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Abstract Background Candida parapsilosis is known to show limited genetic variability, despite different karyotypes and phenotypes have been described. To further investigate this aspect, a collection of 62 sensu strictu C. parapsilosis independent isolates from 4 geographic regions (Italy, n = 19; New Zealand, n = 15; Argentina, n = 14; and Hungary, n = 14) and different body sites (superficial and deep seated) were analysed for their genetic and phenotypic traits. Amplification fragment length polymorphism (AFLP) analysis was used to confirm species identification and to evaluate intraspecific genetic variability. Phenotypic characterisation included clinically relevant traits, such as drug susceptibility, in vitro biofilm formation and aspartyl protease secretion. Results AFLP genotyping showed little variation among isolates, when the presence/absence of bands was considered. However, when AFLP profiles were compared by relative intensity for each fragment, a significant level of variation and geographical clustering was observed. All isolates were found to be susceptible to commonly used antifungals, although a reduced susceptibility to echinocandins was observed in all isolates. C. parapsilosis isolates from different geographic origins varied in the number of biofilm producers, with a higher prevalence of producers isolated in Hungary and Argentina. The frequency of secreted proteinase producers also varied in isolates obtained from different areas, with a higher number of proteinase producers found in Italy and New Zealand. Interestingly, biofilm production and proteinase secretion were negatively correlated. This finding could be explained by assuming that proteinase activity plays a role in detachment and release from a mature biofilm, via degradation of C. parapsilosis adhesins and/or extracellular matrix components, as observed for other microorganisms. Conclusions The low number of polymorphic AFLP bands (18 out of 80) obtained for C. parapsilosis isolates is in agreement with the limited sequence variability described for this species. However, when band intensity was included in the analysis, geographical clustering was observed. Expression of virulence factors varied among strains isolated from different geographical regions, with biofilm and proteinase producers more frequently isolated from Hungary and Italy, respectively.

Ultrametric trees are trees whose leaves lie at the same distance from the root. They are used to model the genealogy of a population of particles co-existing at the same point in time. We show how the boundary of an ultrametric tree, like any compact ultrametric space, can be represented in a simple way via the so-called comb metric. We display a variety of examples of random combs and explain how they can be used in applications. In particular, we review some old and recent results regarding the genetic structure of the population when throwing neutral mutations on the skeleton of the tree.

Background: Significant disparities in substance use severity and treatment persist among women who use drugs compared to men. Thus, we explored how identifying as a woman was related to drug use and treatment experiences., Methods: The study recruited participants for a qualitative interview study in Boston and San Francisco from January-November 2020. Self-identified women, age ≥ 18 years, with nonprescribed opioid use in the past 14 days were eligible for inclusion. The study team developed deductive codes based on intersectionality theory and inductive codes generated from transcript review, and identified themes using grounded content analysis., Results: The study enrolled thirty-six participants. The median age was 46; 58 % were White, 16 % were Black, 14 % were Hispanic, and 39 % were unstably housed. Other drug use was common with 81 % reporting benzodiazepine, 50 % cocaine, and 31 % meth/amphetamine use respectively. We found that gender (i.e., identifying as a woman) intersected with drug use and sex work practices and exacerbated experiences of marginalization. Violence was ubiquitous in drug use environments. Some women reported experiences of gender-based violence in substance use service settings that perpetuated cycles of trauma and reinforced barriers to care. Substance use services that were women-led, safe, and responsive to women's needs were valued and sought after., Conclusion: Women reported a cycle of trauma and drug use exacerbated by oppression in substance use services settings. In addition to increasing access to gender-responsive care, our study highlights the need for greater research and examination of practices within substance use service settings that may be contributing to gender-based violence., Competing Interests: Declaration of competing interest None declared., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Post-overdose outreach programs have proliferated in response to opioid overdose. Implementing these programs is associated with reductions in overdose rates, but the role of specific program characteristics in overdose trends has not been evaluated., Methods: Among 58 Massachusetts municipalities with post-overdose outreach programs, we examined associations between five domains of post-overdose outreach program characteristics (outreach contact rate, naloxone distribution, coercive practices, harm reduction activities, and social service provision or referral) and rates of fatal opioid overdoses and opioid-related emergency medical system responses (i.e., ambulance activations) per calendar quarter from 2013 to 2019 using segmented regression analyses with adjustment for municipal covariates and fixed effects. For both outcomes, each domain was modeled: a) individually, b) with other characteristics, and c) with other characteristics and municipal-level fixed effects., Results: There were no significant associations (p < 0.05) between outreach contact rate, naloxone distribution, coercive practices, or harm reduction activities with municipal fatal overdose trends. Municipalities with programs providing or referring to more social services experienced 21% fewer fatal overdoses compared to programs providing or referring to more social services (Rate Ratio (RR) 0.79, 95% Confidence Interval (CI) 0.66-0.93, p = 0.01). Compared to municipalities in quarters when programs had no outreach contacts, municipalities with some, but less than the median outreach contacts, experienced 14% lower opioid-related emergency responses (RR 0.86, 95% CI 0.78-0.96, p = 0.01). Associations between naloxone distribution, coercive practices, harm reduction practices, or social services and opioid-related emergency responses were not consistently significant across modeling approaches., Conclusion: Municipalities with post-overdose outreach programs providing or referring to more social services had lower fatal opioid overdose rates. Municipalities in quarters when programs outreached to overdose survivors had fewer opioid-related emergency responses, but only among programs with below the median number of outreach contacts. Social service linkage should be core to post-overdose programs. Evaluations should assess program characteristics to optimize program design., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Drs. Kimmel and Walley report financial support from the Massachusetts Department of Public Health as part of the state opioid overdose prevention program., (Copyright © 2023. Published by Elsevier B.V.)

Importance: Nonfatal opioid overdose is the leading risk factor for subsequent fatal overdose and represents a critical opportunity to reduce future overdose and mortality. Postoverdose outreach programs emerged in Massachusetts beginning in 2013 with the main purpose of linking opioid overdose survivors to addiction treatment and harm reduction services., Objective: To evaluate whether the implementation of postoverdose outreach programs among Massachusetts municipalities was associated with lower opioid fatality rates compared with municipalities without postoverdose outreach programs., Design, Setting, and Participants: This retrospective interrupted time-series analysis was performed over 26 quarters (from January 1, 2013, through June 30, 2019) across 93 municipalities in Massachusetts. These 93 municipalities were selected based on a threshold of 30 or more opioid-related emergency medical services (EMS) responses in 2015. Data were analyzed from November 2021 to August 2022., Exposures: The main exposure was municipality postoverdose outreach programs. Municipalities had various program inceptions during the study period., Main Outcomes and Measures: The primary outcome was quarterly municipal opioid fatality rate per 100 000 population. The secondary outcome was quarterly municipal opioid-related EMS response (ambulance trips) rates per 100 000 population., Results: The mean (SD) population size across 93 municipalities was 47 622 (70 307), the mean (SD) proportion of female individuals was 51.5% (1.5%) and male individuals was 48.5% (1.5%), and the mean (SD) age proportions were 29.7% (4.0%) younger than 25 years, 26.0% (4.8%) aged 25 to 44 years, 14.8% (2.1%) aged 45 to 54 years, 13.4% (2.1%) aged 55 to 64 years, and 16.1% (4.4%) aged 65 years or older. Postoverdose programs were implemented in 58 municipalities (62%). Following implementation, there were no significant level changes in opioid fatality rate (adjusted rate ratio [aRR], 1.07; 95% CI, 0.96-1.19; P = .20). However, there was a significant slope decrease in opioid fatality rate (annualized aRR, 0.94; 95% CI, 0.90-0.98; P = .003) compared with the municipalities without the outreach programs. Similarly, there was a significant slope decrease in opioid-related EMS response rates (annualized aRR, 0.93; 95% CI, 0.89-0.98; P = .007). Several sensitivity analyses yielded similar findings., Conclusions and Relevance: In this study, among Massachusetts municipalities with high numbers of opioid-related EMS responses, implementation of postoverdose outreach programs was significantly associated with lower opioid fatality rates over time compared with municipalities that did not implement such programs. Program components, including cross-sectoral partnerships, operational best practices, involvement of law enforcement, and related program costs, warrant further evaluation to enhance effectiveness.

