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Background: The introduction of cyclin-dependent kinase inhibitors (CDK4/6i) was a great advance in therapeutics for patients with estrogen receptor+/human epidermal growth factor receptor (HER2) locally advanced and metastatic breast cancer. Despite the increasing use of these agents, their adverse drug-related events have not yet been fully characterized. We describe the spectrum of cutaneous adverse reactions occurring in advanced breast cancer patients treated with cyclin-dependent kinase inhibitors, analyzing types, severity, time to onset, and possible treatment outcomes., Methods: We performed a multicentric retrospective study including patients with advanced breast cancer who developed cutaneous lesions during treatment with CDK4/6i in the period from June 2020 to June 2021. Patients > 18 years were recruited at eleven onco-dermatology units located in Albania (1), Argentina (1), France (1), Greece (3), Italy (3), and Spain (2). We evaluated patients' epidemiological and clinical characteristics, types of cutaneous adverse events, their time to onset, and treatment outcomes. The severity of the skin reactions was assessed using the Common Terminology Criteria for Adverse Events (CTCAE) version 5.0 score., Results: Seventy-nine patients (median age: 62.3 years; range 39-83 years) were included in the study, and, collectively, we recorded a total of 165 cutaneous adverse events during follow-up visits. The most frequent cutaneous reactions were pruritus (49/79 patients), alopecia (25/79), and eczematous lesions (24/79). Cutaneous toxicities were usually mild in severity (>65%) and occurred after a median of 6.5 months. Only four patients (5%) required treatment discontinuation due to the severity of the skin lesions. The majority of the skin reactions were managed with topical treatments., Conclusions: To the best of our knowledge, we present the largest case series of cutaneous adverse events developing in advanced breast cancer patients treated with CDK4/6i. We showed that cutaneous toxicities are usually mild in severity, and manageable with standard supportive care; however, in selected cases, they can lead to treatment discontinuation with possible implications for patients' clinical outcomes.

Background: The morbidity and mortality of extraparenchymal neurocysticercosis (EP-NC) remain high and effectiveness of current medical treatment is suboptimal. Various factors have been implicated in the severity of EP-NC and in the poor response to treatment, but the possible role of host immune and endocrine systems has not yet been examined thoroughly., Methodology/principal Findings: 42 participants with EP-NC before receiving standard treatment and 25 healthy controls were included in the study. Treatment response was assessed by comparing pre/post treatment parasite volumes from 3D MRI. Prior to treatment among participants with EP-NC, specific stimulation induced an increased specific proliferative response accompanied by a significant increase in IL-4, NK, NKT, Bregs and Tregs cells, whereas in healthy controls, specific stimulation induced a significant increase in IL-1β, IL-5, CCL5, IL-6, TNF-α, NK and Bregs cells. Significant differences between participants with EP-NC and healthy controls in the specific inflammatory response were observed. Participants with EP-NC prior to treatment had significantly weaker responses of proinflammatory cytokines (IL-6, TNF-α) and NK cells, and stronger IL-4 response. Anthelmintic treatment did not promote significant peripheral immunological changes at any time, although inflammation was sustained in the cerebrospinal fluid. Serum estradiol concentration significantly decreased after anthelmintic treatment among males, and cortisol correlated negatively with IL-6 and positively with IFN-γ levels. No pre-treatment immunologic or endocrinologic parameters were significantly associated with response to treatment., Conclusion/significance: Prior to anthelmintic treatment, EP-NC was characterized by low lymphocyte reactivity accompanied by a regulatory response, which may be involved in the lack of peripheral immunological changes during and after treatment, although a central inflammatory response was present. This weak specific peripheral response could favor the chronicity of the infection and the poor response to treatment. Our findings highlight the need for new anti-inflammatory treatment focused on the central nervous system with less systemic immunosuppressive effects., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Romo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Anti-coagulant rodenticides (ARs) are commonly utilized for controlling rodent populations; however, non-target companion and wildlife animals are also exposed. A method was developed for quantitation of seven ARs (chlorophacinone, coumachlor, bromadiolone, brodifacoum, difethialone, diphacinone and warfarin) and dicoumarol (a naturally occurring anti-coagulant) in animal serum. Analytes were extracted with 10% (v/v) acetone in methanol and analyzed by reverse phase high-performance liquid chromatography-tandem mass spectrometry using electrospray ionization (negative mode) combined with multiple reaction monitoring. In-house method validation in the originating laboratory using non-blinded samples revealed method limits of quantitation at 2.5 ng/mL for all analytes. The inter-assay accuracy ranged from 99% to 104%, and the relative standard deviation ranged from 3.5% to 20.5%. Method performance was then verified in the originating laboratory during an exercise organized by an independent party using blinded samples. The method was successfully transferred to two naïve laboratories and further evaluated for reproducibility among three laboratories by means of Horwitz ratio (HorRat(R)) values. Such extensive validation provides a high degree of confidence that the method is rugged, robust, and will perform as expected if used by others in the future., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background/aim: Telemedicine, the remote delivery of healthcare services, represents a great opportunity for cancer pain management. A care model of telemedicine that combines remote visits and hospital access could be an effective and safe strategy for pain management of cancer patients., Patients and Methods: A retrospective study was conducted using the dataset of the telemedicine program at the Istituto Nazionale Tumori of Naples, Italy for assessing the efficacy of a telehealth-based model of care. Demographic, clinical, and process variables were investigated., Results: A total of 226 cases and 489 visits were included in the analysis. The mean age of patients was 63.4 years (SD=12.4 years), and no sex differences were observed. Approximately 55% of patients were ECOG-PS ≤2 and 87% suffered from metastatic disease. More than half of the patients were treated with high doses of opioids. Each patient had a mean of 2 remote visits and half of the patients had more than 1 telehealth consultation. The dropout ratio was 5.3%. Most visits (n=472) were conducted on patients in the Campania Region, Italy. The maximum covered distance from the Cancer Center and the patients' location was 555.22 Km. A significant difference in the overall number of visits (p=0.006) and the number of pro-capita remote visits (p=0.010) was found, in favor of the group of patients treated before the end of the Covid-19 emergency in Italy, compared to those treated after the pandemic., Conclusion: Despite various positive outcomes, the analysis highlights several weaknesses, such as the need to assist patients with advanced and non-advanced disease located outside the regional territory. Overall, the telehealth processes should be adapted to post-pandemic scenarios towards their implementation in routine clinical practice., (Copyright © 2023, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background: The most effective strategy for managing cancer pain remotely should be better defined. There is a need to identify those patients who require increased attention and calibrated follow-up programs. Methods: Machine learning (ML) models were developed using the data prospectively obtained from a single-center program of telemedicine-based cancer pain management. These models included random forest (RF), gradient boosting machine (GBM), artificial neural network (ANN), and the LASSO−RIDGE algorithm. Thirteen demographic, social, clinical, and therapeutic variables were adopted to define the conditions that can affect the number of teleconsultations. After ML validation, the risk analysis for more than one remote consultation was assessed in target individuals. Results: The data from 158 patients were collected. In the training set, the accuracy was about 95% and 98% for ANN and RF, respectively. Nevertheless, the best accuracy on the test set was obtained with RF (70%). The ML-based simulations showed that young age (<55 years), lung cancer, and occurrence of breakthrough cancer pain help to predict the number of remote consultations. Elderly patients (>75 years) with bone metastases may require more telemedicine-based clinical evaluations. Conclusion: ML-based analyses may enable clinicians to identify the best model for predicting the need for more remote consultations. It could be useful for calibrating care interventions and resource allocation.

