191) [5–7], causes constitutive entry into dauer diapause. We demonstrate that expression of the mutant DAF-28 insulin peptide results in endoplasmic reticulum (ER) stress in the ASI pair of chemosensory neurons. The neuronal ER stress does not compromise cellular survival, but activates PEK-1, the C. elegans ortholog of the mammalian eIF2α kinase PERK, which in turn phosphorylates Ser49 of eIF2α, specifically in the ASI neuron pair, to promote entry into dauer diapause. Our data establish a novel role for ER stress and the Unfolded Protein Response in promoting entry into dauer diapause, and suggest that in addition to cell autonomous activities in the maintenance of ER homeostasis, the Unfolded Protein Response may act in a cell non-autonomous manner to promote organismal adaptation to stress during larval development.
PMCID: PMC3870035  PMID: 24316205
3.  Natural Polymorphisms in C. elegans HECW-1 E3 Ligase Affect Pathogen Avoidance Behaviour 
Nature  2011;480(7378):525-529.
Heritable variation in behavioural traits generally has a complex genetic basis1, and thus naturally occurring polymorphisms that influence behaviour have been defined in only rare instances2,3. The isolation of wild strains of Caenorhabditis elegans has facilitated the study of natural genetic variation in this species4 and provided insights into its diverse microbial ecology5. C. elegans responds to bacterial infection with conserved innate immune responses6-8 and, while lacking the immunological memory of vertebrate adaptive immunity, exhibits an aversive learning response to pathogenic bacteria9. Here, we report the molecular characterization of naturally occurring coding polymorphisms in a C. elegans gene encoding a conserved HECT domain-containing E3 ubiquitin ligase, HECW-1. We show that two distinct polymorphisms in neighbouring residues of HECW-1 each affect C. elegans behavioural avoidance of a lawn of Pseudomonas aeruginosa. Neuron-specific rescue and ablation experiments, and genetic interaction analysis suggest that HECW-1 functions in a pair of sensory neurons to inhibit P. aeruginosa lawn avoidance behaviour through inhibition of the neuropeptide receptor NPR-110, which we have previously shown promotes P. aeruginosa lawn avoidance behaviour11. Our data establish a molecular basis for natural variation in a C. elegans behaviour that may undergo adaptive changes in response to microbial pathogens.
PMCID: PMC3245782  PMID: 22089131
4.  Physiological IRE-1-XBP-1 and PEK-1 Signaling in Caenorhabditis elegans Larval Development and Immunity 
PLoS Genetics  2011;7(11):e1002391.
Endoplasmic reticulum (ER) stress activates the Unfolded Protein Response, a compensatory signaling response that is mediated by the IRE-1, PERK/PEK-1, and ATF-6 pathways in metazoans. Genetic studies have implicated roles for UPR signaling in animal development and disease, but the function of the UPR under physiological conditions, in the absence of chemical agents administered to induce ER stress, is not well understood. Here, we show that in Caenorhabditis elegans XBP-1 deficiency results in constitutive ER stress, reflected by increased basal levels of IRE-1 and PEK-1 activity under physiological conditions. We define a dynamic, temperature-dependent requirement for XBP-1 and PEK-1 activities that increases with immune activation and at elevated physiological temperatures in C. elegans. Our data suggest that the negative feedback loops involving the activation of IRE-1-XBP-1 and PEK-1 pathways serve essential roles, not only at the extremes of ER stress, but also in the maintenance of ER homeostasis under physiological conditions.
Author Summary
Proteins destined for secretion outside of eukaryotic cells are trafficked through the endoplasmic reticulum (ER). Protein folding in the ER involves the activity of chaperones, as well as catalysis of post-translational modifications such as disulfide bond formation and glycosylation. When the folding capacity of the ER is exceeded, the resulting accumulation of misfolded proteins activates the Unfolded Protein Response (UPR), a conserved signaling response that functions to restore protein folding homeostasis in the ER. Genetic studies have established that the UPR is required for the development of specific cell types in mammals, such as antibody-secreting plasma cells, and recent studies implicate a critical role for UPR signaling in the pathogenesis of metabolic and inflammatory diseases. In this paper we show that innate immunity and elevated physiological temperatures each necessitate UPR activity for C. elegans survival. Furthermore, we show that, under physiological conditions of larval development, basal activity of the UPR is required for the maintenance of ER homeostasis. Our data support the idea not only that the UPR functions as a “stress response” pathway, protecting against the extremes of unfolded protein accumulation, but also that the UPR plays a more general role in animal physiology and development.