Dryland riparian woodlands are considered to be locally buffered from droughts by shallow and stable groundwater levels. However, climate change is causing more frequent and severe drought events, accompanied by warmer temperatures, collectively threatening the persistence of these groundwater dependent ecosystems through a combination of increasing evaporative demand and decreasing groundwater supply. We conducted a dendro-isotopic analysis of radial growth and seasonal (semi-annual) carbon isotope discrimination (Δ 13 C) to investigate the response of riparian cottonwood stands to the unprecedented California-wide drought from 2012 to 2019, along the largest remaining free-flowing river in Southern California. Our goals were to identify principal drivers and indicators of drought stress for dryland riparian woodlands, determine their thresholds of tolerance to hydroclimatic stressors, and ultimately assess their vulnerability to climate change. Riparian trees were highly responsive to drought conditions along the river, exhibiting suppressed growth and strong stomatal closure (inferred from reduced Δ 13 C) during peak drought years. However, patterns of radial growth and Δ 13 C were quite variable among sites that differed in climatic conditions and rate of groundwater decline. We show that the rate of groundwater decline, as opposed to climate factors, was the primary driver of site differences in drought stress, and trees showed greater sensitivity to temperature at sites subjected to faster groundwater decline. Across sites, higher correlation between radial growth and Δ 13 C for individual trees, and higher inter-correlation of Δ 13 C among trees were indicative of greater drought stress. Trees showed a threshold of tolerance to groundwater decline at 0.5 m year -1 beyond which drought stress became increasingly evident and severe. For sites that exceeded this threshold, peak physiological stress occurred when total groundwater recession exceeded ~3 m. These findings indicate that drought-induced groundwater decline associated with more extreme droughts is a primary threat to dryland riparian woodlands and increases their susceptibility to projected warmer temperatures., (© 2022 The Authors. Global Change Biology published by John Wiley & Sons Ltd.)

Background: Drug overdose mortality continues to increase, now driven by fentanyl. Prevention tools such as naloxone and medications to treat opioid use disorder are not sufficient to control overdose rates; additional strategies are urgently needed., Objective: We sought to adapt a behavioral intervention to prevent opioid overdose (repeated-dose behavioral intervention to reduce opioid overdose [REBOOT]) that had been successfully piloted in San Francisco, California, United States, to the setting of Boston, Massachusetts, United States, and the era of fentanyl for a full efficacy trial., Methods: We used the assessment, decision, adaptation, production, topical experts, integration, training, and testing (ADAPT-ITT) framework for intervention adaptation. We first identified opioid overdose survivors who were actively using opioids as the population of interest and REBOOT as the intervention to be adapted. We then performed theater testing and elicited feedback with 2 focus groups (n=10) in Boston in 2018. All participants had used opioids that were not prescribed to them in the past year and experienced an opioid overdose during their lifetime. We incorporated focus group findings into our initial draft of the adapted REBOOT intervention. The adapted intervention was reviewed by 3 topical experts, and their feedback was integrated into a subsequent draft. We trained study staff on the intervention and made final refinements based on internal piloting. This paper describes the overall ADAPT-ITT process for intervention adaptation, as well as a qualitative analysis of the focus groups. Working independently, 2 authors (VMM and JA) reviewed the focus group transcripts and coded them for salient and common themes using the constant comparison method, meeting to discuss any discrepancies until consensus was reached. Codes and themes were then mapped onto the REBOOT counseling steps., Results: Focus group findings contributed to substantial changes in the counseling intervention to better address fentanyl overdose risk. Participants described the widespread prevalence of fentanyl and said that, although they tried to avoid it, avoidance was becoming impossible. Using alone and lower opioid tolerance were identified as contributors to overdose risk. Slow shots or tester shots were acceptable and considered effective to reduce risk. Naloxone was considered an effective reversal strategy. Although calling emergency services was not ruled out, participants described techniques to prevent the arrival of police on the scene. Expert review and internal piloting improved the intervention manual through increased participant centeredness, clarity, and usability., Conclusions: We successfully completed the ADAPT-ITT approach for an overdose prevention intervention, using theater testing with people who use opioids to incorporate the perspectives of people who use drugs into a substance use intervention. In the current crisis, overdose prevention strategies must be adapted to the context of fentanyl, and innovative strategies must be deployed, including behavioral interventions., Trial Registration: ClinicalTrials.gov NCT03838510; https://clinicaltrials.gov/ct2/show/NCT03838510., (©Vanessa M McMahan, Justine Arenander, Tim Matheson, Audrey M Lambert, Sarah Brennan, Traci C Green, Alexander Y Walley, Phillip O Coffin. Originally published in JMIR Formative Research (https://formative.jmir.org), 07.09.2022.)

Background: Severe mental illness (SMI; schizophrenia, bipolar disorders (BDs), and other nonorganic psychoses) is associated with increased risk of cardiovascular disease (CVD) and CVD-related mortality. To date, no systematic review has investigated changes in population level CVD-related mortality over calendar time. It is unclear if this relationship has changed over time in higher-income countries with changing treatments., Methods and Findings: To address this gap, a systematic review was conducted, to assess the association between SMI and CVD including temporal change. Seven databases were searched (last: November 30, 2021) for cohort or case-control studies lasting ≥1 year, comparing frequency of CVD mortality or incidence in high-income countries between people with versus without SMI. No language restrictions were applied. Random effects meta-analyses were conducted to compute pooled hazard ratios (HRs) and rate ratios, pooled standardised mortality ratios (SMRs), pooled odds ratios (ORs), and pooled risk ratios (RRs) of CVD in those with versus without SMI. Temporal trends were explored by decade. Subgroup analyses by age, sex, setting, world region, and study quality (Newcastle-Ottawa scale (NOS) score) were conducted. The narrative synthesis included 108 studies, and the quantitative synthesis 59 mortality studies (with (≥1,841,356 cases and 29,321,409 controls) and 28 incidence studies (≥401,909 cases and 14,372,146 controls). The risk of CVD-related mortality for people with SMI was higher than controls across most comparisons, except for total CVD-related mortality for BD and cerebrovascular accident (CVA) for mixed SMI. Estimated risks were larger for schizophrenia than BD. Pooled results ranged from SMR = 1.55 (95% confidence interval (CI): 1.33 to 1.81, p < 0.001), for CVA in people with BD to HR/rate ratio = 2.40 (95% CI: 2.25 to 2.55, p < 0.001) for CVA in schizophrenia. For schizophrenia and BD, SMRs and pooled HRs/rate ratios for CHD and CVD mortality were larger in studies with outcomes occurring during the 1990s and 2000s than earlier decades (1980s: SMR = 1.14, 95% CI: 0.57 to 2.30, p = 0.71; 2000s: SMR = 2.59, 95% CI: 1.93 to 3.47, p < 0.001 for schizophrenia and CHD) and in studies including people with younger age. The incidence of CVA, CVD events, and heart failure in SMI was higher than controls. Estimated risks for schizophrenia ranged from HR/rate ratio 1.25 (95% CI: 1.04 to 1.51, p = 0.016) for total CVD events to rate ratio 3.82 (95% CI: 3.1 to 4.71, p < 0.001) for heart failure. Incidence of CHD was higher in BD versus controls. However, for schizophrenia, CHD was elevated in higher-quality studies only. The HR/rate ratios for CVA and CHD were larger in studies with outcomes occurring after the 1990s. Study limitations include the high risk of bias of some studies as they drew a comparison cohort from general population rates and the fact that it was difficult to exclude studies that had overlapping populations, although attempts were made to minimise this., Conclusions: In this study, we found that SMI was associated with an approximate doubling in the rate ratio of CVD-related mortality, particularly since the 1990s, and in younger groups. SMI was also associated with increased incidence of CVA and CHD relative to control participants since the 1990s. More research is needed to clarify the association between SMI and CHD and ways to mitigate this risk., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: CUC has been a consultant and/or advisor to or has received honoraria from: AbbVie, Acadia, Alkermes, Allergan, Angelini, Aristo, Axsome, Damitsa, Gedeon Richter, Hikma, IntraCellular Therapies, Janssen/J&J, Karuna, LB Pharma, Lundbeck, MedAvante-ProPhase, MedInCell, Medscape, Merck, Mitsubishi Tanabe Pharma, Mylan, Neurocrine, Noven, Otsuka, Pfizer, Recordati, Rovi, Servier, Sumitomo Dainippon, Sunovion, Supernus, Takeda, Teva, and Viatris. He provided expert testimony for Janssen and Otsuka. He served on a Data Safety Monitoring Board for Lundbeck, Rovi, Supernus, and Teva. He has received grant support from Janssen and Takeda. He is also a stock option holder of LB Pharma. MS received honoraria/has been a consultant for Angelini, Lundbeck, Otsuka. MLO receives funding from The Liaison Committee for education, research and innovation in Central Norway.