Background: Since cancer pain requires complex modalities of care, the proper strategy for addressing its telemedicine-based management should be better defined. This study aimed to trace a pathway for a progressive implementation of the telemedicine process for the treatment of pain in the setting of cancer patients. Methods: The features of the model were investigated to dissect the dropout from the telemedicine pathway. A cross-sectional patient satisfaction study was conducted. The degree of satisfaction was evaluated through a developed 22-item questionnaire (Likert scale 0−7). Results: A total of 375 video consultations for 164 patients (mean age 62.9 years) were performed through remote consultations for cancer pain management between March 2021 and February 2022. After the exclusion of 72 patients, 92 (56.1%) were included in the analysis. The dropout ratio was 8.7%. The number of visits and pharmacological therapies for neuropathic pain correlated with the risk for readmission (p < 0.05). Overall, the satisfaction was very high (mean > 5.5 for all items). Conclusion: Feedback from patients reflected high satisfaction rates with the care provided. A methodological approach based on the degree of satisfaction combined with the analysis of the pathways can help to implement the quality of a service provided through telemedicine. While not without limitations, our hybrid protocol can be useful for addressing cancer pain through a patient-centered approach.

Poisoning of nontarget species is a major concern with the use of anticoagulant rodenticides (ARs). At postmortem examination, differentiating toxicosis from incidental exposure is sometimes difficult. Clotting profiles cannot be performed on postmortem samples, and clinically significant serum, blood, and liver AR concentrations are not well-established in most species. We chose diphacinone for our study because, at the time, it was the publicly available AR most commonly detected in samples analyzed at the University of Kentucky Veterinary Diagnostic Laboratory. We determined an approximate minimum toxic dosage (MTD) of oral diphacinone in 3 horses and measured corresponding serum, blood, and liver diphacinone concentrations. Diphacinone was administered orally to healthy horses. Prothrombin time (PT), activated partial thromboplastin time (aPTT), and serum and blood diphacinone concentrations were measured daily. At the study endpoint, the horses were euthanized, and diphacinone concentration was measured in each liver lobe. The horse that received 0.2 mg/kg diphacinone developed prolonged (>1.5× baseline) PT and aPTT; the horse that received 0.1 mg/kg did not. This suggests an approximate oral MTD in horses of 0.2 mg/kg diphacinone. Median liver diphacinone concentration at this dosage was 1,780 (range: 1,590-2,000) ppb wet weight. Marginal (model-adjusted) mean diphacinone concentrations of liver lobes were not significantly different from one another ( p  = NS). Diphacinone was present in similar concentrations in both serum and blood at each time after administration, indicating that both matrices are suitable for detection of diphacinone exposure in horses.

Background: By end December of 2021, COVID-19 has infected around 276 million individuals and caused over 5 million deaths worldwide. Infection results in dysregulated systemic inflammation, multi-organ dysfunction, and critical illness. Cells of the central nervous system are also affected, triggering an uncontrolled neuroinflammatory response. Low doses of glucocorticoids, administered orally or intravenously, reduce mortality among moderate and severe COVID-19 patients. However, low doses administered by these routes do not reach therapeutic levels in the CNS. In contrast, intranasally administered dexamethasone can result in therapeutic doses in the CNS even at low doses., Methods: This is an approved open-label, multicenter, randomized controlled trial to compare the effectiveness of intranasal versus intravenous dexamethasone administered in low doses to moderate and severe COVID-19 adult patients. The protocol is conducted in five health institutions in Mexico City. A total of 120 patients will be randomized into two groups (intravenous vs. intranasal) at a 1:1 ratio. Both groups will be treated with the corresponding dexamethasone scheme for 10 days. The primary outcome of the study will be clinical improvement, defined as a statistically significant reduction in the NEWS-2 score of patients with intranasal versus intravenous dexamethasone administration. The secondary outcome will be the reduction in mortality during hospitalization., Conclusions: This protocol is currently in progress to improve the efficacy of the standard therapeutic dexamethasone regimen for moderate and severe COVID-19 patients., Trial Registration: ClinicalTrials.gov NCT04513184 . Registered November 12, 2020. Approved by La Comisión Federal para la Protección contra Riesgos Sanitarios (COFEPRIS) with identification number DI/20/407/04/36. People are currently being recruited., (© 2022. The Author(s).)

