SA Kostas et al. (1999) International C. elegans Meeting "Postembryonic Muscle Patterning"

SA Kostas, AZ Fire

[International C. elegans Meeting, 1999]

We are interested in understanding the genetic and molecular mechanisms required for muscle patterning. The worm has a variety of muscle types that arise through a combination of lineage-specific programs and cell-cell interactions. A subset of bodywall muscles, all of the sex muscles (uterine and vulval) and two non-muscle mesodermal cells are born postembryonically. All of these cells are derived from the lone postembryonic mesodermal blast cell, M. Characterization of several genes active in the M lineage and their mutant phenotypes (e.g. 1, 2, 3, 4) has indicated the usefulness of studying this lineage as a paradigm of mesodermal development and myogenesis. Using green fluorescent protein ( gfp ) reporters that are active in cells derived from the M lineage we examined alterations in cell pattern in a number of characterized mutant strains, and screened for new mutants with altered muscle lineages. Preliminary analysis of these mutants has indicated roles in specific cell-fate decisions within the M lineage. One surprising observation from this analysis is that mutations in mog-1, 2, 4, 6 result in a uterine to vulval muscle transformation. This phenotype is identical to that of three novel mutants isolated from our screens. An analysis of the mog-x focus of action will be described, as will the efforts to map and clone the mutants with similar phenotypes. We are interested in determining the target(s) for Mog action in sex muscle patterning. We will also describe the efforts to clone mutants involved in other M lineage cell fate decisions. Eventually we hope to apply this information to an overall understanding of mesodermal and muscle development. 1. Greenwald, I. et al., Cell 34:435-444 1983. 2. Kenyon, C. Cell 46: 477-487 1986. 3. Harfe, B. et al., Development 125: 421-429 1998. 4. Harfe, B. et al., Genes Dev. 12: 2623-2635 1998.

Kostas SA et al. (1998) East Coast Worm Meeting "Postembryonic Muscle Patterning"

Kostas SA, Fire A

[East Coast Worm Meeting, 1998]

We have used the postembryonic "M" lineage as a microcosm of mesodermal development to investigate genetic and molecular mechanisms regulating muscle patterning. The M mesoblast is born in the embryo and migrates to the posterior of the animal, where several consecutive divisions give rise to 14 body wall muscles, 2 coelomocytes (cc's), and 2 SM cells. Later, the SM's migrate toward the center of the gonad and proliferate to give rise to 8 vulval muscles and 8 uterine muscles. Using green fluorescent protein (gfp) reporters that are active in cells derived from this lineage we 1) examined alterations in cell pattern in a number of characterized mutant strains, and 2) screened for new mutants with altered muscle patterns. In these screens, we have isolated more than a dozen mutations that define at least 5 complementations groups. Our preliminary analysis of mutant phenotypes has focused on the effects on developmental decisions within the lineage (see figure). Previous studies in our lab and others have indicated a role for lin-12, mab-5, and hlh-1 in the cc/SM decision (1,2,3). We have initially focused on mutations that effect other cell fate decisions in the lineage. Efforts to map and clone egl-31, cc569, cc570, and cc571, will be reported. 1. Greenwald, I. et al., Cell 34: 435-444 1983 2. Kenyon, C. Cell 46: 477-487 1986 3. Harfe B. et al., Development, in press.

Kostas SA et al. (1997) International C. elegans Meeting "MUTANTS WITH ALTERED MUSCLE LINEAGES"

Kostas SA, Fire A

[International C. elegans Meeting, 1997]

The goal of this project is to analyze the genetic mechanisms regulating myogenic patterning. C. elegans has a diversity of muscle types that arise through a combination of lineage-specific programs and cell-cell interactions. During embryogenesis, 81 body wall muscles derive from four of the five somatic blastomeres. Additionally, 14 body wall muscles and all of the sex muscles develop post-embryonically from the M mesoblast. Since muscle number and position is invariant in normal development, we reason that mutations affecting muscle lineages will likely display altered muscle number. In order to screen for mutations affecting muscle number, we needed to efficiently count muscle cells in a live animal. We have developed a set of green fluorescent protein (gfp) constructs that allows facile counting of body wall and vulval muscles. A mixture of nuclear- and mitochondrial-localized gfp fusions was used to allow simultaneous observation of nuclei and cell outlines. Several transgenic lines were derived with this mixture. The most highly expressing was then integrated, yielding a strain with virtually uniform muscle-gfp expression. A preliminary EMS mutagenesis screen was performed on the "muscle counting" strain and several classes of mutations have been isolated. A dozen mutations that alter the vulval muscle number have been isolated; these define at least four complementation groups. One mutation has been found that affects both vulval and body wall muscle number. A few mutations have been found that give rise to extra body wall muscles without affecting the sex muscle number. We are in the process of characterizing the mutations found in this screen. Our "muscle counting" strain has also enabled us to look closely at muscle patterns in previously described mutations (largely from other labs) that might be expected to affect body wall or sex muscles. We will present our observations for these mutations as well.