Introduction: We conducted a qualitative study to explore the impact of the COVID-19 pandemic on experiences with addiction treatment and harm reduction services., Methods: The study recruited participants from Boston, Massachusetts, aged 18-65 who had a history of opioid use disorder and overdose, from a parent study (REpeated dose Behavioral intervention to reduce Opioid Overdose, REBOOT) to participate between August and October 2020. In-depth individual interviews explored the impact of the COVID-19 pandemic on addiction service experiences. We conducted a grounded content analysis that examined codes related to addiction service access and engagement during the pandemic to compare and categorize participants according to their experiences., Results: The study enrolled twenty participants. The mean age was 42 years; most identified as white (n = 16); ten participants identified as men, nine as cis-gender women, and one as a trans-gender woman. Participants described their experiences with COVID-19-driven changes to addiction care (methadone take homes, televisits for either buprenorphine or behavioral health services, and syringe service outreach) access and engagement as: 1) liberating (n = 7), 2) destabilizing (n = 8), or 3) unjust (n = 5). Participants in the liberating group found adaptations allowed for increased flexibility, freedom, and safety from COVID-19. This group was mostly housed and had strong social supports that facilitated participation in adapted treatment programs. COVID-19-related changes to addiction treatment disrupted routine and community supports among those in the destabilizing group. Participants in the unjust group felt that adaptations exacerbated inequities as a lack of housing and other social supports prohibited them from benefiting from the relaxed restrictions to methadone or buprenorphine. This group was mostly unhoused and found that adaptations did not adequately mitigate other inequities worsened by public health mandates for unhoused people who use drugs., Conclusion: Relaxed restrictions on medications for opioid use disorder created opportunities for improved patient-centered care. Concrete measures that address service barriers, such as phone or transportation access, may have reduced destabilizing and unjust experiences reported by our participants. However, addiction care inequities will persist if drivers of marginalization, specifically a lack of housing, remain unaddressed., (Copyright © 2021. Published by Elsevier Inc.)

Background: Post-overdose outreach programs engage survivors in harm reduction and treatment to prevent future overdoses. In Massachusetts, these emerging programs commonly deploy teams comprised of police and public health professionals based on 911 call information. Some teams use name/address data to conduct arrest warrant checks prior to outreach visits. We used mixed methods to understand approaches to outreach related to warrant checking, from the perspectives of police and public health outreach agencies and staff., Methods: We analyzed a 2019 statewide survey of post-overdose outreach programs in Massachusetts to classify approaches to warrant checking and identify program and community factors associated with particular approaches. Ethnographic analysis of qualitative interviews conducted with outreach staff helped further contextualize outreach program practices related to warrants., Results: A majority (57% - 79/138) of post-overdose outreach programs in Massachusetts conducted warrant checks prior to outreach. Among programs that checked warrants, we formulated a taxonomy of approaches to handling warrants: 1) performing outreach without addressing warrants (19.6% - 27/138), 2) delaying outreach until warrants are cleared (15.9% - 22/138), 3) arresting the survivor (11/138 - 8.0%), 4) taking a situational approach (10/138 - 7.2%), 5) not performing outreach (9/138 - 6.5%). Program characteristics and staff training did not vary across approaches. From police and public health outreach staff interviews (n = 38), we elicited four major themes: a) diverse motivations precede warrant checking, b) police officers feel tension between dual roles, c) warrants alter approaches to outreach, and d) teams leverage warrants in relationships. Findings from both analyses converged to demonstrate unintended consequences of warrant checking., Conclusion: Checking warrants prior to post-overdose outreach visits can result in arrest, delayed outreach, and barriers to obtaining services for overdose survivors, which can undermine the goal of these programs to engage overdose survivors. With the public health imperative of engaging overdose survivors, programs should consider limiting warrant checking and police participation in field activities., Competing Interests: Declarations of Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: AYW is the medical director of overdose prevention programs at the Massachusetts Department of Public health and signs the statewide naloxone standing order, including multiple police and fire departments in Massachusetts., (Copyright © 2021. Published by Elsevier B.V.)

Background: Naloxone is a prescription medication that reverses opioid overdoses. Allowing naloxone to be dispensed directly by a pharmacist without an individual prescription under a naloxone standing order (NSO) can expand access. The community-level factors associated with naloxone dispensed under NSO are unknown., Methods: Using a dataset comprised of pharmacy reports of naloxone dispensed under NSO from 70% of Massachusetts retail pharmacies, we examined relationships between community-level demographics, rurality, measures of treatment for opioid use disorder, and overdose deaths with naloxone dispensed under NSO per ZIP Code-quarter from 2014 until 2018. We used a multi-variable zero-inflated negative binomial model, assessing odds of any naloxone dispensed under NSO, as well as a multi-variable negative binomial model assessing quantities of naloxone dispensed under NSO., Results: From 2014-2018, quantities of naloxone dispensed under NSO and the number of pharmacies dispensing any naloxone under NSO increased over time. However, communities with greater percentages of people with Hispanic ethnicity (aOR 0.91, 95% CI 0.86-0.96 per 5% increase), and rural communities compared to urban communities (aOR 0.81, 95% CI 0.73-0.90) were less likely to dispense any naloxone by NSO. Communities with more individuals treated with buprenorphine dispensed more naloxone under NSO, as did communities with more opioid-related overdose deaths., Conclusion: Naloxone dispensing has substantially increased, in part driven by standing orders. A lower likelihood of naloxone being dispensed under NSO in communities with larger Hispanic populations and in more rural communities suggests the need for more equitable access to, and uptake of, lifesaving medications like naloxone., (Copyright © 2021 Elsevier B.V. All rights reserved.)

This study examined the performance of Poanes viator (Edwards) (Hesperiidae), a native North American skipper, and Rhizedra lutosa (Hübner) (Noctuidae), an introduced moth, reared on native and non-native, invasive lineages of Phragmites australis . Poanes viator is a generalist on monocots and larvae were also fed leaves of Zizania aquatica , a native macrophyte that the skipper commonly uses as a host plant. Larval survival and duration, pupal weight, and pupation time were compared for P. viator feeding on leaf tissue and R. lutosa feeding on rhizomes of either native or introduced plants. We also tested an artificial diet supplemented with P. australis rhizome powder as a potential food for rearing other stalk and rhizome boring Lepidoptera. In experiments using excised plant tissues, some individuals of both species fed and developed to the pupal stage on native and introduced plants, but overall, larval survival rates were low. Plant species/haplotype identity did not cause strong differences in larval survival for either species. However, P. viator larvae only pupated when feeding on native plants ( Zizania aquatica and native P. australis haplotypes), whereas R. lutosa successfully pupated on both native and introduced P. australis . Although larval survival was low, 100% of P. viator and 95% of R. lutosa that reached the pupal stage emerged as adults. Rhizedra lutosa larvae fed an artificial diet supplemented with P. australis rhizome powder had significantly greater survival and pupal weights, and shorter pupation times than larvae fed rhizomes only. Several specialist Lepidopteran species are being considered for approval as biological control agents for the non-native P. australis haplotype, and the convenience and increased larval performance make this artificial diet a good alternative for rearing organisms.

