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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: 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.

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.)

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.)

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.

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.

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.

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 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.

Plants can adapt their shape to environmental stimuli. This response is mediated by the reorganization of cortical microtubules, a unique element of the cytoskeleton. However, the molecular base of this response has remained obscure so far. In an attempt to solve this problem, signal-dependent changes in the pattern of microtubule-binding proteins were analysed during coleoptile elongation in maize, that is, under the control of the plant photoreceptor phytochrome. Two putative MAPs of 100 kDa (P100) and 50 kDa apparent molecular weights were identified in cytosolic extracts from non-elongating and elongating cells. Both proteins co-assembled with endogenous tubulin, bound to neurotubules and were immunologically related to the neural MAP tau: the P100 protein, depending on the physiological situation, was manifest as a double band and was always found to be heat-stable. In contrast, the 50 kDa MAP was heat-stable only for particular tissues and physiological treatments. The P100 protein was present in all tissues, however in a reduced amount in elongating coleoptiles. The 50 kDa MAP was expressed exclusively upon induction of phytochrome-dependent cell elongation. As shown by immunofluorescence double-staining, an epitope shared by both proteins colocalized with cortical microtubules in situ, but exclusively in elongating cells. In non-elongating cells, only the nuclei were stained. Partially purified nuclei from elongating cells were enriched in P100, whereas the 50 kDa MAP became enriched in a partially purified plasma membrane fraction.

Compelling evidence has been obtained in favour of the idea that the nuclear surface of higher plant cells is a microtubule-nucleating and/or organizing site (MTOC), in the absence of defined centrosomes. How these plant MTOC proteins are redistributed and function during the progression of the cell cycle remains entirely unknown. Using a monoclonal antibody (mAb 6C6) raised against isolated calf thymus centrosomes and showing apparent reaction with the plant nuclear surface, we followed the targeted antigen distribution during mitosis and meiosis of higher plants. Immunoblot analysis of protein fractions from Allium root meristematic cell extracts probed with mAb 6C6 reveals a polypeptide of an apparent Mr of 78000. In calf centrosome extracts, a polypeptide of comparable molecular mass is found in addition to a major antigen of Mr 180000 after mAb 6C6 immunoblotting. During mitotic initiation, the plant antigen is prominent on the periphery of the prophase nucleus. When the nuclear envelope breaks down, the antigen suddenly becomes associated with the centromere-kinetochores until late anaphase. In telophase, when the nuclear envelope is being reconstructed, it is no longer detected at the kinetochores but is solely associated again with the nuclear surface. This antigen displays a unique spatial and temporal distribution, which may reflect the pathway of plant protein(s) between the nuclear surface and the kinetochores under cell cycle control. So far, such processes have not been described in higher plant cells. These observations shed light on the putative activity of the plant kinetochore as a protein transporter.(ABSTRACT TRUNCATED AT 250 WORDS)

In most eukaryotic cells, microtubules (MTs) are assembled at identified nucleating sites, such as centrosomes or spindle pole bodies. Higher plant cells do not possess such centrosome-like structures. Thus, the fundamental issues of where and how the intracellular plant MTs are nucleated remain highly debatable. A large body of evidence indicates that plant MTs emerge from the nuclear periphery. In this study, we developed an in vitro assay in which isolated maize nuclei nucleate MT assembly at a tubulin concentration (14 [mu]M of neurotubulin) that is not efficient for spontaneous MT assembly. No MT-stabilizing agents, such as taxol or dimethyl sulfoxide, were used. Our model provides evidence that the nuclear surface functions as a MT-nucleating site in higher plant cells. A monoclonal antibody raised against a pericentriolar antigen immunostained the surface of isolated nuclei, and a 100-kD polypeptide in 4 M urea-treated nuclear extracts was detected.