PMCID: PMC3219621  PMID: 22125500
5.  A Decline in p38 MAPK Signaling Underlies Immunosenescence in Caenorhabditis elegans 
PLoS Genetics  2011;7(5):e1002082.
The decline in immune function with aging, known as immunosenescence, has been implicated in evolutionarily diverse species, but the underlying molecular mechanisms are not understood. During aging in Caenorhabditis elegans, intestinal tissue deterioration and the increased intestinal proliferation of bacteria are observed, but how innate immunity changes during C. elegans aging has not been defined. Here we show that C. elegans exhibits increased susceptibility to bacterial infection with age, and we establish that aging is associated with a decline in the activity of the conserved PMK-1 p38 mitogen-activated protein kinase pathway, which regulates innate immunity in C. elegans. Our data define the phenomenon of innate immunosenescence in C. elegans in terms of the age-dependent dynamics of the PMK-1 innate immune signaling pathway, and they suggest that a cycle of intestinal tissue aging, immunosenescence, and bacterial proliferation leads to death in aging C. elegans.
Author Summary
Aging is associated with a diminished capacity to resist infection in evolutionarily diverse species including humans, mice, and fruit flies. Few studies have addressed the underlying genetic and molecular basis of this phenomenon. Here we examined immune function during aging in the nematode Caenorhabditis elegans, which we demonstrate exhibits a progressive age-dependent increase in susceptibility to the human pathogen Pseudomonas aeruginosa. Our studies suggest that, during aging in C. elegans, the activity of the PMK-1 p38 mitogen-activated protein kinase pathway, a conserved pathway involved in pathogen defense, sharply declines. We propose a model in which age-related damage to intestinal tissue impairs immune function and therefore promotes infection, which in turn amplifies tissue damage, thus setting in motion a self-perpetuating cycle that gradually erodes host defense.
PMCID: PMC3098197  PMID: 21625567
6.  Oral curcumin supplementation in patients with atopic asthma 
Allergy & Rhinology  2011;2(2):e51-e53.
Oral curcumin is recognized to have anti-inflammatory properties and has been used by ancient traditional medicine for centuries to treat a variety of diseases. In vitro studies have confirmed the ability of curcumin to inhibit allergic inflammatory cytokine responses from lymphocytes; however, there are no in vivo studies of curcumin to treat inflammation associated with allergic asthma. This study was designed to determine the effect of oral curcumin supplementation on patients with stable, persistent, atopic asthma. Adult patients with stable, persistent asthma with evidence of allergic sensitization were randomized to receive 1000 mg of curcumin twice daily or placebo. Subjects were followed for 6 months and performed monthly spirometry (pre- and postbronchodilator); Asthma Control Test (ACT) scoring; and measurements for fractional excretion of nitric oxide (NO), serum eosinophil count, leukocyte count, total IgE, specific IgE to Dermatophagoides pteronyssinus (Der p) and Dermatophagoides farinae (Der f), use of rescue albuterol, and dose of inhaled corticosteroid. Nine patients were randomized into the treatment arm and six were randomized into the placebo group. No differential response was seen in the treatment and placebo groups regarding the primary end point, postbronchodilator forced expiratory volume in 1 second (FEV1). Similarly, all secondary end point evaluations were not significantly different. Despite in vitro evidence that curcumin has anti-inflammatory properties and can inhibit allergic cytokine responses from lymphocytes in vitro, curcumin, 1000-mg, twice daily supplementation did not significantly affect postbronchodilator FEV1, ACT scores, use of rescue bronchodilator, dose of inhaled corticosteroid, exhaled NO, serum IgE, total white blood cell count specific IgE to Der p or Der f, and blood eosinophils in patients with persistent atopic asthma.
PMCID: PMC3390116  PMID: 22852117
Allergy; asthma; curcumin; herbal
7.  Tissue-Specific Activities of an Immune Signaling Module Regulate Physiological Responses to Pathogenic and Nutritional Bacteria in C. elegans 
Cell host & microbe  2009;6(4):321-330.