WNT signalling is important for development in all metazoans and is associated with various human diseases. The ubiquitin-proteasome system (UPS) and regulatory endoplasmic reticulum-associated degradation (ERAD) have been implicated in the production of WNT proteins. Here, we investigated how the WNT secretory factor EVI (also known as WLS) is ubiquitylated, recognised by ERAD components and subsequently removed from the secretory pathway. We performed a focused immunoblot-based RNAi screen for factors that influence EVI/WLS protein stability. We identified the VCP-binding proteins FAF2 and UBXN4 as novel interaction partners of EVI/WLS and showed that ERLIN2 links EVI/WLS to the ubiquitylation machinery. Interestingly, we also found that EVI/WLS is ubiquitylated and degraded in cells irrespective of their level of WNT production. This K11, K48 and K63-linked ubiquitylation is mediated by the E2 ubiquitin-conjugating enzymes UBE2J2, UBE2K and UBE2N, but is independent of the E3 ubiquitin ligases HRD1 (also known as SYVN1) and GP78 (also known as AMFR). Taken together, our study identifies factors that link the UPS to the WNT secretory pathway and provides mechanistic details of the fate of an endogenous substrate of regulatory ERAD in mammalian cells. This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Recognition memory is improved for items produced at study (e.g., by reading them aloud) relative to a non-produced control condition (e.g., silent reading). This production effect is typically attributed to the extra elements in the production task (e.g., motor activation, auditory perception) enhancing item distinctiveness. To evaluate this claim, the present study examined the neural mechanisms underlying the production effect. Prior to a recognition memory test, different words within a study list were read either aloud, silently, or while saying "check" (as a sensorimotor control condition). Production improved recognition, and aloud words yielded higher rates of both recollection and familiarity judgments than either silent or control words. During encoding, fMRI revealed stronger activation in regions associated with motor, somatosensory, and auditory processing for aloud items than for either silent or control items. These activations were predictive of recollective success for aloud items at test. Together, our findings are compatible with a distinctiveness-based account of the production effect, while also pointing to the possible role of other processing differences during the aloud trials as compared to silent and control., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Arundo donax (giant reed) is invasive in Mediterranean, sub-, and tropical riparian systems worldwide. The armored scale Rhizaspidiotus donacis is approved for biocontrol in North America, but an adventive population was recently discovered in southern California. We documented this population's distribution, phylogeny, phenology, potential host spillover to Phragmites spp ., and potential for parasitism by a common biocontrol parasitoid of citrus scale. The adventive scale was found within a single watershed and is genetically closest to Iberian scale genotypes. Rhizaspidiotus donacis developed on Phragmites haplotypes but at much lower densities than Arundo . The adventive population is univoltine, producing crawlers from March-June. Aphytis melinus parasitoids exhibited sustained interest in R. donacis during choice and no-choice trials and oviposition resulted in a small second generation. Rhizaspidiotus donacis appears limited in distribution by its univoltinism and sessile adult females. This presents challenges for broad biocontrol implementation but allows for targeted application. The genetic differentiation between imported biocontrol samples and adventive populations presents an opportunity for exploring benefits of hybrids and/or alternative genotypes where establishment has been difficult. While unlikely to occur in situ, spillover to vulnerable endemic Phragmites or deleterious parasitoid effects on scale biocontrol agents warrants consideration when planning use of R. donacis .

microRNA (miRNA)-mediated gene silencing is enacted through the recruitment of effector proteins that direct translational repression or degradation of mRNA targets, but the relative importance of their activities for animal development remains unknown. Our concerted proteomic surveys identified the uncharacterized GYF-domain encoding protein GYF-1 and its direct interaction with IFE-4, the ortholog of the mammalian translation repressor 4EHP, as key miRNA effector proteins in Caenorhabditis elegans. Recruitment of GYF-1 protein to mRNA reporters in vitro or in vivo leads to potent translation repression without affecting the poly(A) tail or impinging on mRNA stability. Loss of gyf-1 is synthetic lethal with hypomorphic alleles of embryonic miR-35-42 and larval (L4) let-7 miRNAs, which is phenocopied through engineered mutations in gyf-1 that abolish interaction with IFE-4. GYF-1/4EHP function is cascade-specific, as loss of gyf-1 had no noticeable impact on the functions of other miRNAs, including lin-4 and lsy-6. Overall, our findings reveal the first direct effector of miRNA-mediated translational repression in C. elegans and its physiological importance for the function of several, but likely not all miRNAs., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The mariner ( myo7aa -/- ) mutant is a zebrafish model for Usher syndrome type 1 (USH1). To further characterize hair cell synaptic elements in myo7aa -/- mutants, we focused on the ribbon synapse and evaluated ultrastructure, number and distribution of immunolabeled ribbons, and postsynaptic densities. By transmission electron microscopy, we determined that myo7aa -/- zebrafish have fewer glutamatergic vesicles tethered to ribbon synapses, yet maintain a comparable ribbon area. In myo7aa -/- hair cells, immunolocalization of Ctbp2 showed fewer ribbon-containing cells in total and an altered distribution of Ctbp2 puncta compared to wild-type hair cells. myo7aa -/- mutants have fewer postsynaptic densities - as assessed by MAGUK immunolabeling - compared to wild-type zebrafish. We quantified the circular swimming behavior of myo7aa -/- mutant fish and measured a greater turning angle (absolute smooth orientation). It has previously been shown that L-type voltage-gated calcium channels are necessary for ribbon localization and occurrence of postsynaptic density; thus, we hypothesized and observed that L-type voltage-gated calcium channel agonists change behavioral and synaptic phenotypes in myo7aa -/- mutants in a drug-specific manner. Our results indicate that treatment with L-type voltage-gated calcium channel agonists alter hair cell synaptic elements and improve behavioral phenotypes of myo7aa -/- mutants. Our data support that L-type voltage-gated calcium channel agonists induce morphological changes at the ribbon synapse - in both the number of tethered vesicles and regarding the distribution of Ctbp2 puncta - shift swimming behavior and improve acoustic startle response., Competing Interests: Competing InterestsThe content of this article is solely the responsibility of the authors and does not necessarily represent the official view of the National Institutes of Health., (© 2020. Published by The Company of Biologists Ltd.)

Premise of the Study: Hybridization between previously isolated species or lineages can stimulate invasiveness because of increased genetic diversity and inherited traits facilitating competitive and reproductive potential. We evaluated differences in stand characteristics and sexual and vegetative reproduction among native, introduced, and hybrid Phragmites australis lineages in the southwestern United States. We also assessed the degree of hybridization among lineages and backcrossing of hybrids with parental lineages., Methods: Growth and morphological characteristics were measured in native, introduced, and hybrid Phragmites stands to evaluate relative cover and dominance in associated plant communities. Panicles were collected from stands to evaluate germination, dormancy, and differences in seed traits. Seedlings from germination trials were genotyped to determine frequency of crossing and backcrossing among lineages., Key Results: Introduced and hybrid Phragmites stands had significantly greater stem and panicle densities than native stands and were more likely to be dominant members of their respective plant communities. Hybrid seed outputs were significantly greater, but hybrid seeds had lower germination rates than those from native and introduced lineages. We detected a novel hybridization event between native and introduced lineages, but found no strong evidence of hybrids backcrossing with parental lineages., Conclusions: Hybrid Phragmites in the Southwest exhibits reproductive, genetic, and morphological characteristics from both parental lineages that facilitate dispersal, establishment, and aggressive growth, including high reproductive output, rhizome viability, and aboveground biomass, with smaller seeds and greater genetic diversity than its progenitors. Our results show hybrids can inherit traits that confer invasiveness and provide insight for managing this species complex and other cryptic species with native and introduced variants with potential for intraspecific hybridization., (© 2019 Botanical Society of America.)

The production effect reflects a memory advantage for words read aloud versus silently. We investigated how production influences free recall of a single long list of words. In each of 4 experiments, a production effect occurred in a mixed-list group but not across pure-list groups. When compared to the pure-list groups, the mixed-list effects typically reflected a cost to silent words rather than a benefit to aloud words. This cost persisted when participants had to perform a generation or imagery task for the silent items, ruling out a lazy reading explanation. This recall pattern challenges both distinctiveness and strength accounts, but is consistent with an item-order account. By this account, the aloud words in a mixed list disrupt the encoding of item-order information for the silent words, thus impairing silent word recall. However, item-order measures and a forced-choice order test did not provide much evidence that recall was guided by retrieval of item-order information. We discuss our pattern of results in light of another recent study of the effects of production on long-list recall. (PsycINFO Database Record, ((c) 2016 APA, all rights reserved).)

