185 research outputs found
Latrunculin A delays anaphase onset in fission yeast by disrupting an ase1-independent pathway controlling mitotic spindle stability
No full textIt has been proposed previously that latrunculin A, an inhibitor of actin polymerization, delays the onset of anaphase by causing spindle misorientation in fission yeast. However, we show that {Delta}mto1 cells, which are defective in nucleation of cytoplasmic microtubules, have profoundly misoriented spindles but are not delayed in the timing of sister chromatid separation, providing compelling evidence that fission yeast does not possess a spindle orientation checkpoint. Instead, we show that latrunculin A delays anaphase onset by disrupting interpolar microtubule stability. This effect is abolished in a latrunculin A-insensitive actin mutant and exacerbated in cells lacking Ase1, which cross-links antiparallel interpolar microtubules at the spindle midzone both before and after anaphase. These data indicate that both Ase1 and an intact actin cytoskeleton are required for preanaphase spindle stability. Finally, we show that loss of Ase1 activates a checkpoint that requires only the Mad3, Bub1, and Mph1, but not Mad1, Mad2, or Bub3 checkpoint proteins
Precocious activation of APC/C-Cdh1 at pre-anaphase causes genome instability
No full textFaithful chromosome segregation and thereby accurate gene transmission are crucial for all organisms. Until proper attachment of the mitotic spindle to the kinetochore is established, the ubiquitin ligase (E3) Cdc20-activated APC/C (anaphase promoting complex/cyclosome) is repressed by the spindle assembly checkpoint (SAC) and sister chromatin cohesion is protected. Mutants defective in SAC fail to arrest at metaphase even in the presence of damaged microtubules. Interestingly, a similar phenomenon occurs in yeast cells defective in Bub2, a negative factor of the mitotic exit network (MEN), which is required for telophase onset, although its precise molecular mechanism is unknown. Here, we show that chromosome missegregation occurs frequently in bub2∆ cells in the presence of damaged microtubules. The loss of Bub2 caused precocious activation of APC/C-Cdh1/Hct1 at pre-anaphase, leading to securin degradation and then separase-mediated cohesin cleavage. Overexpression of CDH1 and CDC14, encoding Cdc14 phosphatase, at pre-anaphase similarly caused chromosome missegregation. Thus, sequential activation of APC/C-Cdc20 and then APC/C-Cdh1 is critical for precise chromosome segregation and precocious activation of APC/C-Cdh1 at pre-anaphase causes genomic instability. Since degradation of human securin is also mediated by APC/C-Cdc20 and APC/C-Cdh1, this study predicts that precocious activation APC/C-Cdh1 in human cells similarly causes genomic instability, and thereby cell death or tumorigenesis
TACC3-ch-TOG track the growing tips of microtubules independently of clathrin and Aurora-A phosphorylation
No full textThe interaction between TACC3 (transforming acidic coiled coil protein 3) and the microtubule polymerase ch-TOG (colonic, hepatic tumor overexpressed gene) is evolutionarily conserved. Loading of TACC3–ch-TOG onto spindle microtubules requires the phosphorylation of TACC3 by Aurora-A kinase and the subsequent interaction of TACC3 with clathrin to form a microtubule binding surface. Whether there is a pool of TACC3–ch-TOG that is independent of clathrin in human cells, and what is the function of this pool, are open questions. Here, we report that TACC3 is recruited to the plus-ends of microtubules by its association with ch-TOG and that this pool is independent of phosphorylation and binding to clathrin. The plus-end binding of TACC3–ch-TOG persists in interphase and we propose that one cellular function of TACC3–ch-TOG is to modulate cell migration. We also describe the distinct subcellular pools of TACC3, ch-TOG and clathrin. TACC3 is often described as a centrosomal protein, but we show that there is no significant population of TACC3 at centrosomes. The delineation of distinct protein pools reveals a simplified view of how these proteins are organized and controlled by post-translational modification
Clathrin recruits phosphorylated TACC3 to spindle poles for bipolar spindle assembly and chromosome alignment
