Preputial lymphangioma in a stallion: First report.

This short communication describes a case of a 30-year-old stallion with a mass on the base of the penis causing paraphimosis. The patient was submitted to anti-inflammatory and diuretic therapy with no signs of improvement, so 16 days after the lesion was detected, the animal was euthanized. Necropsy was performed, and histopathological assessment of the lesion was conducted. The mass was composed primarily of channels and cavernous structures, lined by elongated cells of vascular origin, located in the preputium. The lesion was diagnosed as a preputial lymphangioma. To the authors' best knowledge, the anatomical location of this neoplasm (which is rare in veterinary medicine) has not been previously reported.

Fatal congenital and traumatic cervical spine injuries in a captive newborn plains zebra (Equus quagga).

Background: In this report, we describe the clinical, macro-, and microscopic findings of a newborn zebra victim of a fatal trauma and its possible causes are discussed in detail. Case Description: A plains zebra (Equus quagga) born in a Portuguese zoo was found in hypothermia and unable to get up. The animal was admitted to the hospital 36 hours later and, after the first hour of treatment and no attempts to get up, it began to convulse and died. At necropsy, subdural hematoma and atlantoaxial dislocation were the main findings. Conclusion: The inability to pinpoint the exact moment of the lesions' onset determines the importance of a thorough surveillance of the periparturient period and parturition of captive animals. Furthermore, literature concerning zebras, their reproduction, and neonatal period is scarce, reinforcing the need to report these cases.

Selective Persulfide Detection Reveals Evolutionarily Conserved Antiaging Effects of S-Sulfhydration.

Life on Earth emerged in a hydrogen sulfide (H2S)-rich environment eons ago and with it protein persulfidation mediated by H2S evolved as a signaling mechanism. Protein persulfidation (S-sulfhydration) is a post-translational modification of reactive cysteine residues, which modulate protein structure and/or function. Persulfides are difficult to label and study due to their reactivity and similarity with cysteine. Here, we report a facile strategy for chemoselective persulfide bioconjugation using dimedone-based probes, to achieve highly selective, rapid, and robust persulfide labeling in biological samples with broad utility. Using this method, we show persulfidation is an evolutionarily conserved modification and waves of persulfidation are employed by cells to resolve sulfenylation and prevent irreversible cysteine overoxidation preserving protein function. We report an age-associated decline in persulfidation that is conserved across evolutionary boundaries. Accordingly, dietary or pharmacological interventions to increase persulfidation associate with increased longevity and improved capacity to cope with stress stimuli.

Purine Homeostasis Is Necessary for Developmental Timing, Germline Maintenance and Muscle Integrity in Caenorhabditis elegans.

Purine homeostasis is ensured through a metabolic network widely conserved from prokaryotes to humans. Purines can either be synthesized de novo, reused, or produced by interconversion of extant metabolites using the so-called recycling pathway. Although thoroughly characterized in microorganisms, such as yeast or bacteria, little is known about regulation of the purine biosynthesis network in metazoans. In humans, several diseases are linked to purine metabolism through as yet poorly understood etiologies. Particularly, the deficiency in adenylosuccinate lyase (ADSL)-an enzyme involved both in the purine de novo and recycling pathways-causes severe muscular and neuronal symptoms. In order to address the mechanisms underlying this deficiency, we established Caenorhabditis elegans as a metazoan model organism to study purine metabolism, while focusing on ADSL. We show that the purine biosynthesis network is functionally conserved in C. elegans Moreover, adsl-1 (the gene encoding ADSL in C. elegans) is required for developmental timing, germline stem cell maintenance and muscle integrity. Importantly, these traits are not affected when solely the de novo pathway is abolished, and we present evidence that germline maintenance is linked specifically to ADSL activity in the recycling pathway. Hence, our results allow developmental and tissue specific phenotypes to be ascribed to separable steps of the purine metabolic network in an animal model.

An automated method for the analysis of food intake behaviour in Caenorhabditis elegans.