Experimental studies to determine the nature of ecological interactions between invasive and native species are necessary for conserving and restoring native species in impacted habitats. Theory predicts that species boundaries along environmental gradients are determined by physical factors in stressful environments and by competitive ability in benign environments, but little is known about the mechanisms by which hydrophytes exclude halophytes and the life history stage at which these mechanisms are able to operate. The ongoing invasion of the South American Spartina densiflora in European marshes is causing concern about potential impacts to native plants along the marsh salinity gradient, offering an opportunity to evaluate the mechanisms by which native hydrophytes may limit, or even prevent, the expansion of invasive halophytes. Our study compared S. densiflora seedling establishment with and without competition with Phragmites australis and Typha domingensis, two hydrophytes differing in clonal architecture. We hypothesized that seedlings of the stress tolerant S. densiflora would be out-competed by stands of P. australis and T. domingensis. Growth, survivorship, biomass patterns and foliar nutrient content were recorded in a common garden experiment to determine the effect of mature P. australis and T. domingensis on the growth and colonization of S. densiflora under fresh water conditions where invasion events are likely to occur. Mature P. australis stands prevented establishment of S. densiflora seedlings and T. domingensis reduced S. densiflora establishment by 38%. Seedlings grown with P. australis produced fewer than five short shoots and all plants died after ca. 2 yrs. Our results showed that direct competition, most likely for subterranean resources, was responsible for decreased growth rate and survivorship of S. densiflora. The presence of healthy stands of P. australis, and to some extent T. domingensis, along river channels and in brackish marshes may prevent the invasion of S. densiflora by stopping the establishment of its seedlings.

The current study assessed the effects of a positive peer reporting procedure known as Tootling on classwide disruptive as well as appropriate behavior with fourth- and fifth-grade students and their teachers in two regular education classrooms. Tootling is a technique that teaches students to recognize and report peers' prosocial behavior rather than inappropriate behavior (i.e., as in tattling), and is also a variation on the expression, "tooting your own horn." Tootling combined with an interdependent group contingency and publicly posted feedback were assessed using an ABAB withdrawal design with a multiple baseline element across classrooms. Results demonstrated decreases in classwide disruptive behavior as well as increases in appropriate behavior compared with baseline and withdrawal phases across both classrooms, with results maintained at follow-up. Tootling was also rated highly acceptable by both teachers. Effect size calculations reflected moderate to strong effects across all comparisons. Limitations of the present study, directions for future research, and implications for practice are discussed., (© The Author(s) 2014.)

Purpose: Nivolumab is a fully human immunoglobulin G4 programmed death-1 immune checkpoint inhibitor antibody that restores T-cell immune activity. This phase II trial assessed the antitumor activity, dose-response relationship, and safety of nivolumab in patients with metastatic renal cell carcinoma (mRCC)., Patients and Methods: Patients with clear-cell mRCC previously treated with agents targeting the vascular endothelial growth factor pathway were randomly assigned (blinded ratio of 1:1:1) to nivolumab 0.3, 2, or 10 mg/kg intravenously once every 3 weeks. The primary objective was to evaluate the dose-response relationship as measured by progression-free survival (PFS); secondary end points included objective response rate (ORR), overall survival (OS), and safety., Results: A total of 168 patients were randomly assigned to the nivolumab 0.3- (n = 60), 2- (n = 54), and 10-mg/kg (n = 54) cohorts. One hundred eighteen patients (70%) had received more than one prior systemic regimen. Median PFS was 2.7, 4.0, and 4.2 months, respectively (P = .9). Respective ORRs were 20%, 22%, and 20%. Median OS was 18.2 months (80% CI, 16.2 to 24.0 months), 25.5 months (80% CI, 19.8 to 28.8 months), and 24.7 months (80% CI, 15.3 to 26.0 months), respectively. The most common treatment-related adverse event (AE) was fatigue (24%, 22%, and 35%, respectively). Nineteen patients (11%) experienced grade 3 to 4 treatment-related AEs., Conclusion: Nivolumab demonstrated antitumor activity with a manageable safety profile across the three doses studied in mRCC. No dose-response relationship was detected as measured by PFS. These efficacy and safety results in mRCC support study in the phase III setting., (© 2014 by American Society of Clinical Oncology.)

Fullerenes and their structure and stability have been a major topic of discussion and research since their discovery nearly 30 years ago. The isolated pentagon rule (IPR) has long served as a guideline for predicting the most stable fullerene cages. More recently, endohedral metallofullerenes have been discovered that violate the IPR. This article presents a systematic, temperature dependent, statistical thermodynamic study of the 24 possible IPR isomers of C84 as well as two of the experimentally known non-IPR isomers (51365 and 51383), at several different charges (0, -2, -4, and -6). From the results of this study, we conclude that the Hückel rule is a valid simpler explanation for the stability of fused pentagons in endohedral metallofullerenes., (© 2014 Wiley Periodicals, Inc.)

Transient receptor potential, melastatin-like 7 (Trpm7) is a combined ion channel and kinase implicated in the differentiation or function of many cell types. Early lethality in mice and frogs depleted of the corresponding gene impedes investigation of the functions of this protein particularly during later stages of development. By contrast, zebrafish trpm7 mutant larvae undergo early morphogenesis normally and thus do not have this limitation. The mutant larvae are characterized by multiple defects including melanocyte cell death, transient paralysis, and an ion imbalance that leads to the development of kidney stones. Here we report a requirement for Trpm7 in differentiation or function of dopaminergic neurons in vivo. First, trpm7 mutant larvae are hypomotile and fail to make a dopamine-dependent developmental transition in swim-bout length. Both of these deficits are partially rescued by the application of levodopa or dopamine. Second, histological analysis reveals that in trpm7 mutants a significant fraction of dopaminergic neurons lack expression of tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis. Third, trpm7 mutants are unusually sensitive to the neurotoxin 1-methyl-4-phenylpyridinium, an oxidative stressor, and their motility is partially rescued by application of the iron chelator deferoxamine, an anti-oxidant. Finally, in SH-SY5Y cells, which model aspects of human dopaminergic neurons, forced expression of a channel-dead variant of TRPM7 causes cell death. In summary, a forward genetic screen in zebrafish has revealed that both melanocytes and dopaminergic neurons depend on the ion channel Trpm7. The mechanistic underpinning of this dependence requires further investigation., (© 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Premise of the Study: Rare plants may be rare, in part, because they are more susceptible to damage by predispersal insect seed predators than widespread congeneric species; thus, seed predation may be an important determinant of plant rarity. Scant evidence exists to either support or reject this hypothesis, limiting our ability to predict herbivore effects on plant rarity. We used a comparative framework to test this hypothesis in rare-common, sympatric congeners., Methods: We compared seed consumption by insect type and seed production among a rare Astragalus (Fabaceae) species and two sympatric, widespread congeners. We measured plant traits and tested whether traits were correlated with seed damage within and among rare-common species., Key Results: Seed predation was significantly higher in a rare species than in common congeners over 2 yr. Seed production per pod was significantly lower in the rare species. Seed weevils exclusively consumed seeds of the rare species. Higher seed predation in the rare species is related to a combination of factors: plant phenology, dispersal ability, and vigor; seed predator identity; and insect phenology., Conclusions: Our results support the hypothesis that a rare plant is more susceptible to seed predators than two common, sympatric congeners. Seed predator reduction is a promising management tool to increase seed-set, recruitment, and survival of the rare species. Further studies that compare rare-common, sympatric congeners are greatly needed to broadly evaluate the hypothesis concerning rarity and susceptibility to seed predators and to inform conservation plans for rare species.

The most conserved part of the vertebrate dopaminergic system is the orthopedia (otp)-expressing diencephalic neuronal population that constitutes the dopaminergic diencephalospinal tract (DDT). Although studies in the neonatal murine spinal cord in vitro suggest an early locomotor role of the DDT, the function of the DDT in developing vertebrates in vivo remains unknown. Here, we investigated the role of the DDT in the locomotor development of zebrafish larvae. To assess the development of the behavioral and neural locomotor pattern, we used high-throughput video tracking in combination with peripheral nerve recordings. We found a behavioral and neural correspondence in the developmental switch from an immature to mature locomotor pattern. Blocking endogenous dopamine receptor 4 (D(4)R) signaling in vivo either before or after the developmental switch prevented or reversed the switch, respectively. Spinal transections of post-switch larvae reestablished the immature locomotor pattern, which was rescued to a mature-like pattern via spinal D(4)R agonism. Selective chemogenetic ablation of otp b (otpb) neurons that contribute to the DDT perpetuated the immature locomotor pattern in vivo. This phenotype was recapitulated by diencephalic transections that removed the dopaminergic otpb population and was rescued to a mature-like locomotor pattern by D(4)R agonism. We conclude that the dopaminergic otpb population, via the DDT, is responsible for spinal D(4)R signaling to mediate the developmental switch to the mature locomotor pattern of zebrafish. These results, integrated with the mammalian literature, suggest that the DDT represents an evolutionarily conserved neuromodulatory system that is necessary for normal vertebrate locomotor development.