Glioblastoma (GB) is the most common and aggressive primary brain tumour in adult humans. Therapeutic resistance and tumour recurrence after surgical removal contribute to poor prognosis for glioblastoma patients. Men are known to be more likely than women to develop an aggressive form of GB, and differences in sex steroids have emerged as a leading explanation for this finding. Studies indicate that the metabolism and proliferation of GB-derived cells are increased by sex steroids, the expression of androgen receptors (ARs) and the synthesis of androgens and oestrogens, suggesting that these hormones have a role in the tumour pathogenesis. The expression of aromatase, the enzyme that converts androgens to oestrogens, has been reported in glial cells and GB cell lines. Thus, it was necessary to test whether the steroidogenic enzymes involved in androgen synthesis are expressed in GB cells. Therefore, here, we investigated the expression of four key enzymes involved in androgen synthesis in human-derived GB cells. U87 cells were cultured in Dulbecco's modified Eagle medium plus foetal bovine serum and antibiotics on slides for immunocytochemistry or immunofluorescence. U87, LN229 and C6 cells were also cultured in multi-well chambers to obtain proteins for Western blotting. We used primary antibodies against 3β-hydroxysteroid dehydrogenase (3β-HSD), 17α-hydroxilase/17,20-lyase (P450c17), 17β-hydroxysteroid dehydrogenase (17β-HSD) and 5α-reductase. Immunocytochemistry, and immunofluorescence results revealed that glioblastoma cells express 3β-HSD, P450c17, 17β-HSD and 5α-reductase proteins in their cytoplasm. Moreover, Western blot analyses revealed bands corresponding to the molecular weight of these four enzymes in the three GB cell lines. Thus, glioblastoma cells have the key enzymatic machinery necessary to synthesize androgens, and these enzymes might be useful targets for new therapeutic approaches., (© 2021 The Authors. Endocrinology, Diabetes & Metabolism published by John Wiley & Sons Ltd.)

Fatal sodium intoxication can occur in many species, including cattle, and postmortem confirmation often includes brain sodium concentration determination. Published information regarding brain sodium distribution in cattle was not found in a literature review. Our study was designed to determine whether sodium is uniformly distributed throughout the bovine brain. Eight whole bovine brains were collected from adult cattle with no neurologic signs or history suggestive of sodium intoxication, and with a non-neurologic cause of death diagnosed on gross examination. Brains were divided mid-sagittally. One hemisphere of each brain was homogenized. Subsamples were obtained from the remaining hemisphere (rostral, caudal, and dorsal cerebral cortices; brainstem, thalamus, and cerebellum). Sodium concentrations of regions and homogenates were measured by inductively coupled plasma-mass spectrometry. Data were analyzed using repeated measures ANOVA with a pairwise post-test to compare mean sodium concentration of each region to mean homogenate sodium concentration. Brain sodium was not uniformly distributed; sodium concentrations in different regions of the same brain varied somewhat unpredictably. Homogenization of an entire brain hemisphere appears to be the ideal method of sample preparation to ensure accurate brain sodium concentration measurement in adult cattle.

One of the most common tools in conservation physiology is the assessment of environmental stress via glucocorticoid measurement. However, little is known of its relationship with other stress-related biomarkers, and how the incidence of an immune challenge during long-term stress could affect an individual's overall stress response. We investigated here the relationship between basal and post-acute stress fecal cortisol metabolite (FC) with different antioxidant enzymes, oxidative damage and immune parameters in the fish-eating bat, Myotis vivesi We found that in both basal and post-stress conditions, FC was highly related with a number of antioxidant enzymes and immune parameters, but not to oxidative damage. We also assessed changes of FC through the seasons. Basal FC samples and stress reactivity after short-duration stress displayed similar levels during summer, autumn and early winter, but lower concentrations in late winter. Stress reactivity after long-duration stress was greater in summer and early winter. Finally, we tested the effect of a simultaneous exposure to a long, strong stress stimulus with an immune response stimulation by administrating adrenocorticotropic hormone (ACTH) and phytohemagglutinin (PHA) after 42 h. Results showed that when both stimuli were administrated, FC concentrations, inflammation and some antioxidant activity were lowered in comparison with the control and individual administration of the challenges. Our findings support the idea that animals maintain constant basal glucocorticoid levels when living in challenging environments, but response to acute stress differs seasonally and immune defense mechanisms and stress responses might be compromised when confronted with multiple challenges., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Taeniosis-cysticercosis caused by Taenia crassiceps (Zeder, 1800) is a useful experimental model for biomedical research, in substitution of Taenia solium Linnaeus, 1758, studied during decades to develop effective vaccination, novel anti-helminthic drugs and diagnostic tools. Cysticercosis in mouse (Mus musculus Linnaeus) is achieved by the larval subculturing of the Wake Forest University (WFU) strain of T. crassiceps. Golden hamster, Mesocricetus auratus (Waterhouse), has been shown to be the most suitable host for adult forms of parasite in experimental taeniosis. Metacestodes of T. crassiceps WFU multiply by budding without restrictions once inoculated into the mouse, while the number of tapeworms developed from these larvae in hamsters remains highly variable. Three objectives have been proposed to improve the infection of T. crassiceps WFU in hamsters: (1) to re-evaluate the need of immune suppression; (2) to investigate the advantage of infecting hamsters with metacestodes with in vitro protruded scolices; and (3) to compare a number of tapeworms developed from metacestodes subcultured in hamsters against those proliferated in mice. Our results demonstrated that when the evagination of murine metacestodes was high, the number of T. crassiceps WFU adults obtained from hamsters was also high. Immunosuppressive treatment remains relevant for this experimental rodent model. The hamster-to-hamster cysticercosis-taeniosis by T. crassiceps overcame the mouse-to-hamster model in the yield of adult specimens. In vitro scolex evagination and metacestode asexual proliferation in hamsters place this rodent model by T. crassiceps WFU as the most affordable experimental models with taeniids.