The study of mechanisms that govern feeding behaviour and its related disorders is a matter of global health interest. The roundworm Caenorhabditis elegans is becoming a model organism of choice to study these conserved pathways. C. elegans feeding depends on the contraction of the pharynx (pumping). Thanks to the worm transparency, pumping can be directly observed under a stereoscope. Therefore, C. elegans feeding has been historically investigated by counting pharyngeal pumping or by other indirect approaches. However, those methods are short-term, time-consuming and unsuitable for independent measurements of sizable numbers of individuals. Although some particular devices and long-term methods have been lately reported, they fail in the automated, scalable and/or continuous aspects. Here we present an automated bioluminescence-based method for the analysis and continuous monitoring of worm feeding in a multi-well format. We validate the method using genetic, environmental and pharmacological modulators of pharyngeal pumping. This flexible methodology allows studying food intake at specific time-points or during longer periods of time, in single worms or in populations at any developmental stage. Additionally, changes in feeding rates in response to differential metabolic status or external environmental cues can be monitored in real time, allowing accurate kinetic measurements.

Microtubule severing by the katanin complex is activated by PPFR-1-dependent MEI-1 dephosphorylation.

Katanin is an evolutionarily conserved microtubule (MT)-severing complex implicated in multiple aspects of MT dynamics. In Caenorhabditis elegans, the katanin homologue MEI-1 is required for meiosis, but must be inactivated before mitosis. Here we show that PPFR-1, a regulatory subunit of a trimeric protein phosphatase 4 complex, enhanced katanin MT-severing activity during C. elegans meiosis. Loss of ppfr-1, similarly to the inactivation of MT severing, caused a specific defect in meiosis II spindle disassembly. We show that a fraction of PPFR-1 was degraded after meiosis, contributing to katanin inactivation. PPFR-1 interacted with MEL-26, the substrate recognition subunit of the CUL-3 RING E3 ligase (CRL3(MEL-26)), which also targeted MEI-1 for post-meiotic degradation. Reversible protein phosphorylation of MEI-1 may ensure temporal activation of the katanin complex during meiosis, whereas CRL3(MEL-26)-mediated degradation of both MEI-1 and its activator PPFR-1 ensure efficient katanin inactivation in the transition to mitosis.

The CRL2LRR-1 ubiquitin ligase regulates cell cycle progression during C. elegans development.

The molecular mechanisms that regulate cell cycle progression in a developmental context are poorly understood. Here, we show that the leucine-rich repeat protein LRR-1 promotes cell cycle progression during C. elegans development, both in the germ line and in the early embryo. Our results indicate that LRR-1 acts as a nuclear substrate-recognition subunit of a Cullin 2-RING E3 ligase complex (CRL2(LRR-1)), which ensures DNA replication integrity. LRR-1 contains a typical BC/Cul-2 box and binds CRL2 components in vitro and in vivo in a BC/Cul-2 box-dependent manner. Loss of lrr-1 function causes cell cycle arrest in the mitotic region of the germ line, resulting in sterility due to the depletion of germ cells. Inactivation of the DNA replication checkpoint signaling components ATL-1 and CHK-1 suppresses this cell cycle arrest and, remarkably, restores lrr-1 mutant fertility. Likewise, in the early embryo, loss of lrr-1 function induces CHK-1 phosphorylation and a severe cell cycle delay in P lineage division, causing embryonic lethality. Checkpoint activation is not constitutive in lrr-1 mutants but is induced by DNA damage, which may arise due to re-replication of some regions of the genome as evidenced by the accumulation of single-stranded DNA-replication protein A (ssDNA-RPA-1) nuclear foci and the increase in germ cell ploidy in lrr-1 and lrr-1; atl-1 double mutants, respectively. Collectively, these observations highlight a crucial function of the CRL2(LRR-1) complex in genome stability via maintenance of DNA replication integrity during C. elegans development.

Using RNA interference to identify specific modifiers of a temperature-sensitive, embryonic-lethal mutation in the Caenorhabditis elegans ubiquitin-like Nedd8 protein modification pathway E1-activating gene rfl-1.

The essential Caenorhabditis elegans gene rfl-1 encodes one subunit of a heterodimeric E1-activating enzyme in the Nedd8 ubiquitin-like protein conjugation pathway. This pathway modifies the Cullin scaffolds of E3 ubiquitin ligases with a single Nedd8 moiety to promote ligase function. To identify genes that influence neddylation, we used a synthetic screen to identify genes that, when depleted with RNAi, enhance or suppress the embryonic lethality caused by or198ts, a temperature-sensitive (ts) mutation in rfl-1. We identified reproducible suppressor and enhancer genes and employed a systematic specificity analysis for each modifier using four unrelated ts embryonic lethal mutants. Results of this analysis highlight the importance of specificity controls in identifying genetic interactions relevant to a particular biological process because 8/14 enhancers and 7/21 suppressors modified lethality in other mutants. Depletion of the strongest specific suppressors rescued the early embryonic cell division defects in rfl-1(or198ts) mutants. RNAi knockdown of some specific suppressors partially restored Cullin neddylation in rfl-1(or198ts) mutants, consistent with their gene products normally opposing neddylation, and GFP fusions to several suppressors were detected in the cytoplasm or the nucleus, similar in pattern to Nedd8 conjugation pathway components in early embryonic cells. In contrast, depletion of the two strongest specific enhancers did not affect the early embryonic cell division defects observed in rfl-1(or198ts) mutants, suggesting that they may act at later times in other essential processes. Many of the specific modifiers are conserved in other organisms, and most are nonessential. Thus, when controlled properly for specificity, modifier screens using conditionally lethal C. elegans mutants can identify roles for nonessential but conserved genes in essential processes.

