Elucidating the Regulation of the Metaphase-to-Anaphase Transition During Mitosis in Candida albicans

Candida albicans is an important fungal pathogen of humans. Understanding the regulation of its cell cycle and mechanisms governing the metaphase-to-anaphase transition may reveal new targets for therapeutic development. This process is dependent on several factors including; separase, a conserved cohesin protease, the separase regulator securin, the Anaphase Promoting Complex/Cyclosome (APC/C) and its cofactors Cdc20p and Cdh1p, as well as the cohesin complex. Although Cdc20p and Cdh1p have been previously characterized in C. albicans, a detailed framework of metaphase-to-anaphase progression remains elusive. We provide the first characterization of separase in C. albicans, and demonstrate its putative interactors, collectively implying conserved and novel functions. We hypothesized one putative interactor; a divergent securin called Eip1p (Esp1-Interacting Protein 1). Subsequent characterization demonstrated that Eip1p is Candida-specific, important for chromosome segregation, and exhibited conserved securin-like features including stabilization in the presence of DNA damage/stress agents. Moreover, Eip1p depletion partially suppressed a metaphase block induced by these agents. Eip1p was reduced upon Cdc5p depletion, suggesting regulation via degradation, a diagnostic feature of securins. A proportion of Eip1p-depleted cells also exhibited novel phenotypes including misoriented spindles and maintenance of elongated spindles. However, Eip1p was not consistently or strongly enriched in the absence of Cdc20p unlike other securins, and its mechanisms of action remained unclear. Since Eip1p is functioning like a securin, we hypothesized that its regulation includes degradation mediated by the APC/C Cdc20p, and inconsistent modulation in Cdc20p-depleted cells may be due to an incomplete Cdc20p-arrest phenotype. To test this, we investigated the APC/C subunit Cdc16p and provide the first characterization of an APC/C subunit in C. albicans. Bioinformatic analyses revealed sequence conservation of Cdc16p. Its depletion caused an early mitotic arrest, and showed enrichment of the mitotic cyclin Clb2p. Eip1p was strongly enriched upon Cdc16p depletion, suggesting conserved function of Cdc16p in C. albicans. Mechanisms of action of Eip1p were addressed through affinity purification of Eip1p and mass spectrometry, revealing some unique putative protein interactors. Overall, we present evidence of a new securin, a potential drug target, and novel insights on mitotic regulation in C. albicans

Characterization of a putative separase Esp1p and a novel interacting protein in regulating mitosis in the fungal pathogen Candida albicans

C. albicans is an important fungal pathogen of humans, and an understanding of the regulation of its cell cycle may reveal new targets for anti-fungal therapies. We previously characterized the C. albicans Anaphase Promoting Complex/Cyclosome regulatory co-factors Cdc20p and Cdh1p, and showed that they are important for the metaphase-to-anaphase transition and mitotic exit. In order to determine the mechanisms of action of Cdc20p, we searched the genome for factors that bind it in other systems, and found that C. albicans lacks the conserved Cdc20p target, securin. Securins bind and prevent separase from cleaving cohesin, and must be targeted for degradation by Cdc20p for separase-dependent sister chromatid separation. We hypothesized that C. albicans contains a divergent securin, which may be uncovered by identifying proteins that bind separase. We demonstrated that the C. albicans separase homologue, ESP1, is required for chromosome segregation. We then identified Esp1p-interacting factors using affinity purification and mass spectrometry, and uncovered Orf19.955p, a protein of unknown function with homologues only in Candida species. Orf19.955p contains Destruction and a KEN box, like other securins. While not essential, depletion impairs cell growth and phenotype. Chromosomes could segregate; unlike in Esp1p-depleted cells, but chromosome organization and microtubules were adversely affected. In contrast, depletion of securin Pds1p in S. cerevisiae prevented chromosome separation and phenocopied absence of Esp1p. Future experiments will clarify if Orf19.955p is a securin. Collectively our results provide new insights on factors involved in the metaphase-to-anaphase transition, and thus cell proliferation in C. albicans, including a novel fungal-specific protein

