miR-786 Regulation of a Fatty-Acid Elongase Contributes to Rhythmic Calcium-Wave Initiation in \u3cem\u3eC. elegans\u3c/em\u3e

Background: Rhythmic behaviors are ubiquitous phenomena in animals. In C. elegans, defecation is an ultradian rhythmic behavior: every ∼50 s a calcium wave initiating in the posterior intestinal cells triggers the defecation motor program that comprises three sequential muscle contractions. Oscillatory calcium signaling is central to the periodicity of defecation. The posteriormost intestinal cells function as the pacemaker for this rhythmic behavior, although it is unclear how the supremacy of these cells for calcium-wave initiation is controlled. Results: We describe how the loss of the mir-240/786 microRNA cluster, which results in arrhythmic defecation, causes ectopic intestinal calcium-wave initiation. mir-240/786 expression in the intestine is restricted to the posterior cells that function as the defecation pacemaker. Genetic data indicate that mir-240/786 functions upstream of the inositol 1,4,5-trisphosphate (IP3) receptor. Through rescue analysis, it was determined that miR-786 functions to regulate defecation. Furthermore, we identified elo-2, a fatty-acid elongase with a known role in defecation cycling, as a direct target for miR-786. We propose that the regulation of palmitate levels through repression of elo-2 activity is the likely mechanistic link to defecation. Conclusions: Together, these data indicate that miR-786 confers pacemaker status on posterior intestinal cells for the control of calcium-wave initiation through the regulation of elo-2 and, subsequently, palmitate levels. We propose that a difference in fatty-acid composition in the posterior intestinal cells may alter the activities of membrane proteins, such as IP3-receptor or TRPM channels, that control pacemaker activity in the C. elegans intestine

The inositol 1,4,5-trisphosphate receptor in C. elegans

The soil nematode Caenorhabditis elegans is a genetic model organism whose cellular physiology is closely related to that of mammals, with many signaling cascades and second messengers mirroring those found in higher organisms. Due to the genetic, anatomical, and behavioral simplicity of worms, integrative physiological techniques are relatively straightforward and represent a powerful approach to understand the molecular mechanisms underlying more complex system functions. Studies of the nematode inositol 1,4,5-trisphosphate receptor (InsP 3 R) have led to advances in our understanding of its role in development and behavior

Artemisinin-resistant K13 mutations rewire Plasmodium falciparum's intra-erythrocytic metabolic program to enhance survival

The emergence and spread of artemisinin resistance, driven by mutations in Plasmodium falciparum K13, has compromised antimalarial efficacy and threatens the global malaria elimination campaign. By applying systems-based quantitative transcriptomics, proteomics, and metabolomics to a panel of isogenic K13 mutant or wild-type P. falciparum lines, we provide evidence that K13 mutations alter multiple aspects of the parasite's intra-erythrocytic developmental program. These changes impact cell-cycle periodicity, the unfolded protein response, protein degradation, vesicular trafficking, and mitochondrial metabolism. K13-mediated artemisinin resistance in the Cambodian Cam3.II line was reversed by atovaquone, a mitochondrial electron transport chain inhibitor. These results suggest that mitochondrial processes including damage sensing and anti-oxidant properties might augment the ability of mutant K13 to protect P. falciparum against artemisinin action by helping these parasites undergo temporary quiescence and accelerated growth recovery post drug elimination

Increased circulation time of Plasmodium falciparum underlies persistent asymptomatic infection in the dry season

The dry season is a major challenge for Plasmodium falciparum parasites in many malaria endemic regions, where water availability limits mosquito vectors to only part of the year. How P. falciparum bridges two transmission seasons months apart, without being cleared by the human host or compromising host survival, is poorly understood. Here we show that low levels of P. falciparum parasites persist in the blood of asymptomatic Malian individuals during the 5- to 6-month dry season, rarely causing symptoms and minimally affecting the host immune response. Parasites isolated during the dry season are transcriptionally distinct from those of individuals with febrile malaria in the transmission season, coinciding with longer circulation within each replicative cycle of parasitized erythrocytes without adhering to the vascular endothelium. Low parasite levels during the dry season are not due to impaired replication but rather to increased splenic clearance of longer-circulating infected erythrocytes, which likely maintain parasitemias below clinical and immunological radar. We propose that P. falciparum virulence in areas of seasonal malaria transmission is regulated so that the parasite decreases its endothelial binding capacity, allowing increased splenic clearance and enabling several months of subclinical parasite persistence