During protein synthesis, charged tRNAs deliver amino acids to translating ribosomes, and are then re-charged by tRNA synthetases (aaRS). In humans, mutant aaRS cause a diversity of neurological disorders, but their molecular aetiologies are incompletely characterised. To understand system responses to aaRS depletion, the yeast glutamine aaRS gene (GLN4) was transcriptionally regulated using doxycycline by tet-off control. Depletion of Gln4p inhibited growth, and induced a GCN4 amino acid starvation response, indicative of uncharged tRNA accumulation and Gcn2 kinase activation. Using a global model of translation that included aaRS recharging, Gln4p depletion was simulated, confirming slowed translation. Modelling also revealed that Gln4p depletion causes negative feedback that matches translational demand for Gln-tRNAGln to aaRS recharging capacity. This maintains normal charged tRNAGln levels despite Gln4p depletion, confirmed experimentally using tRNA Northern blotting. Model analysis resolves the paradox that Gln4p depletion triggers a GCN4 response, despite maintenance of tRNAGln charging levels, revealing that normally, the aaRS population can sequester free, uncharged tRNAs during aminoacylation. Gln4p depletion reduces this sequestration capacity, allowing uncharged tRNAGln to interact with Gcn2 kinase. The study sheds new light on mutant aaRS disease aetiologies, and explains how aaRS sequestration of uncharged tRNAs can prevent GCN4 activation under non-starvation conditions., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The cytoplasm is a densely packed environment filled with macromolecules with hindered diffusion. Molecular simulation of the diffusion of biomolecules under such macromolecular crowding conditions requires the definition of a simulation cell with a cytoplasmic-like composition. This has been previously done for prokaryote cells ( E. coli ) but not for eukaryote cells such as yeast as a model organism. Yeast proteomics datasets vary widely in terms of cell growth conditions, the technique used to determine protein composition, the reported relative abundance of proteins, and the units in which abundances are reported. We determined that the gene ontology profiles of the most abundant proteins across these datasets are similar, but their abundances vary greatly. To overcome this problem, we chose five mass spectrometry proteomics datasets that fulfilled the following criteria: high internal consistency, consistency with published experimental data, and freedom from GFP-tagging artifacts. Using these datasets, the contents of a simulation cell containing a single 80S ribosome were defined, such that the macromolecular density and the mass ratio of ribosomal-to-cytoplasmic proteins were consistent with experiment and chosen datasets. Finally, multiple tRNAs were added, consistent with their experimentally-determined number in the yeast cell. The resulting composition can be readily used in molecular simulations representative of yeast cytoplasmic macromolecular crowding conditions to characterize a variety of phenomena, such as protein diffusion, protein-protein interactions and biological processes such as protein translation., (Copyright © 2019 Kompella, Stansfield, Romano and Mancera.)

Diagnosing mushroom poisoning in dogs can be difficult and often includes identification of suspect mushrooms. Visual identification may be hindered by mastication, oral medications, or poor quality of environmental mushroom samples. Other analytical techniques may thus be necessary to aid in mushroom identification. A 5-y-old neutered male Labrador Retriever dog developed acute onset of vomiting, diarrhea, tremors, seizures, and somnolence. The dog was treated at a veterinary clinic and was briefly stabilized, but died during transport to an emergency clinic. On postmortem examination at the University of Kentucky Veterinary Diagnostic Laboratory, the dog's stomach was full of mushrooms covered with activated charcoal. Mushrooms were damaged, fragmented, and discolored, precluding accurate visual identification. Mushroom pieces were sent to the Department of Plant Pathology at the University of California-Davis for PCR identification; the neurotoxic mushroom Amanita muscaria was identified. A qualitative liquid chromatography-mass spectrometry (LC-MS) method was developed to detect ibotenic acid and muscimol, the toxic compounds present in A. muscaria. Mushrooms, stomach contents, and urine were analyzed by LC-MS; ibotenic acid and muscimol were detected in all samples. Because identification of ingested mushrooms is sometimes necessary to confirm mushroom poisoning, PCR can identify ingested mushrooms when visual identification is unreliable.

The aim of this study was to evaluate the role of environmental (dry versus wet season) and individual (sex, body mass and reproductive status) factors in the levels of faecal cortisol metabolites (FGCs) in Gracilinanus agilis faecal samples as an index of stress levels in this species; as well as its association with abundance of Eimeria spp, as an indicator of immunocompetence against parasites. Our study found that FGCFGCs are a reliable indicator of adrenal activity in G. agilis . We found that FGCFGCs increase considerably by environmental stressors like the dry season. Moreover, the observed positive association between FGCs and body mass is the result of the effect of season and reproduction in both variables. We also demonstrated that an increase in FGC levels among G. agilis during the dry season is associated with a rise in the probability of being infected by Eimeria spp. Hence, our finding supports the corticosteroid-fitness hypothesis, which predicts that increased glucocorticoids as a response to stressors usually results in decreased fitness of individuals, translated into low future survival and reproductive success, and higher parasite infection. To our knowledge, this is the first study that integrates environmental changes, hormone responses and parasite loads in a US marsupial in both empirical and experimental approaches.