The biology of vasopressin

Here, we review the biology of AVP through examination of its normal physiological roles in kidney and heart, neurological and behavioral effects, and of how AVP dysfunction contributes to pathologies such as polycystic kidney disease (PKD) and heart failure (HF), and the pharmacological manipulation of AVP-dependent pathways in these diseases

The impact of anaphylaxis on the absorption of intranasal epinephrine in anaesthetized non-naive beagle dogs

Background: Epinephrine delivery via an intranasal spray (neffy) is being evaluated as an additional option to treat severe allergic reaction and may provide clinical benefit by reducing the time to dosing in community settings by avoiding needles. Given that hypotension is a hallmark symptom of severe allergic reactions, a preclinical study was conducted to evaluate the impact of this factor on epinephrine absorption via neffy. Objective: The objective of this study was to evaluate the absorption of epinephrine via neffy in a dog model of anaphylaxis with severe hypotension. Methods: Epinephrine absorption via neffy was evaluated in anesthetized beagle dogs under both normal conditions and hypotension associated with anaphylaxis. A total of 14 dogs (10 males and 4 females) were dosed with neffy, 1.0 mg, under normal conditions, followed by neffy, 1.0 mg, under conditions of anaphylaxis. Results: The mean maximum concentration of epinephrine was higher during anaphylaxis than under normal conditions (2,670 ± 2,150 pg/mL and 1,330 ± 739 pg/mL [P < .05]). Relative to normal conditions, anaphylaxis resulted in higher overall epinephrine exposure (area under the curve from 0 to 45 minutes = 54,400 ± 18,100 min × pg/mL and 34,300 ± 21,500 minutes × pg/mL [P < .05]), which is likely due to the increase in vascular permeability commonly observed during severe allergic reactions. Conclusion: Taken together with real-world evidence from nasal naloxone treatment for opioid overdose demonstrating that the reduced blood flow or hypotension associated with overdose does not appear to suppress naloxone’s efficacy, the current findings demonstrate that epinephrine is well absorbed following neffy delivery during the hypotension associated with severe anaphylaxis reactions

Self-regulation predicts companionship in children with autism

 Self-regulation is associated with many positive outcomes in children with and without autism, including increased mental health and academic achievement, and decreased problem behavior. Less is known regarding whether and how self-regulation and symptoms of mental health challenges (internalizing and externalizing problems) relate to social outcomes, such as friendship quality and loneliness. Parents and teachers of 106 children with autism aged 5-12 reported on children's self-regulation difficulties and externalizing and internalizing symptoms. Four-to-five months later, children reported on the quality of their friendship with their best friend (companionship, conflict, helpfulness, sense of relationship security, closeness), and their feelings of loneliness. Linear regression was used to examine the effects of self-regulation and symptoms of mental health challenges on friendship quality and loneliness. Less self-regulation difficulties predicted stronger companionship and girls had better quality friendships with their best friend than did boys, in terms of companionship, helpfulness, security and closeness, confirming that they have a protective advantage in friendship development. Autism symptoms, IQ, and age were not associated with friendship quality or loneliness. Results highlight the importance of self-regulation and mental health interventions for school-aged children with autism

The Biology of Vasopressin

Vasopressins are evolutionarily conserved peptide hormones. Mammalian vasopressin functions systemically as an antidiuretic and regulator of blood and cardiac flow essential for adapting to terrestrial environments. Moreover, vasopressin acts centrally as a neurohormone involved in social and parental behavior and stress response. Vasopressin synthesis in several cell types, storage in intracellular vesicles, and release in response to physiological stimuli are highly regulated and mediated by three distinct G protein coupled receptors. Other receptors may bind or cross-bind vasopressin. Vasopressin is regulated spatially and temporally through transcriptional and post-transcriptional mechanisms, sex, tissue, and cell-specific receptor expression. Anomalies of vasopressin signaling have been observed in polycystic kidney disease, chronic heart failure, and neuropsychiatric conditions. Growing knowledge of the central biological roles of vasopressin has enabled pharmacological advances to treat these conditions by targeting defective systemic or central pathways utilizing specific agonists and antagonists