Analysis of zebrafish mutants that demonstrate abnormal locomotive behavior can elucidate the molecular requirements for neural network function and provide new models of human disease. Here, we show that zebrafish quetschkommode (que) mutant larvae exhibit a progressive locomotor defect that culminates in unusual nose-to-tail compressions and an inability to swim. Correspondingly, extracellular peripheral nerve recordings show that que mutants demonstrate abnormal locomotor output to the axial muscles used for swimming. Using positional cloning and candidate gene analysis, we reveal that a point mutation disrupts the gene encoding dihydrolipoamide branched-chain transacylase E2 (Dbt), a component of a mitochondrial enzyme complex, to generate the que phenotype. In humans, mutation of the DBT gene causes maple syrup urine disease (MSUD), a disorder of branched-chain amino acid metabolism that can result in mental retardation, severe dystonia, profound neurological damage and death. que mutants harbor abnormal amino acid levels, similar to MSUD patients and consistent with an error in branched-chain amino acid metabolism. que mutants also contain markedly reduced levels of the neurotransmitter glutamate within the brain and spinal cord, which probably contributes to their abnormal spinal cord locomotor output and aberrant motility behavior, a trait that probably represents severe dystonia in larval zebrafish. Taken together, these data illustrate how defects in branched-chain amino acid metabolism can disrupt nervous system development and/or function, and establish zebrafish que mutants as a model to better understand MSUD.

Background: The Deadly Trio programme offered cardiovascular health checks to men over 40 in inner-city Birmingham. The aim was to increase diagnosis of diabetes, cardiovascular and kidney disease among this deprived and ethnically diverse population. Either patients' own general practitioners (GPs) were paid to provide health checks or patients were invited to an alternative provider., Methods: Routine data were sought from 68 participating practices. Logistic regression analysis was undertaken to determine the patient and practice factors associated with being screened and with being added to a disease register., Results: Data were obtained from 58 practices; 5871 (24.3%) of 24 166 eligible men were screened. Screening uptake was higher in those with a recorded phone number, South Asians and Blacks but lower in smokers. Compared to the alternative provider, uptake was higher among men registered with single-handed (but not multi-partner) GPs paid to provide health checks. South Asian, older and screened men were more often added to disease registers. Men with missing information and GP-screened men were less likely to be added to registers., Conclusions: The programme achieved higher screening uptake and diagnosis of disease among minority ethnic men. Single-handed GPs paid to provide screening (and their patients) were more responsive than multi-partner practices.

Premise of the Study: Climate change has affected species worldwide, including alterations in phenology, migration patterns, distribution, and survival. Because Erythronium grandiflorum is an early-season bloomer, alterations in its phenology may have serious implications for many North American Rocky Mountain communities, including changes in resource availability for pollinators and herbivores. •, Methods: We investigated whether changes in the snowmelt date, summer temperature, and summer precipitation have altered the timing and abundance of flowering in E. grandiflorum by collecting long-term data on floral abundance from 1975-2008 in a series of 2 × 2 m plots at the Rocky Mountain Biological Laboratory (RMBL) in Gothic, Colorado in the United States. •, Key Results: Snowmelt date and mean summer temperature were negatively correlated. Over the 30-yr study, the snowmelt date advanced by 4.14 d/decade, and mean summer temperature increased by 0.38°C/decade. Summer precipitation was variable, showing no change. The first, peak, and last flowering dates of E. grandiflorum advanced an average of 3.2 d/decade. Furthermore, earlier snowmelt and greater summer precipitation in the previous year led to earlier flowering in E. grandiflorum. There was no change in flowering abundance in this species, indicating it may be controlled by a complex set of abiotic and biotic variables. •, Conclusions: Our study indicates that snowmelt is arriving earlier at the RMBL, which has caused earlier flowering in E. grandiflorum. Because alterations in phenology can disrupt important ecological interactions, information on potential phenological shifts in species that interact with E. grandiflorum is essential in determining the net effect of climate-driven alterations in phenology.

Topical anesthesia is routinely employed to facilitate diagnostic and therapeutic procedures that involve the oropharynx. Although this practice is usually safe, there is always the potential that methemoglobinemia might be induced by the topical anesthetic agent. The clinician should consider this possible complication when the patient experiences signs or symptoms of oxygen desaturation in the absence of another explanation. Because methemoglobinemia can be life-threatening, early recognition and treatment are extremely important.

An aggressive, non-native haplotype (distinct genetic lineage within a species) of Phragmites australis is invading brackish and freshwater systems in the eastern United States, potentially displacing native haplotypes. We studied the differential susceptibility of native and non-native populations collected from sites throughout North America to the non-native aphid, Hyalopterus pruni. In a greenhouse study, we found significantly higher aphid populations on native haplotypes than on the non-native haplotype 2 mo after infestation. Aphid feeding caused chlorosis and death of native stems, and in some cases, killed whole native genets. The non-native plants remained relatively undamaged. In a field study, non-native plants had significantly lower aphid densities than native plants or remained aphid free. There was an interactive effect in which aphid populations increased on the native plants over the 1-mo study period but remained low on non-native plants over the same period. The susceptibility of native North American populations of P. australis to non-native aphid infestation may indirectly affect the ability of these native plants to compete with non-native plant populations, ultimately contributing to the decline of native haplotypes.

Suppression of reactions to one noxious stimulus by a spatially distant noxious stimulus is termed heterotopic antinociception. In lightly anesthetized rats, a noxious visceral stimulus, colorectal distension (CRD), suppressed motor withdrawals but not blood pressure or heart rate changes evoked by noxious hindpaw heat. Microinjection of muscimol, a GABA(A) receptor agonist, into raphe magnus (RM) reduced CRD-evoked suppression of withdrawals, evidence that RM neurons contribute to this heterotopic antinociception. To understand how brain stem neurons contribute to heterotopic antinociception, RM neurons were recorded during CRD-elicited suppression of hindpaw withdrawals. Although subsets of RM neurons that were excited (on cells) or inhibited (off cells) by noxious cutaneous stimulation were either excited or inhibited by CRD, on cells were inhibited and off cells excited by an intracerebroventricularly administered opioid, evidence that the nociception-facilitating and -inhibiting functions of on and off cells, respectively, are predicted by the cellular response to noxious cutaneous stimulation alone and not by the response to CRD. When recorded during CRD-elicited antinociception, RM cell discharge resembled the pattern observed in response to CRD stimulation alone. However, when hindpaw withdrawal suppression was incomplete, RM cell discharge resembled the pattern observed in response to heat alone. We propose that on cells excited by CRD facilitate responses to CRD itself, which in turn augments excitation of off cells that then act to suppress cutaneous nociception. RM cells may thereby contribute to the dominance of quiet recuperative reactions evoked by potentially life-threatening visceral stimuli over transient somatomotor activity elicited by less-injurious noxious cutaneous stimuli.

Background: Up to 3% of patients undergoing heart surgery suffer from an intraabdominal complication (IAC). These complications carry a high mortality besides adding to the morbidity and cost. This review was undertaken to see if a subset of patients with increased risk of IAC could be identified., Methods: Medical records of 7,731 consecutive patients undergoing heart surgery in a single center were screened for identification of postoperative IAC. One hundred and twenty (120) cases were found. One hundred and six (106) cases were compared with the same number of matched controls., Results: Significant predictors of the development of IAC were increased cardiopulmonary bypass times (> 99 minutes), peripheral vascular disease, chronic steroid use, and low left ventricular ejection fraction. Patients on postoperative antiplatelet therapy or warfarin had a lower risk of IAC. Significant predictors of mortality in IAC were increased cardiopulmonary bypass times (> or = 120 minutes.), use of inotropes, cerebral vascular disease, and incremental age., Conclusions: A subset of patients can be identified who are at higher risk for IAC and an associated adverse outcome. Patients who have prolonged cardiopulmonary bypass, have a low left ventricular ejection fraction, are on steroids, and suffer from other vascular disease should be observed carefully for development of IAC. Postoperative anticlotting strategies may be helpful. Early diagnosis and intervention are essential for improving outcomes in cases of IAC.