In higher-plant cells, the different cell-cycle-dependent microtubule arrays are involved in a wide range of activities including chromosome segregation, cell-plate formation and cellulose microfibril distribution and orientation. A wealth of data, obtained using animal cells, has indicated that the differential stability and function of microtubules during cell-cycle and/or differentiation could be primarily regulated by selective microtubule-associated proteins (MAP). Compared to animal MAP, our knowledge of plant MAP is so far very limited. In this study, we have identified a maize heat-stable protein with apparent molecular mass 100 kDa (P-100) which binds to taxol-stabilized neurotubules and copolymerizes in vitro with purified neural tubulin. Moreover, P-100 cross-reacts with affinity-purified tau antibodies like a maize 83-kDa putative MAP described previously [Vantard, M., Schellenbaum, P., Fellous, A. & Lambert, A. M. (1991) Biochemistry 30, 9334-9340]. Polyclonal antibodies directed against P-100 were obtained and indicated that this protein is found in diverse higher-plant cultured cells suggesting the ubiquitous nature of this protein. P-100 can be phosphorylated in vitro by protein kinases present in a maize cytosol extract. Together, our data suggest that P-100 could be a higher plant MAP.

We have used monoclonal antibodies raised against isolated native calf thymus centrosomes to probe the structure and composition of the pericentriolar material. To distinguish prospective antibodies as specific to conserved elements of this material, we screened clones by their identification of microtubule organizing centers (MTOCs) in different animal and plant cells. Among the clonal antibodies that reacted with MTOCs in both plant and mammalian cells, we describe one (mAb 6C6) that was found to immunostain centrosomes in a variety of bovine and human cells. In cycling cells this signal persisted through the entire cell cycle. Microscopy showed that the mAb 6C6 antigen was a component of the pericentriolar material and this was confirmed by biochemical analysis of centrosomes. Using immunoblot analysis of protein fractions derived from purified components of centrosomes, we have characterized the mAb 6C6 antigen as a 180 kDa polypeptide. We conclude that we have identified a protein component permanently associated with the pericentriolar material. Surprisingly, monoclonal antibody 6C6 also stained other mitotic organelles in mammalian cells, in a cell-cycle-dependent manner. During prometaphase and metaphase the antibody stained both centrosomes and kinetochores. At the onset of anaphase the kinetochore-specific staining dissociated from chromosomes and was subsequently redistributed onto a newly characterized organelle, the telophase disc while the centrosomal stain remained intact. It is not known if the 180 kDa centrosomal protein itself redistributes during mitosis, or if the pattern observed represents other antigens with shared epitopes. The pericentriolar material is thought to be composed of conserved elements, which appeared very early during the evolution of eukaryotes. Our results strongly suggest that mAb 6C6 identifies one of these elements.

Incorporation of Paramecium axonemal tubulin into lysed endosperm cells of the higher plant Haemanthus enabled us to identify sites of microtubule assembly. This exogenous Paramecium tubulin could be traced by specific antibodies that do not stain endogenous plant microtubules. Intracellular copolymerization of protozoan and higher plant tubulins gave rise to hybrid polymers that were visualized by immunofluorescence and by immunoelectron microscopy. The addition of exogenous tubulin revealed many free ends of endogenous microtubules that were competent to assemble ciliate tubulin. The functional roles of the nuclear surface and the equatorial region of the phragmoplast as plant microtubule-organizing centers, which were revealed by the intense incorporation of exogenous tubulin, are discussed. These data shed light on the present debate on higher plant microtubule organizing centers.

Endosperm mitotic cells microinjected with fluorescent phalloidin enabled us to follow the in vivo dynamics of the F-actin cytoskeleton. The fluorescent probe immediately bound to plant microfilaments. First, we investigated the active rearrangement of F-actin during chromosome migration, which appeared to be slowed down in the presence of phalloidin. These findings were compared with the actin patterns observed in mitotic cells fixed at different stages. Our second aim was to determine the origin of the actin filaments that appear at the equator during anaphase-telophase transition. It is not clear whether this F-actin is newly assembled at the end of mitosis and could control plant cytokinesis or whether it corresponds to a passive redistribution of broken polymers in response to microtubule dynamics. We microinjected the same cells twice, first in metaphase with rhodamine-phalloidin and then in late anaphase with fluorescein isothiocyanate-phalloidin. This technique enabled us to visualize two F-actin populations that are not co-localized, suggesting that actin is newly assembled during cell plate development. These in vivo data shed new light on the role of actin in plant mitosis and cytokinesis.