No full textTransforming acidic coiled-coil-containing protein 3 (TACC3) has been implicated in mitotic spindle assembly, although the mechanisms involved are largely unknown. Here we identify that clathrin heavy chain (CHC) binds specifically to phosphorylated TACC3 and recruits it to spindle poles for proper spindle assembly and chromosome alignment. Phosphorylation of Xenopus TACC3 at serine 620 (S620) and S626, but not S33, is required for its binding with CHC. Knockdown of CHC by RNA interference (RNAi) abolishes the targeting of TACC3 to spindle poles and results in abnormal spindle assembly and chromosome misalignment, similar to the defects caused by TACC3 knockdown. Furthermore, the binding of CHC with phosphorylated TACC3 is inhibited by importin. and this inhibition is reversed by the presence of the GTP-binding nuclear protein Ran in the GTP-bound state. Together, these results indicate that the recruitment of phosphorylated TACC3 to spindle poles by CHC ensures proper spindle assembly and chromosome alignment, and is regulated by Ran.</p
Pins homolog LGN regulates meiotic spindle organization in mouse oocytes
No full textMouse oocytes undergo polarization during meiotic maturation, and this polarization is essential for asymmetric cell divisions that maximize retention of maternal components required for early development. Without conventional centrosomes, the meiotic spindle has less focused poles and is barrel-shaped. The migration of meiotic spindles to the cortex is accompanied by a local reorganization and polarization of the cortex. LGN is a conserved protein involved in cell polarity and regulation of spindle organization. In the present study, we characterized the localization dynamics of LGN during mouse oocyte maturation and analyzed the effects of LGN upregulation and downregulation on meiotic spindle organization. At the germinal vesicle stage, LGN is distributed both cytoplasmically and at the cortex. During maturation, LGN localizes to the meiotic spindle apparatus and cortical LGN becomes less concentrated at the actin cap region. Excessive LGN induces meiotic spindle organization defects by elongating the spindle and enhancing pole focusing, whereas depletion of LGN by RNA interference results in meiotic spindle deformation and chromosome misalignment. Furthermore, the N-terminus of LGN has the ability of full-length LGN to regulate spindle organization, whereas the C-terminus of LGN controls cortical localization and polarization. Our results reveal that LGN is cortically polarized in mouse oocytes and is critical for meiotic spindle organization.Cell BiologySCI(E)PubMed中国科技核心期刊(ISTIC)中国科学引文数据库(CSCD)11ARTICLE7838-8481
Product Design Features, Packaging, and Online Retailer Marketing of Cannabis Products Containing Cannabinoids Other than THC and CBD: Opportunities for Cannabis Regulation
No full textBackground
Cannabis product characteristics affect product safety, and communication of these characteristics through product packaging and retailers informs consumers about potential risks, benefits, and psychoactive effects of cannabis products. Little is known about product characteristics, labeling, and marketing of cannabis products containing cannabinoids other than delta-9 THC and CBD, which remain unregulated in many jurisdictions. This dissertation assesses characteristics, packaging, availability, and retailer marketing practices for these products.
Methods
A content analysis of 140 delta-8 THC product packages from the International Cannabis Policy Study was conducted to explore product characteristics, cannabinoid content labels, health warnings, and marketing appeals (Aim 1). Availability of products containing cannabinoids other than delta-9 THC and CBD was determined for 39 online CBD/hemp retailers in Maryland, and cannabinoid content information for 175 products from these retailers was reviewed (Aim 2). A content analysis of 20 CBD/hemp retailer websites in Maryland was conducted to assess age verification, health claims, warnings, promotions, and engagement strategies (Aim 3).
Results
Most ingestible products contained more than 10mg of intoxicating cannabinoids per serving. Cannabinoid content labels for packages and product listings varied substantially, with many lacking sufficient information to calculate cannabinoid concentration. Lab results often differed from information in product listings. Explicit and implicit health claims were present on most retailer websites, with 50 health conditions mentioned across websites. Warnings on packaging and retailer websites were often small, inconspicuously placed (e.g., on the back or bottom), or absent altogether. Packages and retailer websites frequently included language describing products as “hemp,” “legal,” or “natural.” Half of retailer websites lacked age verification.