Microbes represent both an essential source of nutrition and a potential source of lethal infection to the nematode Caenorhabditis elegans. Immunity in C. elegans requires a signaling module comprised of orthologs of the mammalian Toll-Interleukin-1 Receptor (TIR) domain protein SARM, the mitogen-activated protein kinase kinase kinase (MAPKKK) ASK1, and MAPKK MKK3, which activates p38 MAPK. We determined that the SARM-ASK1-MKK3 module has dual tissue-specific roles in the C. elegans response to pathogens—in the cell autonomous regulation of innate immunity, and the neuroendocrine regulation of serotonin-dependent aversive behavior. SARM-ASK1-MKK3 signaling in the sensory nervous system also regulates egg-laying behavior that is dependent on bacteria provided as a nutrient source. Our data demonstrate that these physiological responses to bacteria share a common mechanism of signaling through the SARM-ASK1-MKK3 module and suggest the co-option of ancestral immune signaling pathways in the evolution of physiological responses to microbial pathogens and nutrients.
PMCID: PMC2772662  PMID: 19837372
8.  An Essential Role for XBP-1 in Host Protection against Immune Activation in C. elegans 
Nature  2010;463(7284):1092-1095.
The detection and compensatory response to the accumulation of unfolded proteins in the endoplasmic reticulum (ER), termed the Unfolded Protein Response (UPR), represents a conserved cellular homeostatic mechanism with important roles in normal development and in the pathogenesis of disease1. The IRE1-XBP1/Hac1p pathway is a major branch of the UPR that has been conserved from yeast to human2,3,4,5,6. XBP-1 is required for the differentiation of the highly secretory plasma cells of the mammalian adaptive immune system7,8, but recent work also points to reciprocal interactions between the UPR and other aspects of immunity and inflammation9,10,11. We have been studying innate immunity in the nematode Caenorhabditis elegans, having established a key role for a conserved PMK-1 p38 mitogen-activated protein kinase (MAPK) pathway in mediating resistance to microbial pathogens12. Here, we show that during C. elegans development, XBP-1 has an essential role in protecting the host during activation of innate immunity. Activation of the PMK-1-mediated response to infection with Pseudomonas aeruginosa induces the XBP-1-dependent UPR. Whereas a loss-of-function xbp-1 mutant develops normally in the presence of relatively non-pathogenic bacteria, infection of the xbp-1 mutant with P. aeruginosa leads to disruption of ER morphology and larval lethality. Unexpectedly, the larval lethality phenotype on pathogenic P. aeruginosa is suppressed by loss of PMK-1-mediated immunity. Furthermore, hyperactivation of PMK-1 causes larval lethality in the xbp-1 mutant even in the absence of pathogenic bacteria. Our data establish innate immunity as a physiologically relevant inducer of ER stress during C. elegans development and suggest that an ancient, conserved role for XBP-1 may be to protect the host organism from the detrimental effects of mounting an innate immune response to microbes.
PMCID: PMC2834299  PMID: 20182512
9.  Phosphorylation of the Conserved Transcription Factor ATF-7 by PMK-1 p38 MAPK Regulates Innate Immunity in Caenorhabditis elegans 
PLoS Genetics  2010;6(4):e1000892.
Innate immunity in Caenorhabditis elegans requires a conserved PMK-1 p38 mitogen-activated protein kinase (MAPK) pathway that regulates the basal and pathogen-induced expression of immune effectors. The mechanisms by which PMK-1 p38 MAPK regulates the transcriptional activation of the C. elegans immune response have not been identified. Furthermore, in mammalian systems the genetic analysis of physiological targets of p38 MAPK in immunity has been limited. Here, we show that C. elegans ATF-7, a member of the conserved cyclic AMP–responsive element binding (CREB)/activating transcription factor (ATF) family of basic-region leucine zipper (bZIP) transcription factors and an ortholog of mammalian ATF2/ATF7, has a pivotal role in the regulation of PMK-1–mediated innate immunity. Genetic analysis of loss-of-function alleles and a gain-of-function allele of atf-7, combined with expression analysis of PMK-1–regulated genes and biochemical characterization of the interaction between ATF-7 and PMK-1, suggest that ATF-7 functions as a repressor of PMK-1–regulated genes that undergoes a switch to an activator upon phosphorylation by PMK-1. Whereas loss-of-function mutations in atf-7 can restore basal expression of PMK-1–regulated genes observed in the pmk-1 null mutant, the induction of PMK-1–regulated genes by pathogenic Pseudomonas aeruginosa PA14 is abrogated. The switching modes of ATF-7 activity, from repressor to activator in response to activated PMK-1 p38 MAPK, are reminiscent of the mechanism of regulation mediated by the corresponding ancestral Sko1p and Hog1p proteins in the yeast response to osmotic stress. Our data point to the regulation of the ATF2/ATF7/CREB5 family of transcriptional regulators by p38 MAPK as an ancient conserved mechanism for the control of innate immunity in metazoans, and suggest that ATF2/ATF7 may function in a similar manner in the regulation of mammalian innate immunity.