Pregnant Sprague-Dawley rats were exposed to ethylbenzene (EB; 0, 250, or 1000 ppm) and methylethylketone (MEK; 0, 1000, or 3000 ppm), alone and in combination, by inhalation, for 6h/day, during days 6-20 of gestation. Maternal toxicity, evidenced by decreased in body weight gain and food consumption, tended to be greater after simultaneous exposures to the high concentrations of 1000 ppm EB and 3000 ppm MEK, when compared to the treatments with individual compounds. No significant increase in embryo/fetal lethality or incidence of malformations and variations was observed in any of the treatment groups. Fetal body weight was significantly reduced after individual treatment with 1000 ppm EB or 3000 ppm MEK, and in the combined groups. There was no evidence of interaction between EB and MEK in causing developmental toxicity.

The current experiments were undertaken to determine whether or not styrene-induced hearing loss in the rat depends more on the existence of a critical period between 14 and 21 weeks of age than on body weight. For these purposes, two experiments were carried out with mature Long-Evans rats. In the first experiment, two groups of 5-month old rats, but having different body weight (slim: 314 g vs. fat: 415 g) were exposed to 700 ppm styrene for 4 consecutive weeks, 5 days per week, 6 hours per day. In the second experiment, two groups of rats having the same weight: 345 g, but different ages (14- vs. 21- week old) were exposed to styrene in strictly identical experimental conditions. Auditory sensitivity was tested by recording evoked potentials from the inferior colliculus. Surface preparations of the organ of Corti were also performed to complete the investigation. At the end of the six week recovery period following the styrene exposure, a 7 dB permanent threshold shift (PTS) was obtained with the same age animals regardless of the body weight. Consequently, weight was not a major factor in styrene-induced hearing loss. Age was a more critical factor in determining higher sensitivity to styrene. Indeed, the three months old group had 23.5 dB PTS, whereas the five months old group had only a 7.7 dB PTS at 16 kHz. Thus, a 15 dB difference of PTS was obtained between the rats having the same weight but different age. While the weight does not play a major role in styrene ototoxicity, there is a critical period whose duration lasts more than three months and for which the susceptibility to styrene is enhanced.

We describe here a new alpha-tubulin isoform from sunflower we named alpha(pi)-tubulin. Alpha(pi)-tubulin is the most divergent higher-plant alpha-tubulin described so far, having an unusual deletion in the H1/B2 loop and a glutamine-rich C-terminus. We constructed a three-dimensional model and discuss its implications. Using specific antibodies, we show that alpha(pi)-tubulin expression is restricted to the male gametophyte. Alpha(pi)-tubulin mRNA represents 90% of alpha-tubulin mRNA and a small percentage of total pollen mRNA. Among the plants tested, alpha(pi)-tubulin was only detected in sunflower and in Cosmos. Since both plants are Asteraceae, we propose that alpha(pi)-tubulin is specific to this family. Our results suggest that alpha(pi)-tubulin can inhibit tubulin assembly in pollen. This hypothesis is reinforced by the fact that alpha(pi)-tubulin is found in a complex with beta-tubulin in mature sunflower pollen.

The molecular basis of microtubule nucleation is still not known in higher plant cells. This process is better understood in yeast and animals cells. In the yeast spindle pole body and the centrosome in animal cells, gamma-tubulin small complexes and gamma-tubulin ring complexes, respectively, nucleate all microtubules. In addition to gamma-tubulin, Spc98p or its homologues plays an essential role. We report here the characterization of rice and Arabidopsis homologues of SPC98. Spc98p colocalizes with gamma-tubulin at the nuclear surface where microtubules are nucleated on isolated tobacco nuclei and in living cells. AtSpc98p-GFP also localizes at the cell cortex. Spc98p is not associated with gamma-tubulin along microtubules. These data suggest that multiple microtubule-nucleating sites are active in plant cells. Microtubule nucleation involving Spc98p-containing gamma-tubulin complexes could then be conserved among all eukaryotes, despite differences in structure and spatial distribution of microtubule organizing centers.

The assembly of the higher plant cytoskeleton poses several fundamental questions. Since different microtubule arrays are successively assembled during the cell cycle in the absence of centrosomes, we can ask how these arrays are assembled and spatially organized. Two hypotheses are under debate. Either multiple nucleation sites are responsible for the assembly and organization of microtubule arrays or microtubule nucleation takes place at one site, the nuclear surface. In the latter case, microtubule nucleation and organization would be two distinct but coregulated processes. During recent years, novel approaches have provided entirely new insights to understand the assembly and dynamics of the plant cytoskeleton. In the present review, we summarize advances made in microscopy and in molecular biology which lead to novel hypotheses and open up new fields of investigation. From the results obtained, it is clear that the higher plant cell is a powerful model system to investigate cytoskeletal organization in acentrosomal eukaryotic cells., (Copyright 2000 Wiley-Liss, Inc.)

Centrin - higher plants - MTOCs - microtubules nucleation In most eukaryotic cells, the Ca(2+)-binding protein centrin is associated with structured microtubule-organizing centers (MTOCs) such as centrosomes. In these cells, centrin either forms centrosome-associated contractile fibers, or is involved in centrosome biogenesis. Our aim was to investigate the functions of centrin in higher plant cells which do not contain centrosome-like MTOCs. We have cloned two tobacco BY-2 centrin cDNAs and we show that higher plant centrins define a phylogenetic group of proteins distinct from centrosome-associated centrins. In addition, tobacco centrins were found primarily associated with microsomes and did not colocalize with gamma-tubulin, a known MTOC marker. While the overall level of centrin did not vary during the cell cycle, centrin was prominently detected at the cell plate during telophase. Our results suggest that in tobacco, the major portion of centrin is not MTOC-associated and could be involved in the formation of the cell plate during cytokinesis.

The aim of this work on rat hepatic P450 2E1 activity was to seek the most suitable experimental model to study the role of cytochrome P450 2E1 in the metabolism of industrial chemicals. Two sets of experiments were devoted to selecting the age and sex of animals and to estimating the response of male and female rats to different inducers. In the first set, the effect of three inducers (fasting; ethanol; acetone) was studied in male rats aged 5, 7 and 9 weeks. In the second set, the effect of different inducers, namely beta-naphthoflavone (BNF), phenobarbital (PB), ethanol, acetone and pyridine, on PNP and chlorzoxazone (CLZO) hydroxylase activities was studied in 7 week old male and female rats. The results demonstrate firstly that microsomal p-nitrophenol (PNP) hydroxylase activity significantly decreases in control male rats in inverse function of age, and secondly that induction by ethanol decreases with age. The PNP hydroxylase activity level of controls and the significant increases in PNP hydroxylase activity observed in 7 week old male rats show that this is the most suitable age for the second set of experiments. In this second set, it was shown that P450 1A (induced by BNF) is involved in CLZO hydroxylase activity only. PB increased the hydroxylase activities in male and female rats by about 1.5 and 1.7 times those of the controls, respectively. The effects of P450 2E1 inducers in function of sex show that male rats exhibited more significant increases in PNP and CLZO hydroxylase activities than female. The specificity of these two substrates is discussed. Neither of these two reactions was specifically catalysed by P450 2E1, but PNP may be considered as the most specific and the least sensitive substrate. In addition, the linear relationship observed between the two substrates (PNP and CLZO) showed a good correlation between their activities (r = 0.90, P < 0.001). In conclusion, these results suggest the use of the 7 week old male rat as the experimental model to study the role of cytochrome P450 2E1 in the hepatotoxicity of low molecular weight industrial chemicals.

The structure and the molecular composition of the microtubule-organizing centers in acentriolar higher plant cells remain unknown. We developed an in vitro complementation assay where tobacco BY-2 extracts can restore the microtubule-nucleating activity of urea-inactivated mammalian centrosomes. Our results provide first evidence that soluble microtubule-nucleating factors are present in the plant cytosolic fraction. The implication for microtubule nucleation in higher plants is discussed.