Discussion
The range of cannabinoids found in this study highlights the need for regulations requiring standardized cannabinoid content labels that communicate dose clearly and consistently. The presence of unsubstantiated health claims—both explicit and implicit—highlight the need for regulations around what the industry and retailers can communicate to consumers about the potential effects of cannabis products. Large, concise, rotating health warnings are needed at the top of product packages and retailer webpages to inform consumers about potential risks of cannabis, and effective age verification is needed to curb youth access
Applications of Clinical Pharmacology to the Study of Emerging Psychoactive Substances
No full textChapter I: Emerging psychoactive substances have poorly-understood human exposure-response relationships. Applied clinical pharmacology tools can help understand the health outcomes associated with emerging psychoactive substance use.
Chapter II: Delta-8 tetrahydrocannabinol (Δ-8 THC) is considered less psychoactive than Δ-9 THC. A population pharmacokinetic analysis and model was developed from two clinical trials (total N=23) comparing Δ-8 THC (10mg, 20mg, 40mg), Δ-9 THC (20mg), and placebo after oral and vaporized administration. Despite a similar pharmacokinetic pattern, Δ-8 THC was less metabolized to its more active metabolite than Δ-9 THC. Participant inhalation behaviors influenced the relative bioavailability of vaped Δ-8 THC compared to oral, with decreasing bioavailability at increasing doses captured by the model using covariates from measured puff topography. The validated model supports future analyses to compare Δ-8 and Δ-9 THC effects.
Chapter III: The pharmacokinetics of fentanyl are well-studied, but the clinical phenomena associated with illicitly manufactured fentanyl exposure are understudied in persons who use drugs. This narrative review applies fentanyl’s known absorption, distribution, metabolism, and excretion properties to illicit fentanyl. A notable gap in research exists due to differences between medicinal fentanyl and illicit fentanyl use patterns and composition. However, its properties suggest sustained, supratherapeutic use lends to the peripheral accumulation of fentanyl, leading to prolonged elimination in persons who use drugs.
Chapter IV: The pharmacology of fentanyl and xylazine is not characterized in persons regularly exposed to illicit fentanyl. This case series presents individual-level urine pharmacokinetics of fentanyl, its metabolite norfentanyl, and xylazine in persons with opioid use disorder. Participants (N=11) provided urine samples (n=95) for quantitative analysis. Urinary half-life ranges were: fentanyl 1.5-28.7 hours, norfentanyl 5.2-27.4 hours, xylazine 1.1-25.2 hours. Predicted detection window ranges were: fentanyl 13.0-130.9 hours (0.5-5.5 days), norfentanyl 64.8-255.9 hours (2.7-10.7 days), xylazine 13.8-123.4 hours (0.6-5.1 days). Individuals with a high magnitude of drug exposure are likely to evidence an extended duration of urinary detection.
Chapter V: Pharmacokinetic and pharmacokinetic-pharmacodynamic modeling and simulation techniques are promising translational tools to study the exposure-response relationship of emerging psychoactive substances. Potential uses of these techniques are discussed, including clinical applications to inform drug testing guidelines using real-world data
Applications of Clinical Pharmacology to the Study of Emerging Psychoactive Substances
No full textChapter I: Emerging psychoactive substances have poorly-understood human exposure-response relationships. Applied clinical pharmacology tools can help understand the health outcomes associated with emerging psychoactive substance use.
Chapter II: Delta-8 tetrahydrocannabinol (Δ-8 THC) is considered less psychoactive than Δ-9 THC. A population pharmacokinetic analysis and model was developed from two clinical trials (total N=23) comparing Δ-8 THC (10mg, 20mg, 40mg), Δ-9 THC (20mg), and placebo after oral and vaporized administration. Despite a similar pharmacokinetic pattern, Δ-8 THC was less metabolized to its more active metabolite than Δ-9 THC. Participant inhalation behaviors influenced the relative bioavailability of vaped Δ-8 THC compared to oral, with decreasing bioavailability at increasing doses captured by the model using covariates from measured puff topography. The validated model supports future analyses to compare Δ-8 and Δ-9 THC effects.