Kostas SA et al. (1996) East Coast Worm Meeting "A screen for mutations that affect the founding of the germline"

Kostas SA, Fire A

[East Coast Worm Meeting, 1996]

We are interested in studying genes involved in determining cell fate during early development. The first four cleavages of the germline progenitors are asymmetric, resulting in both somatic (AB, EMS, C, and D) and gerrnline (P1-P4) daughter cells. The last division gives a body wall muscle precursor (D) and the germline precursor (P4). In normal development the adult worm has ninetyfive body wall muscles with the majority (94/95) produced by the MS, C, and D lineages. There are twenty progeny of the D blastomere, all of which form body wall muscle. An additional unequal division has been observed in the germ lineage of Parascaris univalens. This division of P. generates a germline blastomere (termed Ps) and a presumed somatic daughter (S5). We are screening for mutations that result in a change in the number of divisions leading to the germline founder cell. Initially, we will screen for viable animals with altered numbers of body wall muscle cells. The underlying expectation is that transformations leading P3 to commit early to a germ cell fate, or asymmetric P4 division to give an extra somatic founder cell would lead to altered renumbers of body wall muscles. To our knowledge, no screen has been reported which would have identified viable, nonsterile mutations with either transformation. Our mutagenic screen will be performed on transgenic lines in which muscle cells can be counted by examining expression of gfp driven by a body wall musclespecific promoter. It is critical in this screen to minimize the level of mosaicism observed in expression of the transgene. Several approaches will been combined to achieve this. We will use a promoter segment (myo-3) with minimal intrinsic variegation, include genomic DNA during transgene injection to avoid the fonnation of highly repetitive structures, and integrate the arrays. Initially, we will be screening for viable mutants, some of which may be sterile. As a control, we might expect to find mutations in several genes that affect body wall muscle cell number (eg. mes-1, lin-4). As mutations with altered muscle number are isolated they will be tested to see if there are alterations in the early germline and/or muscle founder lineages. 1 Esteban, M.R., Giovinazzo, G., Goday, C. 1995. Journal of Cell Science, 108: 23932404 2 Kelly, W., Xu, S., Fire, A. 1995. Worrn Breeders Gazette, 14(1): 6465

Kostas SA et al. (2002) Genes Dev "The T-box factor MLS-1 acts as a molecular switch during specification of nonstriated muscle in C. elegans."

Fire A, Kostas SA

[Genes Dev, 2002]

We have isolated mutations in a gene mls-1 that is required for proper specification of nonstriated muscle fates in Caenorhabditis elegans. Loss of MLS-1 activity causes uterine muscle precursors to forego their normal fates, instead differentiating as vulval muscles. We have cloned mls-1 and shown that the product is a member of the T-box family of transcriptional regulators. MLS-1 acts as a cell fate determinant in that ectopic expression can transform other cell types to uterine muscle precursors. Uterine muscle patterning is executed by regulation of MLS-1 at several different levels. The mls-1 promoter is activated by the C. elegans orthologs of Twist and Daughterless, but is only active in a subset of the lineage where these two transcription factors are present. mls-1 activity also appears to be regulated by posttranscriptional processes, as expression occurs in both uterine and vulval muscle precursors.

Prithiviraj B et al. (2005) Infect Immun "Down regulation of virulence factors of Pseudomonas aeruginosa by salicylic acid attenuates its virulence on Arabidopsis thaliana and Caenorhabditis elegans."