Compelling evidence supports the idea that actin filaments play an active role in the cytokinetic process of higher plant cells. However, the mechanisms that control the growth of the cell plate and its stabilization remain so far unknown. We show that a novel population of short actin filaments continuously assembles in the phragmoplast at the growing cell plate. Microinjection of rhodamine-phalloidin during these final stages of telophase revealed the dynamic assembly and organization of these actin filaments during vesicle fusion. Comparable data were obtained in endosperm syncytia during the development of the cell plate between non sister nuclei, i.e. independently of the formation of the mitotic phragmoplast. Concomitantly, plant polypeptides sharing epitopes with human vinculin are revealed within the forming cell plate, suggesting their recruitment during cytokinesis-associated actin assembly. These vinculin-like antigens may participate in membrane/F-actin anchorage protein complexes. Our data, in addition to the identification of plant integrin homologues reported by several authors, suggest the existence of a cell wall/extracellular matrix/plasma membrane/actin cytoskeleton continuum. Such an architecture may control cell-cell interactions during cell plate formation and may contribute to the establishment of polarity in higher plants.

Moderate nephrotoxicity was induced in male and female rats exposed to o-xylene for 4 h at atmospheric concentrations of approximately 3000 ppm. The xylene in vivo nephrotoxicity resulted in low enzyme leakage from the kidney into the urine. This low leakage was confirmed in 24-h urine by an increase in gamma-glutamyltranspeptidase (gammaGT), N-acetyl-beta-D-glucosaminidase (NAG) and alkaline phosphatase (ALP) activities. Compared to the control, both the 24-h urine output and the glucose excretion increased in male and female rats. These increases were probably a result of damage to the renal proximal tubules. The role of the metabolic pathway of glutathione in the emergence of the renal damage observed with o-xylene was investigated in rats. Recent studies indicate that the metabolic pathway of glutathione may be a bioactivation pathway, which is responsible for nephrotoxic effects with several drugs or chemicals. The renal toxicity of three synthesized o-xylene thio-conjugates was investigated in several groups of female rats. Administration of S-(o-methylbenzyl)glutathione (i.p., 1 mmol/kg), S-(o-methylbenzyl)cysteine (per os, 1 mmol/kg) or N-acetyl-S-(o-methylbenzyl)cysteine (i.p., 0.75 mmol/kg) to female rats did not induce renal toxicity, as monitored by urinary biochemical parameters (gammaGT, NAG, ALP, glucose). The data obtained suggest that the glutathione pathway would appear to be only detoxication, and probably does not contribute to the renal toxicity of o-xylene in female rats. Thus, either another metabolic pathway or other intermediate metabolites are probably involved in the nephrotoxic action of o-xylene.

Male Sprague-Dawley rats were administered a single intraperitoneal injection of N, N-dimethylformamide (DMF, 0.01-1.5 g kg-1) or were exposed for 4 h to DMF vapours (75-900 ppm). The serum activities of the enzymes sorbitol deshydrogenase and glutamate deshydrogenase were used as indicators of liver damage, and were determined at 24, 48 or 72 h post-treatment. Following either route of administration DMF caused concentration-dependent elevations in enzyme activities, the maxima of which occurred later after administration of higher concentrations of DMF than after lower concentrations.

The propagation of several modes in an optical fiber is not easy to study. The experiment that we propose permits us to measure the difference in time propagation between two successive modes of a multimode fiber. The same laser beam is coupled into the fiber to be tested and into the reference single-mode fiber. The correlation of output electric fields of the modes propagated by each fiber is realized by an interferometric system.

Recognition of homologous chromosomes during meiotic prophase is associated in most cases with the formation of the synaptonemal complex along the length of the chromosome. Telomeres, located at the nuclear periphery, are preferential initiation sites for the assembly of the synaptonemal complex. In most eukaryotic cells, telomeres cluster in a restricted area, leading to the "bouquet" configuration in leptotene-zygotene, while this typical organization progressively disappears in late zygotene-pachytene. We wondered whether such striking changes in the intranuclear ordering and pairing of meiotic chromosomes during the progression of prophase I could be correlated with activity of the centrosome and/or microtubule-organizing center (MTOC). Plant cells may be used as a model of special interest for this study as the whole nuclear surface acts as an MTOC, unlike other cell types where MTOCs are restricted to centrosomes or spindle pole bodies. Using a monoclonal antibody (mAb 6C6) raised against isolated calf centrosomes we found that the 6C6 antigen is present over the entire surface of the plant meiotic nucleus, in early prophase I, before chromosomal pairing. At zygotene, short fragments of chromosomes become stained near the nuclear envelope and within the nucleus. At pachytene, after complete synapsis, the labeling specifically concentrates within the synaptonemal complexes, although the nuclear surface is no longer reactive. Ultrastructural localization using immunogold labeling indicates that the 6C6 antigen is colocalized with the synaptonemal complex structures. Later in metaphase I, the antigen is found at the kinetochores. Our data favor the idea that the 6C6 antigen may function as a particular "chromosomal passenger-like" protein. These observations shed new light on the molecular organization of the plant synaptonemal complex and on the redistribution of cytoskeleton-related antigens during initiation of meiosis. They suggest that antigens of MTOCs are relocated to chromosomes during the synapsis process starting at telomeres and contribute to the spatial arrangement of meiotic chromosomes. Such cytoskeleton-related antigens may acquire different functions depending on their localization, which is cell-cycle regulated.

Ethylene glycol monomethyl ether (EGME) exhibits testicular toxicity and ethylene glycol monobutyl ether (EGBE) is a solvent with haemolytic effects in rats. The study of the interaction of two glycol ethers (EGME and EGBE) and three alcohols (ethanol, n-propanol and n-butanol, 10 or 30 mmol/kg), orally co-administered in male rats, was carried out from a toxicodynamic and toxicokinetic point of view. Administered alone, EGME (10 mmol/kg) caused a 30- and 5-fold increase in the urinary creatine/creatinine ratio at 24 and 48 h, respectively, and 24 h urinary excretion of methoxyacetic acid was of 0.71 +/- 0.042 mmol 24 h (mean +/- SE). The simultaneous administration of one of the three alcohols at either of the doses mentioned above did not significantly modify the urinary creatine/creatinine ration (24 and 48 h), or the 24 h urinary excretion of methoxyacetic acid. Administered alone, EGBE (5 mmol/kg) caused an average decrease of 26% in the number of circulating red blood cells and a strong (250 times) increase in the level of plasma haemoglobin 4 h after treatment. Urinary excretion of butoxyacetic acid in rats treated with EGBE (1 mmol/kg) was 0.083 +/- 0.0039 mmol/24 h (mean +/- SE). The simultaneous injection of 30 mmol/kg alcohol (ethanol, n-propanol or n-butanol) almost totally inhibits the haemolytic effect of EGBE, and decreases the urinary excretion of butoxyacetic acid by 43-31%. A strong dose of alcohol (30 mmol/kg) decreases the haemolytic effect due to EGBE, and reduces the urinary excretion of butoxyacetic acid. In contrast, the coadministration of alcohol did not modify the testicular toxicity of EGME, or the 24 h urinary excretion of methoxyacetic acid. It is possible that competitive inhibition of alcohol dehydrogenase by alcohols results in the diversion of EGBE metabolism.

In this study, we investigated the effect of plant microtubule-associated proteins (MAPs) on microtubule nucleation and growth in vitro. Since it has recently been demonstrated that plant nuclear surface acts as a microtubule-organizing center (MTOC), we tested the effects of plant MAPs using a nucleus-mediated microtubule nucleation assay. Nuclei were isolated from interphase tobacco BY-2 cells, and MAPs were isolated from tobacco BY-2 cells at different stages of the cell cycle. The effects of tobacco MAPs on microtubule nucleation at mammalian centrosomes were also analyzed. Under our experimental conditions, both interphase and mitotic tobacco MAPs promoted microtubule assembly around tobacco nuclei and at mammalian centrosomes below the critical tubulin concentration for spontaneous assembly. Interphase tobacco MAPs increase the mean length of nucleated microtubules in proportion to its molar ratio to tubulin. In contrast, mitotic tobacco MAPs do not induce nucleus- and centrosome-mediated nucleation of microtubules in a dose-dependent manner. Both MAP-fractions possessed microtubule bundling activity. The implications of these plant MAP properties on microtubule nucleation in living cells are discussed.