Chapter III: The pharmacokinetics of fentanyl are well-studied, but the clinical phenomena associated with illicitly manufactured fentanyl exposure are understudied in persons who use drugs. This narrative review applies fentanyl’s known absorption, distribution, metabolism, and excretion properties to illicit fentanyl. A notable gap in research exists due to differences between medicinal fentanyl and illicit fentanyl use patterns and composition. However, its properties suggest sustained, supratherapeutic use lends to the peripheral accumulation of fentanyl, leading to prolonged elimination in persons who use drugs.
Chapter IV: The pharmacology of fentanyl and xylazine is not characterized in persons regularly exposed to illicit fentanyl. This case series presents individual-level urine pharmacokinetics of fentanyl, its metabolite norfentanyl, and xylazine in persons with opioid use disorder. Participants (N=11) provided urine samples (n=95) for quantitative analysis. Urinary half-life ranges were: fentanyl 1.5-28.7 hours, norfentanyl 5.2-27.4 hours, xylazine 1.1-25.2 hours. Predicted detection window ranges were: fentanyl 13.0-130.9 hours (0.5-5.5 days), norfentanyl 64.8-255.9 hours (2.7-10.7 days), xylazine 13.8-123.4 hours (0.6-5.1 days). Individuals with a high magnitude of drug exposure are likely to evidence an extended duration of urinary detection.
Chapter V: Pharmacokinetic and pharmacokinetic-pharmacodynamic modeling and simulation techniques are promising translational tools to study the exposure-response relationship of emerging psychoactive substances. Potential uses of these techniques are discussed, including clinical applications to inform drug testing guidelines using real-world data
Product Design Features, Packaging, and Online Retailer Marketing of Cannabis Products Containing Cannabinoids Other than THC and CBD: Opportunities for Cannabis Regulation
No full textBackground
Cannabis product characteristics affect product safety, and communication of these characteristics through product packaging and retailers informs consumers about potential risks, benefits, and psychoactive effects of cannabis products. Little is known about product characteristics, labeling, and marketing of cannabis products containing cannabinoids other than delta-9 THC and CBD, which remain unregulated in many jurisdictions. This dissertation assesses characteristics, packaging, availability, and retailer marketing practices for these products.
Methods
A content analysis of 140 delta-8 THC product packages from the International Cannabis Policy Study was conducted to explore product characteristics, cannabinoid content labels, health warnings, and marketing appeals (Aim 1). Availability of products containing cannabinoids other than delta-9 THC and CBD was determined for 39 online CBD/hemp retailers in Maryland, and cannabinoid content information for 175 products from these retailers was reviewed (Aim 2). A content analysis of 20 CBD/hemp retailer websites in Maryland was conducted to assess age verification, health claims, warnings, promotions, and engagement strategies (Aim 3).
Results
Most ingestible products contained more than 10mg of intoxicating cannabinoids per serving. Cannabinoid content labels for packages and product listings varied substantially, with many lacking sufficient information to calculate cannabinoid concentration. Lab results often differed from information in product listings. Explicit and implicit health claims were present on most retailer websites, with 50 health conditions mentioned across websites. Warnings on packaging and retailer websites were often small, inconspicuously placed (e.g., on the back or bottom), or absent altogether. Packages and retailer websites frequently included language describing products as “hemp,” “legal,” or “natural.” Half of retailer websites lacked age verification.
Discussion
The range of cannabinoids found in this study highlights the need for regulations requiring standardized cannabinoid content labels that communicate dose clearly and consistently. The presence of unsubstantiated health claims—both explicit and implicit—highlight the need for regulations around what the industry and retailers can communicate to consumers about the potential effects of cannabis products. Large, concise, rotating health warnings are needed at the top of product packages and retailer webpages to inform consumers about potential risks of cannabis, and effective age verification is needed to curb youth access
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