Use of the neurotoxic rodenticide bromethalin has steadily increased since 2011, resulting in an increased incidence of bromethalin intoxications in pets. Presumptive diagnosis of bromethalin toxicosis relies on history of possible rodenticide exposure coupled with compatible neurologic signs or sudden death, and postmortem examination findings that eliminate other causes of death. Diagnosis is confirmed by detecting the metabolite desmethylbromethalin (DMB) in tissues. In experimental models, spongiform change in white matter of the central nervous system (CNS) is the hallmark histologic feature of bromethalin poisoning. We describe fatal bromethalin intoxication in 3 cats and 2 dogs with equivocal or no CNS white matter spongiform change, illustrating that the lesions described in models can be absent in clinical cases of bromethalin intoxication. Cases with history and clinical signs compatible with bromethalin intoxication warrant tissue analysis for DMB even when CNS lesions are not evident.

Neurophysiological testing of the pelvic floor is recognized as an essential tool to identify pathophysiological mechanisms of pelvic floor disorders, support clinical diagnosis, and aid in therapeutic decisions. Nevertheless, the diagnostic value of these tests in specific neurological diseases of the pelvic floor is not completely clarified. Seeking to fill this gap, the members of the Neurophysiology of the Pelvic Floor Study Group of the Italian Clinical Neurophysiology Society performed a systematic review of the literature to gather available evidence for and against the utility of neurophysiological tests. Our findings confirm the utility of some tests in specific clinical conditions [e.g. concentric needle electromyography, evaluation of sacral reflexes and of pudendal somatosensory evoked potentials (pSEPs) in cauda equina and conus medullaris lesions, and evaluation of pSEPs and perineal sympathetic skin response in spinal cord lesions], and support their use in clinical practice. Other tests, particularly those not currently supported by high-level evidence, when employed in individual patients, should be evaluated in the overall clinical context, or otherwise used for research purposes.

The well established phenomenon of ribosome drop-off plays crucial roles in translational accuracy and nutrient starvation responses during protein translation. When cells are under stress conditions, such as amino acid starvation or aminoacyl-tRNA depletion due to a high level of recombinant protein expression, ribosome drop-off can substantially affect the efficiency of protein expression. Here we introduce a mathematical model that describes the effects of ribosome drop-off on the ribosome density along the mRNA and on the concomitant protein synthesis rate. Our results show that ribosome premature termination may lead to non-intuitive ribosome density profiles, such as a ribosome density which increases from the 5' to the 3' end. Importantly, the model predicts that the effects of ribosome drop-off on the translation rate are mRNA-specific, and we quantify their resilience to drop-off, showing that the mRNAs which present ribosome queues are much less affected by ribosome drop-off than those which do not. Moreover, among those mRNAs that do not present ribosome queues, resilience to drop-off correlates positively with the elongation rate, so that sequences using fast codons are expected to be less affected by ribosome drop-off. This result is consistent with a genome-wide analysis of S. cerevisiae, which reveals that under favourable growth conditions mRNAs coding for proteins involved in the translation machinery, known to be highly codon biased and using preferentially fast codons, are highly resilient to ribosome drop-off. Moreover, in physiological conditions, the translation rate of mRNAs coding for regulatory, stress-related proteins, is less resilient to ribosome drop-off. This model therefore allows analysis of variations in the translational efficiency of individual mRNAs by accounting for the full range of known ribosome behaviours, as well as explaining mRNA-specific variations in ribosome density emerging from ribosome profiling studies.

tRNA gene copy number is a primary determinant of tRNA abundance and therefore the rate at which each tRNA delivers amino acids to the ribosome during translation. Low-abundance tRNAs decode rare codons slowly, but it is unclear which genes might be subject to tRNA-mediated regulation of expression. Here, those mRNA targets were identified via global simulation of translation. In-silico mRNA translation rates were compared for each mRNA in both wild-type and a [Formula: see text] sup70-65 mutant, which exhibits a pseudohyphal growth phenotype and a 75% slower CAG codon translation rate. Of 4900 CAG-containing mRNAs, 300 showed significantly reduced in silico translation rates in a simulated tRNA mutant. Quantitative immunoassay confirmed that the reduced translation rates of sensitive mRNAs were [Formula: see text] concentration-dependent. Translation simulations showed that reduced [Formula: see text] concentrations triggered ribosome queues, which dissipated at reduced translation initiation rates. To validate this prediction experimentally, constitutive gcn2 kinase mutants were used to reduce in vivo translation initiation rates. This repaired the relative translational rate defect of target mRNAs in the sup70-65 background, and ameliorated sup70-65 pseudohyphal growth phenotypes. We thus validate global simulation of translation as a new tool to identify mRNA targets of tRNA-specific gene regulation., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The rapid loss, fragmentation and degradation of tropical forests threaten the survival of many animal species. However, the way in which these phenomena affect animal health has been poorly explored, thus limiting the design of appropriate conservation strategies. To address this, here we identified using linear mixed models the effect of proximal (diet, activity pattern, hunting and logging) and distal (sum of the basal areas of fruiting-tree species [SBAFS], landscape forest cover and degree of forest fragmentation) variables over fecal glucocorticoid metabolite (fGCM) levels-hormones associated with animal health and fitness-of six groups of spider monkeys (Ateles geoffroyi) inhabiting six landscapes with different spatial structures in Mexico. Proximal variables showed a stronger predictive power over fGCMs than distal. In this sense, increases in travel time, the occurrence of hunting, and reductions in rest time and fruit consumption resulted in higher fGCM levels. Regarding distal variables, increases in SBAFS were negatively related to fGCM levels, thus suggesting that food scarcity increases stress hormone levels. Nevertheless, contrary to theoretical expectations, spider monkeys living in smaller tracts of forest spent less time travelling, but the same time feeding on fruit as those in more forested areas. The lower net energy return associated with this combination of factors would explain why, contrary to theoretical expectations, increased forest cover was associated with increased levels of fGCMs in these groups. Our study shows that, at least in the short term, spider monkeys in fragmented landscapes do not always present higher levels of stress hormones compared to those inhabiting continuous forest, and the importance of preserving fruit sources and controlling hunting for reducing the levels of stress hormones in free ranging spider monkeys.