Author Summary
We have investigated mechanisms of how the soil nematode Caenorhabditis elegans interacts with pathogenic bacteria. Previously, we have established that a conserved PMK-1 p38 mitogen-activated protein kinase (MAPK) pathway regulates immunity in C. elegans, establishing the conservation of key innate immune signaling pathways of mammals in the immune response of C. elegans. Whereas multiple proteins have been identified as potential targets of p38 MAPK in immunity, the identification of physiological substrates of p38 MAPK in mammalian organisms has been challenging. Here, using a forward genetic approach to identify downstream regulators of the C. elegans innate immune response, we have characterized the transcription factor ATF-7, a conserved member of the basic-region leucine zipper (bZIP) transcription factor family orthologous to mammalian ATF2. We find that ATF-7 functions as a transcriptional regulator of PMK-1 MAPK–mediated innate immunity, functioning as a repressor of immune gene expression that undergoes a switch to an activator upon activation by PMK-1. Our data point to the regulation of the ATF2/ATF7/CREB5 family of transcriptional regulators by p38 MAPK as an ancient conserved mechanism for the control of innate immunity in metazoans and suggests a mechanism by which the protean effects of p38 MAPK on the mammalian innate immune response may be mediated.
PMCID: PMC2848548  PMID: 20369020
10.  A Polymorphism in npr-1 is a Behavioral Determinant of Pathogen Susceptibility in C. elegans 
Science (New York, N.Y.)  2009;323(5912):382-384.
The nematode Caenorhabditis elegans responds to pathogenic bacteria with conserved innate immune responses and pathogen avoidance behaviors. We investigated natural variation in C. elegans resistance to pathogen infection. With the use of quantitative genetic analysis, we determined that the pathogen susceptibility difference between the laboratory wild-type strain N2 and the wild isolate CB4856 is caused by a polymorphism in the npr-1 gene, which encodes a homolog of the mammalian neuropeptide Y receptor. We show that the mechanism of NPR-1-mediated pathogen resistance is through oxygen-dependent behavioral avoidance rather than direct regulation of innate immunity. For C. elegans, bacteria represent food but also a potential source of infection. Our data underscore the importance of behavioral responses to oxygen levels in finding an optimal balance between these potentially conflicting cues.
PMCID: PMC2748219  PMID: 19150845
11.  Transcriptional responses to pathogens in Caenorhabditis elegans 
Current opinion in microbiology  2008;11(3):251-256.
Evolutionarily conserved signaling pathways, such as the p38 and ERK MAPK pathways, the TGF-β pathway, and the insulin signaling pathway are required for resistance to pathogens in C. elegans. Recent microarray expression profiling studies have identified both candidate immune effector genes which may recognize and eliminate microbial pathogens as well as uncharacterized gene classes that are broadly induced in response to pathogen. Comparative analysis of these microarray studies is suggestive of basal versus induced components of the ancient innate immune response in C. elegans. In particular, whereas the PMK-1 p38 MAPK pathway regulates genes that are induced by pathogen, the Forkhead family transcription factor DAF-16 confers pathogen resistance through the regulation of genes that are non-overlapping with pathogen-induced genes.
PMCID: PMC2497333  PMID: 18567532
12.  Penile Corporeal Reconstruction during Difficult Placement of a Penile Prosthesis 
Advances in Urology  2008;2008:370947.