Van Gogh and Frizzled act redundantly in the Drosophila sensory organ precursor cell to orient its asymmetric division.

Drosophila sensory organ precursor cells (SOPs) divide asymmetrically along the anterior-posterior (a-p) body axis to generate two different daughter cells. Planar Cell Polarity (PCP) regulates the a-p orientation of the SOP division. The localization of the PCP proteins Van Gogh (Vang) and Frizzled (Fz) define anterior and posterior apical membrane domains prior to SOP division. Here, we investigate the relative contributions of Vang, Fz and Dishevelled (Dsh), a membrane-associated protein acting downstream of Fz, in orienting SOP polarity. Genetic and live imaging analyses suggest that Dsh restricts the localization of a centrosome-attracting activity to the anterior cortex and that Vang is a target of Dsh in this process. Using a clone border assay, we provide evidence that the Vang and fz genes act redundantly in SOPs to orient its polarity axis in response to extrinsic local PCP cues. Additionally, we find that the activity of Vang is dispensable for the non-autonomous polarizing activity of fz. These observations indicate that both Vang and Fz act as cues for downstream effectors orienting the planar polarity axis of dividing SOPs.

The role of protein phosphatase 4 in regulating microtubule severing in the Caenorhabditis elegans embryo.

MEI-1, the catalytic subunit of the Caenorhabditis elegans "katanin" microtubule-severing complex, is required for meiotic spindle formation. However, MEI-1 must be inactivated after the completion of meiosis to allow formation of the first mitotic spindle. Recent work demonstrated that post-meiotic MEI-1 undergoes ubiquitin-dependent degradation mediated by two independent pathways. Here we describe another level of MEI-1 regulation involving the protein phosphatase 4 (PP4) complex. The PP4 R1 regulatory subunit protein phosphatase four regulatory subunit 1 (ppfr-1) was identified in an RNA interference (RNAi) screen for suppressors of a mei-1(gf) allele that is refractory to post-meiotic degradation. RNAi to the PP4 catalytic subunit PPH-4.1 or to the alpha4 regulatory PPFR-4 also suppressed lethality of ectopic MEI-1. These results suggest that PP4(+) activates MEI-1, and therefore loss of PP4 decreases ectopic MEI-1(gf) activity. PPH-4.1 and MEI-1 co-immunoprecipitate with one another, indicating that the PP4 complex likely regulates MEI-1 activity directly rather than through an intermediate. The ppfr-1 mutant has subtle meiotic defects indicating that PPFR-1 also regulates MEI-1 during meiosis. MBK-2 is the only kinase known to phosphorylate MEI-1 and triggers post-meiotic MEI-1 degradation. However, genetic interactions between PP4 and mbk-2 were not consistent with an antagonistic relationship between the phosphatase and kinase. Additionally, reducing PP4 in mei-1(gf) did not change the level or localization of post-meiotic MEI-1. Thus, by making use of a genetic background where MEI-1 is ectopically expressed, we have uncovered a third mechanism of MEI-1 regulation, one based on phosphorylation but independent of degradation. The redundant regulatory pathways likely contribute in different ways to the rapid and precise post-meiotic inactivation of MEI-1 microtubule-severing activity.

Heads or tails: cell polarity and axis formation in the early Caenorhabditis elegans embryo.

In C. elegans, the first embryonic axis is established shortly after fertilization and requires both the microtubule and microfilament cytoskeleton. Cues from sperm-donated centrosomes result in a cascade of events that polarize the distribution of widely conserved PAR proteins at the cell cortex. The PAR proteins in turn polarize the cytoplasm and position mitotic spindles. Lessons learned from C. elegans should improve our understanding of how cells become polarized and divide asymmetrically during development.