The major fungal pathogen of humans, Candida albicans, mounts robust responses to oxidative stress that are critical for its virulence. These responses counteract the reactive oxygen species (ROS) that are generated by host immune cells in an attempt to kill the invading fungus. Knowledge of the dynamical processes that instigate C. albicans oxidative stress responses is required for a proper understanding of fungus-host interactions. Therefore, we have adopted an interdisciplinary approach to explore the dynamical responses of C. albicans to hydrogen peroxide (H2O2). Our deterministic mathematical model integrates two major oxidative stress signalling pathways (Cap1 and Hog1 pathways) with the three major antioxidant systems (catalase, glutathione and thioredoxin systems) and the pentose phosphate pathway, which provides reducing equivalents required for oxidative stress adaptation. The model encapsulates existing knowledge of these systems with new genomic, proteomic, transcriptomic, molecular and cellular datasets. Our integrative approach predicts the existence of alternative states for the key regulators Cap1 and Hog1, thereby suggesting novel regulatory behaviours during oxidative stress. The model reproduces both existing and new experimental observations under a variety of scenarios. Time- and dose-dependent predictions of the oxidative stress responses for both wild type and mutant cells have highlighted the different temporal contributions of the various antioxidant systems during oxidative stress adaptation, indicating that catalase plays a critical role immediately following stress imposition. This is the first model to encapsulate the dynamics of the transcriptional response alongside the redox kinetics of the major antioxidant systems during H2O2 stress in C. albicans.

In many cases parasites display highly complex life cycles that include the penetration and permanence of the larva or adults within host organs, but even in those that only have one host, reciprocal, intricate interactions occur. Evidence indicates that steroid hormones have an influence on the development and course of parasitic infections. The host gender's susceptibility to infection, and the related differences in the immune response are good examples of the host-parasite interplay. However, the capacity of these organisms to synthesize their own steroidogenic hormones still has more questions than answers. It is now well-known that many parasites synthesize ecdysteroids, but limited information is available on sex steroid and corticosteroid synthesis. This review intends to summarize some of the existing information in the field. In most, but not all parasitosis the host's hormonal environment determines the susceptibility, the course, and severity of parasite infections. In most cases the infection disturbs the host environment, and activates immune responses that end up affecting the endocrine system. Furthermore, sex steroids and corticosteroids may also directly modify the parasite reproduction and molting. Available information indicates that parasites synthesize some steroid hormones, such as ecdysteroids and sex steroids, and the presence and activity of related enzymes have been demonstrated. More recently, the synthesis of corticosteroid-like compounds has been shown in Taenia solium cysticerci and tapeworms, and in Taenia crassiceps WFU cysticerci. In-depth knowledge of the parasite's endocrine properties will contribute to understand their reproduction and reciprocal interactions with the host, and may also help designing tools to combat the infection in some clinical situations.

The follicle destiny towards ovulation or atresia is multi-factorial in nature and involves outcries, paracrine and endocrine factors that promote cell proliferation and survival (development) or unchain apoptosis as part of the atresia process. In several types of cells, sphingosine-1-phospate (S1P) promotes cellular proliferation and survival, whereas ceramide (CER) triggers cell death, and the S1P/CER ratio may determine the fate of the cell. The aim of present study was to quantify S1P and CER concentrations and their ratio in bovine antral follicles of 8 to 17 mm classified as healthy and atretic antral follicles. Follicles were dissected from cow ovaries collected from a local abattoir. The theca cell layer, the granulosa cells and follicular fluid were separated, and 17β-estradiol (E2) and progesterone (P4) concentrations were measured in the follicular fluid by radioimmunoassay. Based on the E2/P4 ratio, the follicles were classified as healthy (2.2±0.3) or atretic (0.2±0.3). In both follicular compartments (granulosa and theca cell layer), sphingolipids were extracted and S1P and CER concentrations were quantified by HPLC (XTerra RP18; 5 µm, 3.0×150 mm column). Results showed that in both follicular compartments, S1P concentrations were higher in healthy antral follicles than in atretic antral follicles (P<0.05). The concentration of CER in the granulosa cells was higher in atretic antral follicles than in healthy antral follicles, but no differences were observed in the theca cell layer. The S1P/CER ratio in both follicular compartments was also higher in healthy antral follicles. Interestingly, in these follicles, there was a 45-fold greater concentration of S1P than CER in the granulosa cells (P<0.05), whereas in the theca cell layer, S1P had only a 14-fold greater concentration than CER when compared with atretic antral follicles. These results suggest that S1P plays a role in follicle health, increasing cellular proliferation and survival. In contrast, reduction of S1P and the S1P/CER in the antral follicle could trigger cellular death and atresia.