For some patients with impotence and concomitant severe tunical/corporeal tissue fibrosis, insertion of a penile prosthesis is the only option to restore erectile function. Closing the tunica over an inflatable penile prosthesis in these patients can be challenging. We review our previous study which included 15 patients with severe corporeal or tunical fibrosis who underwent corporeal reconstruction with autologous rectus fascia to allow placement of an inflatable penile prosthesis. At a mean follow-up of 18 months (range 12 to 64), all patients had a prosthesis that was functioning properly without evidence of separation, herniation, or erosion of the graft. Sexual activity resumed at a mean time of 9 weeks (range 8 to 10). There were no adverse events related to the graft or its harvest. Use of rectus fascia graft for coverage of a tunical defect during a difficult penile prosthesis placement is surgically feasible, safe, and efficacious.
PMCID: PMC2581727  PMID: 19009028
13.  Review of the Surgical Approaches for Peyronie's Disease: Corporeal Plication and Plaque Incision with Grafting 
Advances in Urology  2008;2008:263450.
The understanding and management of Peyronie's disease (PD) has improved but elucidating the exact etiology of the disease has yet to be achieved. In this paper, we review the historical and clinical aspects of PD. We focus on the evolution of surgical management for PD and review recent published articles that compare popular surgical techniques such as plication and plaque incision with vein graft. These two techniques have been reported to be equivalent with respect to patient satisfaction; however, each technique has its own advantages and disadvantages.
PMCID: PMC2581725  PMID: 19009027
14.  p38 MAPK Regulates Expression of Immune Response Genes and Contributes to Longevity in C. elegans 
PLoS Genetics  2006;2(11):e183.
The PMK-1 p38 mitogen-activated protein kinase pathway and the DAF-2–DAF-16 insulin signaling pathway control Caenorhabditis elegans intestinal innate immunity. pmk-1 loss-of-function mutants have enhanced sensitivity to pathogens, while daf-2 loss-of-function mutants have enhanced resistance to pathogens that requires upregulation of the DAF-16 transcription factor. We used genetic analysis to show that the pathogen resistance of daf-2 mutants also requires PMK-1. However, genome-wide microarray analysis indicated that there was essentially no overlap between genes positively regulated by PMK-1 and DAF-16, suggesting that they form parallel pathways to promote immunity. We found that PMK-1 controls expression of candidate secreted antimicrobials, including C-type lectins, ShK toxins, and CUB-like genes. Microarray analysis demonstrated that 25% of PMK-1 positively regulated genes are induced by Pseudomonas aeruginosa infection. Using quantitative PCR, we showed that PMK-1 regulates both basal and infection-induced expression of pathogen response genes, while DAF-16 does not. Finally, we used genetic analysis to show that PMK-1 contributes to the enhanced longevity of daf-2 mutants. We propose that the PMK-1 pathway is a specific, indispensable immunity pathway that mediates expression of secreted immune response genes, while the DAF-2–DAF-16 pathway appears to regulate immunity as part of a more general stress response. The contribution of the PMK-1 pathway to the enhanced lifespan of daf-2 mutants suggests that innate immunity is an important determinant of longevity.
The innate immune system provides the first line of defense against pathogen infection and relies upon pathways conserved across mammals, insects, and nematodes. Here, the authors have analyzed the transcriptional response of the nematode Caenorhabditis elegans to infection by the human pathogen Pseudomonas aeruginosa. They investigated this transcriptional response in the context of two conserved pathways involved in pathogen defense: the PMK-1 p38 mitogen-activated protein kinase (p38 MAPK) pathway and the DAF-2–DAF-16 insulin-signaling pathway. Specifically, the authors found that the p38 MAPK pathway plays a critical role in the infection-induced expression of secreted immune response genes. These genes include C-type lectins, lysozymes, and antimicrobial peptides that fight off infection in many species. In contrast, they found that the DAF-16 pathway is not required for immune response gene expression and may regulate immunity as part of a general stress response that functions in parallel to p38 MAPK. In addition, the authors observed that p38 MAPK contributes to the enhanced longevity of daf-2 mutants, implicating p38 MAPK signaling in the regulation of longevity, possibly through its role in immunity.
PMCID: PMC1635533  PMID: 17096597

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