Study of the light scattered from an unclad optical fiber illuminated by a laser beam perpendicular to its axis gives a valuation of little defects of the fiber cross section. The shifts of some bright fringes that occur when the fiber is rotated are observed. Comparison between experimental measurements and ray interference computations shows that the cross section of the fiber under test is neither circular nor elliptical, but can be described with good approximation by a limited Fourier series, the coefficients of which are estimated.

We have identified an F-actin cytoskeletal network that remains throughout interphase, mitosis, and cytokinesis of higher plant endosperm cells. Fluorescent labeling was obtained using actin monoclonal antibodies and/or rhodamine-phalloidin. Video-enhanced microscopy and ultrastructural observations of immunogold-labeled preparations illustrated microfilament-microtubule co-distribution and interactions. Actin was also identified in cell crude extract with Western blotting. During interphase, microfilament and microtubule arrays formed two distinct networks that intermingled. At the onset of mitosis, when microtubules rearranged into the mitotic spindle, microfilaments were redistributed to the cell cortex, while few microfilaments remained in the spindle. During mitosis, the cortical actin network remained as an elastic cage around the mitotic apparatus and was stretched parallel to the spindle axis during poleward movement of chromosomes. This suggested the presence of dynamic cross-links that rearrange when they are submitted to slow and regular mitotic forces. At the poles, the regular network is maintained. After midanaphase, new, short microfilaments invaded the equator when interzonal vesicles were transported along the phragmoplast microtubules. Colchicine did not affect actin distribution, and cytochalasin B or D did not inhibit chromosome transport. Our data on endosperm cells suggested that plant cytoplasmic actin has an important role in the cell cortex integrity and in the structural dynamics of the poorly understood cytoplasm-mitotic spindle interface. F-actin may contribute to the regulatory mechanisms of microtubule-dependent or guided transport of vesicles during mitosis and cytokinesis in higher plant cells.

In this study we have examined the immunocytochemical distribution of calmodulin during mitosis of higher plant endosperm cells. Spindle development in these cells occurs without centrioles. Instead, asterlike microtubule converging centers appear to be involved in establishing spindle polarity. By indirect immunofluorescence and immunogold staining methods with anti-calmodulin antibodies, we found endosperm calmodulin to be associated with the mitotic apparatus, particularly with asterlike and/or polar microtubule converging centers and kinetochore microtubules, in an immunocytochemical pattern distinct from that of tubulin. In addition, endosperm calmodulin and calcium showed analogous distribution profiles during mitosis. Previous reports have demonstrated that calmodulin is associated with the mitotic apparatus in animal cells. The present observation that calmodulin is also associated with the mitotic apparatus in acentriolar, higher plant endosperm cells suggests that some of the regulatory mechanisms involved in spindle formation, microtubule disassembly, and chromosome movement in plant cells may be similar to those in animal cells. However, unlike animal cell calmodulin, endosperm calmodulin appears to associate with kinetochore microtubules throughout mitosis, which suggests a specialized role for higher plant calmodulin in the regulation of kinetochore microtubule dynamics.

In this paper, the intrinsic diameter of the noncircular cross section of an unclad optical fiber is defined and determines the fiber dimension. Its value can be determined by studying the forwardscattering pattern from the fiber illuminated by a laser beam perpendicular to its axis. A knowledge of the shape of the fiber is not necessary. The suggested method is nondestructive. Moreover, it provides an estimate of the deformation of the fiber cross section.

A procedure is presented for the immunocytochemical visualization of microtubules in interphase and mitotic cells of Haemanthus endosperm. It includes preservation of microtubules (MTs) with glutaraldehyde and uses colloidal gold, coated with secondary antibodies, in a novel indirect-light microscopic technique: the immuno-gold staining method. This immunocytochemical stain allows us to follow the changes in distribution of MTs during mitosis with greater precision and specificity than allowed by other light microscopic techniques. Many aspects of MT arrangements, as reported from ultrastructural studies, are corroborated and extended. This demonstrates the reliability of the technique. In addition, a number of significant observations were made. These concern (i) the presence of a network of MTs in interphase cells, (ii) the transformation of this network into a spindle-like cage of MTs (the clear zone) surrounding the nucleus during prophase, (iii) the drastic rearrangement of MT distribution during prometaphase, (iv) new evidence for the formation of aster-like arrays of polar MTs during anaphase, and (v) the development of the phragmoplast.