During eukaryotic cellular protein synthesis, ribosomal translation is made more efficient through interaction between the two ends of the messenger RNA (mRNA). Ribosomes reaching the 3' end of the mRNA can thus recycle and begin translation again on the same mRNA, the so-called 'closed-loop' model. Using a driven diffusion lattice model of translation, we study the effects of ribosome recycling on the dynamics of ribosome flow and density on the mRNA. We show that ribosome recycling induces a substantial increase in ribosome current. Furthermore, for sufficiently large values of the recycling rate, the lattice does not transition directly from low to high ribosome density, as seen in lattice models without recycling. Instead, a maximal current phase becomes accessible for much lower values of the initiation rate, and multiple phase transitions occur over a wide region of the phase plane. Crucially, we show that in the presence of ribosome recycling, mRNAs can exhibit a peak in protein production at low values of the initiation rate, beyond which translation rate decreases. This has important implications for translation of certain mRNAs, suggesting that there is an optimal concentration of ribosomes at which protein synthesis is maximal, and beyond which translational efficiency is impaired., (© 2014 The Author(s) Published by the Royal Society. All rights reserved.)

Motor enzymes are remarkable molecular machines that use the energy derived from the hydrolysis of a nucleoside triphosphate to generate mechanical movement, achieved through different steps that constitute their kinetic cycle. These macromolecules, nowadays investigated with advanced experimental techniques to unveil their molecular mechanisms and the properties of their kinetic cycles, are implicated in many biological processes, ranging from biopolymerization (e.g., RNA polymerases and ribosomes) to intracellular transport (motor proteins such as kinesins or dyneins). Although the kinetics of individual motors is well studied on both theoretical and experimental grounds, the repercussions of their stepping cycle on the collective dynamics still remains unclear. Advances in this direction will improve our comprehension of transport process in the natural intracellular medium, where processive motor enzymes might operate in crowded conditions. In this work, we therefore extend contemporary statistical kinetic analysis to study collective transport phenomena of motors in terms of lattice gas models belonging to the exclusion process class. Via numerical simulations, we show how to interpret and use the randomness calculated from single particle trajectories in crowded conditions. Importantly, we also show that time fluctuations and non-Poissonian behavior are intrinsically related to spatial correlations and the emergence of large, but finite, clusters of comoving motors. The properties unveiled by our analysis have important biological implications on the collective transport characteristics of processive motor enzymes in crowded conditions., (Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The cell cycle is a sequence of biochemical events that are controlled by complex but robust molecular machinery. This enables cells to achieve accurate self-reproduction under a broad range of different conditions. Environmental changes are transmitted by molecular signalling networks, which coordinate their action with the cell cycle. The cell cycle process and its responses to environmental stresses arise from intertwined nonlinear interactions among large numbers of simpler components. Yet, understanding of how these pieces fit together into a coherent whole requires a systems biology approach. Here, we present a novel mathematical model that describes the influence of osmotic stress on the entire cell cycle of S. cerevisiae for the first time. Our model incorporates all recently known and several proposed interactions between the osmotic stress response pathway and the cell cycle. This model unveils the mechanisms that emerge as a consequence of the interaction between the cell cycle and stress response networks. Furthermore, it characterises the role of individual components. Moreover, it predicts different phenotypical responses for cells depending on the phase of cells at the onset of the stress. The key predictions of the model are: (i) exposure of cells to osmotic stress during the late S and the early G2/M phase can induce DNA re-replication before cell division occurs, (ii) cells stressed at the late G2/M phase display accelerated exit from mitosis and arrest in the next cell cycle, (iii) osmotic stress delays the G1-to-S and G2-to-M transitions in a dose dependent manner, whereas it accelerates the M-to-G1 transition independently of the stress dose and (iv) the Hog MAPK network compensates the role of the MEN network during cell division of MEN mutant cells. These model predictions are supported by independent experiments in S. cerevisiae and, moreover, have recently been observed in other eukaryotes.

To understand the complex relationship governing transcript abundance and the level of the encoded protein, we integrate genome-wide experimental data of ribosomal density on mRNAs with a novel stochastic model describing ribosome traffic dynamics during translation elongation. This analysis reveals that codon arrangement, rather than simply codon bias, has a key role in determining translational efficiency. It also reveals that translation output is governed both by initiation efficiency and elongation dynamics. By integrating genome-wide experimental data sets with simulation of ribosome traffic on all Saccharomyces cerevisiae ORFs, mRNA-specific translation initiation rates are for the first time estimated across the entire transcriptome. Our analysis identifies different classes of mRNAs characterised by their initiation rates, their ribosome traffic dynamics, and by their response to ribosome availability. Strikingly, this classification based on translational dynamics maps onto key gene ontological classifications, revealing evolutionary optimisation of translation responses to be strongly influenced by gene function.

In Saccharomyces cerevisiae, the SUP70 gene encodes the CAG-decoding tRNA(Gln)(CUG). A mutant allele, sup70-65, induces pseudohyphal growth on rich medium, an inappropriate nitrogen starvation response. This mutant tRNA is also a UAG nonsense suppressor via first base wobble. To investigate the basis of the pseudohyphal phenotype, 10 novel sup70 UAG suppressor alleles were identified, defining positions in the tRNA(Gln)(CUG) anticodon stem that restrict first base wobble. However, none conferred pseudohyphal growth, showing altered CUG anticodon presentation cannot itself induce pseudohyphal growth. Northern blot analysis revealed the sup70-65 tRNA(Gln)(CUG) is unstable, inefficiently charged, and 80% reduced in its effective concentration. A stochastic model simulation of translation predicted compromised expression of CAG-rich ORFs in the tRNA(Gln)(CUG)-depleted sup70-65 mutant. This prediction was validated by demonstrating that luciferase expression in the mutant was 60% reduced by introducing multiple tandem CAG (but not CAA) codons into this ORF. In addition, the sup70-65 pseudohyphal phenotype was partly complemented by overexpressing CAA-decoding tRNA(Gln)(UUG), an inefficient wobble-decoder of CAG. We thus show that introducing codons decoded by a rare tRNA near the 5' end of an ORF can reduce eukaryote translational expression, and that the mutant tRNA(CUG)(Gln) constitutive pseudohyphal differentiation phenotype correlates strongly with reduced CAG decoding efficiency., (© 2012 Blackwell Publishing Ltd.)