Bais HP, Schweizer HP, Dayakar BV, Najarro EH, Vivanco JM, Prithiviraj B, Weir T, Suresh B

[Infect Immun, 2005]

Salicylic acid (SA) is a phenolic metabolite produced by plants and is known to play an important role in several physiological processes, such as the induction of plant defense responses against pathogen attack. Here, using the Arabidopsis thaliana-Pseudomonas aeruginosa pathosystem, we provide evidence that SA acts directly on the pathogen, down regulating fitness and virulence factor production of the bacteria. Pseudomonas aeruginosa PA14 showed reduced attachment and biofilm formation on the roots of the Arabidopsis mutants lox2 and cpr5-2, which produce elevated amounts of SA, as well as on wild-type Arabidopsis plants primed with exogenous SA, a treatment known to enhance endogenous SA concentration. Salicylic acid at a concentration that did not inhibit PA14 growth was sufficient to significantly affect the ability of the bacteria to attach and form biofilm communities on abiotic surfaces. Furthermore, SA down regulated three known virulence factors of PA14: pyocyanin, protease, and elastase. Interestingly, P. aeruginosa produced more pyocyanin when infiltrated into leaves of the Arabidopsis transgenic line NahG, which accumulates less SA than wild-type plants. This finding suggests that endogenous SA plays a role in down regulating the synthesis and secretion of pyocyanin in vivo. To further test if SA directly affects the virulence of P. aeruginosa, we used the Caenorhabiditis elegans-P. aeruginosa infection model. The addition of SA to P. aeruginosa lawns significantly diminished the bacterium''s ability to kill the worms, without affecting the accumulation of bacteria inside the nematodes'' guts, suggesting that SA negatively affects factors that influence the virulence of P. aeruginosa. We employed microarray technology to identify SA target genes. These analyses showed that SA treatment affected expression of 331 genes. It selectively repressed transcription of exoproteins and other virulence factors, while it had no effect on expression of housekeeping genes. Our results indicate that in addition to its role as a signal molecule in plant defense responses, SA works as an anti-infective compound by affecting the physiology of P. aeruginosa and ultimately attenuating its virulence.

Nguyen TT et al. (2016) Aging (Albany NY) "Scavengers of reactive -ketoaldehydes extend Caenorhabditis elegans lifespan and healthspan through protein-level interactions with SIR-2.1 and ETS-7."

West JD, Zhu S, Aschner M, Roberts LJ, Fessel JP, Nguyen TT, Caito SW, Zackert WE

[Aging (Albany NY), 2016]

Isoketals (IsoKs) are highly reactive -ketoaldehyde products of lipid peroxidation that covalently adduct lysine side chains in proteins, impairing their function. Using C. elegans as a model organism, we sought to test the hypothesis that IsoKs contribute to molecular aging through adduction and inactivation of specific protein targets, and that this process can be abrogated using salicylamine (SA), a selective IsoK scavenger. Treatment with SA extends adult nematode longevity by nearly 56% and prevents multiple deleterious age-related biochemical and functional changes. Testing of a variety of molecular targets for SA's action revealed the sirtuin SIR-2.1 as the leading candidate. When SA was administered to a SIR-2.1 knockout strain, the effects on lifespan and healthspan extension were abolished. The SIR-2.1-dependent effects of SA were not mediated by large changes in gene expression programs or by significant changes in mitochondrial function. However, expression array analysis did show SA-dependent regulation of the transcription factor ets-7 and associated genes. In ets-7 knockout worms, SA's longevity effects were abolished, similar to sir-2.1 knockouts. However, SA dose-dependently increases ets-7 mRNA levels in non-functional SIR-2.1 mutant, suggesting that both are necessary for SA's complete lifespan and healthspan extension.

Prasansuklab A et al. (2017) BMC Complement Altern Med "Ethanolic extract of Streblus asper leaves protects against glutamate-induced toxicity in HT22 hippocampal neuronal cells and extends lifespan of Caenorhabditis elegans."

Tencomnao T, Prasansuklab A, Sobhon P, Meemon K

[BMC Complement Altern Med, 2017]