Integrated calcium and proton signaling during a rhythmic behavior in C. elegans

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Pharmacology and Physiology, 2014.Biological rhythms are intrinsic to nearly all organisms; yet, they can also be very complex. The genetic model organism C. elegans exhibits several behavioral rhythms that have been studied using integrative physiology. One rhythm in particular, the defecation motor program (DMP), has been shown to be timed by cell-autonomous calcium oscillations. Subsequent trans-epithelial proton fluxes contribute to the DMP’s behavioral output as well as to physiologic events such as development and lifespan. In addition to defining mechanisms that contribute to oscillatory calcium signaling, studies focused on this behavior have emphasized the importance of pH homeostasis and have led to the identification of a novel role for protons in signaling. The work presented here demonstrates that the establishment of an apical intestinal proton gradient, driven by a proton V-ATPase, is necessary for nutrient uptake and development. Proton movement across the basolateral membrane, through the Na+/H+ exchanger NHX-7, is also critical for proper execution of the DMP but not required for pH homeostasis or development. This flux signals posterior body wall muscle contraction and coincides with the underlying IP3R-mediated calcium wave, suggesting calcium-dependent regulation. Structure-function analyses of NHX-7 revealed that its activity is impacted by calcium signaling and feedback from the membrane proton/sodium gradient. Interestingly, the structure-function analysis also indicated that disruption of a consensus binding site for a calcineurin homologous protein, PBO-1, altered the distribution of NHX-7. Genetic analyses further revealed that loss of PBO-1 also affects the localization of the NHX-2 isoform in the apical membrane, which helps to reestablish pH homeostasis following defecation. This ultimately results in a phenotype that resembles a global disruption of intestinal proton transport. The localization deficit appears to be an effect on membrane stability/retention rather than forward protein trafficking. Collectively, these studies support the idea that proton and calcium oscillations synergize to execute the DMP as a means to maintain the intestinal membrane proton motive force. Thus, in addition to contributing to our understanding of oscillatory calcium signaling, pH homeostasis, and proton signaling, our results show that the defecation motor program is an appropriate model to study and potentially target biological rhythms therapeutically

Calcineurin homologous proteins regulate the membrane localization and activity of sodium/proton exchangers in C. elegans

Calcineurin B homologous proteins (CHP) are N-myristoylated, EF-hand Ca2+-binding proteins that bind to and regulate Na+/H+ exchangers, which occurs through a variety of mechanisms whose relative significance is incompletely understood. Like mammals, Caenorhabditis elegans has three CHP paralogs, but unlike mammals, worms can survive CHP loss-of-function. However, mutants for the CHP ortholog PBO-1 are unfit, and PBO-1 has been shown to be required for proton signaling by the basolateral Na+/H+ exchanger NHX-7 and for proton-coupled intestinal nutrient uptake by the apical Na+/H+ exchanger NHX-2. Here, we have used this genetic model organism to interrogate PBO-1\u27s mechanism of action. Using fluorescent tags to monitor Na+/H+ exchanger trafficking and localization, we found that loss of either PBO-1 binding or activity caused NHX-7 to accumulate in late endosomes/lysosomes. In contrast, NHX-2 was stabilized at the apical membrane by a nonfunctional PBO-1 protein and was only internalized following its complete loss. Additionally, two pbo-1 paralogs were identified, and their expression patterns were analyzed. One of these contributed to the function of the excretory cell, which acts like a kidney in worms, establishing an alternative model for testing the role of this protein in membrane transporter trafficking and regulation. These results lead us to conclude that the role of CHP in Na+/H+ exchanger regulation differs between apical and basolateral transporters. This further emphasizes the importance of proper targeting of Na+/H+ exchangers and the critical role of CHP family proteins in this process

Calcineurin homologous proteins regulate the membrane localization and activity of sodium/proton exchangers in C. elegans

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Age validation of yellowfin (Thunnus albacares) and bigeye (Thunnus obesus) tuna of the northwestern Atlantic Ocean

Estimates of age and growth of yellowfin (Thunnus albacares) and bigeye (Thunnus obesus) tuna remain problematic because validation of growth zone deposition (opaque and translucent) has not been properly evaluated. Otolith growth structure (zone clarity) can be poorly defined for tropical tunas, but the use of bomb radiocarbon dating has validated age estimates to 16–18 years for yellowfin and bigeye tuna. Use of the radiocarbon decline period — defined by regional coral and otoliths — provided valid ages through ontogeny. Yellowfin tuna aged 2–18 years (n = 34, 1029–1810 mm FL) and bigeye tuna aged 3–17 years (n = 12, 1280–1750 mm FL) led to birth years that were coincident with the bomb radiocarbon decline. The results indicate there was no age reading bias for yellowfin tuna and that age estimates of previous studies were likely underestimated for both species.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

A Calcineurin Homologous Protein Is Required For Sodium-proton Exchange Events In The C. Elegans Intestine

Caenorhabditis elegans defecation is a rhythmic behavior, composed of three sequential muscle contractions, with a 50-s periodicity. The motor program is driven by oscillatory calcium signaling in the intestine. Proton fluxes, which require sodium-proton exchangers at the apical and basolateral intestinal membranes, parallel the intestinal calcium flux. These proton shifts are critical for defecation-associated muscle contraction, nutrient uptake, and longevity. How sodium-proton exchangers are activated in time with intestinal calcium oscillation is not known. The posterior body defecation contraction mutant (pbo-1) encodes a calcium-binding protein with homology to calcineurin homologous proteins, which are putative cofactors for mammalian sodium-proton exchangers. Loss of pbo-1 function results in a weakened defecation muscle contraction and a caloric restriction phenotype. Both of these phenotypes also arise from dysfunctions in pH regulation due to mutations in intestinal sodium-proton exchangers. Dynamic, in vivo imaging of intestinal proton flux in pbo-1 mutants using genetically encoded pH biosensors demonstrates that proton movements associated with these sodium-proton exchangers are significantly reduced. The basolateral acidification that signals the first defecation motor contraction is scant in the mutant compared with a normal animal. Luminal and cytoplasmic pH shifts are much reduced in the absence of PBO-1 compared with control animals. We conclude that pbo-1 is required for normal sodium-proton exchanger activity and may couple calcium and proton signaling events