BACKGROUND: Although such local herb as Streblus asper (family Moraceae) has long been recognized for traditional folk medicines and important ingredient of traditional longevity formula, its anti-neurodegeneration or anti-aging activity is little known. This study aimed to investigate the neuroprotective effect of S. asper leaf extracts (SA-EE) against toxicity of glutamate-mediated oxidative stress, a crucial factor contributing to the neuronal loss in age-associated neurodegenerative diseases and the underlying mechanism as well as to evaluate its longevity effect. METHODS: Using mouse hippocampal HT22 as a model for glutamate oxidative toxicity, we carried out MTT and LDH assays including Annexin V-FITC/propidium iodide staining to determine the SA-EE effect against glutamate-induced cell death. Antioxidant activities of SA-EE were evaluated using the radical scavenging and DCFH-DA assays. To elucidate the underlying mechanisms, SA-EE treated cells were analyzed for the expressions of mRNA and proteins interested by immunofluorescent staining, western blot analysis and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) techniques. The longevity effect of SA-EE was examined on C. elegans by lifespan assay. RESULTS: We demonstrate that a concentration-dependent reduction of glutamate-induced cytotoxicity was significant after SA-EE treatment as measured by MTT and LDH assays. Annexin V-FITC/propidium iodide and immunofluorescent staining showed that co-treatment of glutamate with SA-EE significantly reduced apoptotic-inducing factor (AIF)-dependent apoptotic cell death. DCFH-DA assay revealed that this extract was capable of dose dependently attenuating the ROS caused by glutamate. Western blot analysis and qRT-PCR showed that nuclear factor erythroid 2-related factor 2 (Nrf2) protein levels in the nucleus, as well as mRNA levels of antioxidant-related genes under Nrf2 regulation were significantly increased by SA-EE. Furthermore, this extract was capable of extending the lifespan of C. elegans. CONCLUSIONS: SA-EE possesses both longevity effects and neuroprotective activity against glutamate-induced cell death, supporting its therapeutic potential for the treatment of age-associated neurodegenerative diseases.

Shamalnasab M et al. (2018) Aging Cell "A salicylic acid derivative extends the lifespan of Caenorhabditis elegans by activating autophagy and the mitochondrial unfolded protein response."

Jager S, Shamalnasab M, Breton L, Gravel SP, St-Pierre J, Aguilaniu H

[Aging Cell, 2018]

Plant extracts containing salicylates are probably the most ancient remedies to reduce fever and ease aches of all kind. Recently, it has been shown that salicylates activate adenosine monophosphate-activated kinase (AMPK), which is now considered as a promising target to slow down aging and prevent age-related diseases in humans. Beneficial effects of AMPK activation on lifespan have been discovered in the model organism Caenorhabditis elegans (C.elegans). Indeed, salicylic acid and acetylsalicylic acid extend lifespan in worms by activating AMPK and the forkhead transcription factor DAF-16/FOXO. Here, we investigated whether another salicylic acid derivative 5-octanoyl salicylic acid (C8-SA), developed as a controlled skin exfoliating ingredient, had similar properties using C. elegans as a model. We show that C8-SA increases lifespan of C. elegans and that a variety of pathways and genes are required for C8-SA-mediated lifespan extension. C8-SA activates AMPK and inhibits TOR both in nematodes and in primary human keratinocytes. We also show that C8-SA can induce both autophagy and the mitochondrial unfolded protein response (UPR^mit ) in nematodes. This induction of both processes is fully required for lifespan extension in the worm. In addition, we found that the activation of autophagy by C8-SA fails to occur in worms with compromised UPR^mit , suggesting a mechanistic link between these two processes. Mutants that are defective in the mitochondrial unfolded protein response exhibit constitutive high autophagy levels. Taken together, these data therefore suggest that C8-SA positively impacts longevity in worms through induction of autophagy and the UPR^mit .

Penna B et al. (2021) Sci Rep "Comparative genomics of MRSA strains from human and canine origins reveals similar virulence gene repertoire."

Silva-Carvalho MC, Ferreira FA, Bandeira PT, Figueiredo AMS, Vieira-da-Motta O, Silva MB, Vasconcelos ATR, de Souza VS, Botelho AMN, Planet PJ, de Sousa VS, Soares AER, Rabello RF, Ramundo MS, Penna B

[Sci Rep, 2021]

Methicillin-resistant Staphylococcus aureus (MRSA) is an important pathogen associated with a wide variety of infections in humans. The ability of MRSA to infect companion animals has gained increasing attention in the scientific literature. In this study, 334 dogs were screened for MRSA in two cities located in Rio de Janeiro State. The prevalence of MRSA in dogs was 2.7%. Genotyping revealed isolates from sequence types (ST) 1, 5, 30, and 239 either colonizing or infecting dogs. The genome of the canine ST5 MRSA (strain SA112) was compared with ST5 MRSA from humans-the main lineage found in Rio de Janeiro hospitals-to gain insights in the origin of this dog isolate. Phylogenetic analysis situated the canine genome and human strain CR14-035 in the same clade. Comparative genomics revealed similar virulence profiles for SA112 and CR14-035. Both genomes carry S. aureus genomic islands SA, SA, and SA. The virulence potential of the canine and human strains was similar in a Caenorhabditis elegans model. Together, these results suggest a potential of canine MRSA to infect humans and vice versa. The circulation in community settings of a MRSA lineage commonly found in hospitals is an additional challenge for public health surveillance authorities.