Background: New chemotherapic agents and new protocols in oncology have led to an increasing survival rate in patients affected by tumors. However, this increased use has been accompanied by a growth in the incidence of cutaneous side effects and a worsening of patients' quality of life. Appropriate management of skin toxicity associated with chemotherapic agents is therefore necessary for suitable drug administration and to improve quality of life and clinical outcomes., Methods: We have clinically examined 100 patients affected by cancer, determining type, frequency, treatment, and evolution of side effects related to chemotherapy., Results: The prevalent cutaneous side effects in patients undergoing chemotherapy are skin rash, xerosis, pruritus, paronychia, hair abnormality, and mucositis. The clinical cases are reported in detail., Conclusion: Oncological therapies have become more selective and have low systemic toxicity because of their high specificity, but cutaneous side effects are common and may worsen the quality of life of these patients.

Background: Saccharomyces cerevisiae senses hyperosmotic conditions via the HOG signaling network that activates the stress-activated protein kinase, Hog1, and modulates metabolic fluxes and gene expression to generate appropriate adaptive responses. The integral control mechanism by which Hog1 modulates glycerol production remains uncharacterized. An additional Hog1-independent mechanism retains intracellular glycerol for adaptation. Candida albicans also adapts to hyperosmolarity via a HOG signaling network. However, it remains unknown whether Hog1 exerts integral or proportional control over glycerol production in C. albicans., Results: We combined modeling and experimental approaches to study osmotic stress responses in S. cerevisiae and C. albicans. We propose a simple ordinary differential equation (ODE) model that highlights the integral control that Hog1 exerts over glycerol biosynthesis in these species. If integral control arises from a separation of time scales (i.e. rapid HOG activation of glycerol production capacity which decays slowly under hyperosmotic conditions), then the model predicts that glycerol production rates elevate upon adaptation to a first stress and this makes the cell adapts faster to a second hyperosmotic stress. It appears as if the cell is able to remember the stress history that is longer than the timescale of signal transduction. This is termed the long-term stress memory. Our experimental data verify this. Like S. cerevisiae, C. albicans mimimizes glycerol efflux during adaptation to hyperosmolarity. Also, transient activation of intermediate kinases in the HOG pathway results in a short-term memory in the signaling pathway. This determines the amplitude of Hog1 phosphorylation under a periodic sequence of stress and non-stressed intervals. Our model suggests that the long-term memory also affects the way a cell responds to periodic stress conditions. Hence, during osmohomeostasis, short-term memory is dependent upon long-term memory. This is relevant in the context of fungal responses to dynamic and changing environments., Conclusions: Our experiments and modeling have provided an example of identifying integral control that arises from time-scale separation in different processes, which is an important functional module in various contexts.

We study the elongation stage of mRNA translation in eukaryotes and find that, in contrast to the assumptions of previous models, both the supply and the demand for tRNA resources are important for determining elongation rates. We find that increasing the initiation rate of translation can lead to the depletion of some species of aa-tRNA, which in turn can lead to slow codons and queueing. Particularly striking "competition" effects are observed in simulations of multiple species of mRNA which are reliant on the same pool of tRNA resources. These simulations are based on a recent model of elongation which we use to study the translation of mRNA sequences from the Saccharomyces cerevisiae genome. This model includes the dynamics of the use and recharging of amino acid tRNA complexes, and we show via Monte Carlo simulation that this has a dramatic effect on the protein production behaviour of the system.

Background: The yeast Saccharomyces cerevisiae responds to amino acid starvation by inducing the transcription factor Gcn4. This is mainly mediated via a translational control mechanism dependent upon the translation initiation eIF2·GTP·Met-tRNAiMet ternary complex, and the four short upstream open reading frames (uORFs) in its 5' mRNA leader. These uORFs act to attenuate GCN4 mRNA translation under normal conditions. During amino acid starvation, levels of ternary complex are reduced. This overcomes the GCN4 translation attenuation effect via a scanning/reinitiation control mechanism dependent upon uORF spacing., Results: Using published experimental data, we have developed and validated a probabilistic formulation of GCN4 translation using the Chemical Master Equation (Model 1). Model 1 explains GCN4 translation's nonlinear dependency upon uORF placements, and predicts that an as yet unidentified factor, which was proposed to regulate GCN4 translation under some conditions, only has pronounced effects upon GCN4 translation when intercistronic distances are unnaturally short. A simpler Model 2 that does not include this unidentified factor could well represent the regulation of a natural GCN4 mRNA. Using parameter values optimised for this algebraic Model 2, we performed stochastic simulations by Gillespie algorithm to investigate the distribution of ribosomes in different sections of GCN4 mRNA under distinct conditions. Our simulations demonstrated that ribosomal loading in the 5'-untranslated region is mainly determined by the ratio between the rates of 5'-initiation and ribosome scanning, but was not significantly affected by rate of ternary complex binding. Importantly, the translation rate for codons starved of cognate tRNAs is predicted to be the most significant contributor to the changes in ribosomal loading in the coding region under repressing and derepressing conditions., Conclusions: Our integrated probabilistic Models 1 and 2 explained GCN4 translation and helped to elucidate the role of a yet unidentified factor. The ensuing stochastic simulations evaluated different factors that may impact on the translation of GCN4 mRNA, and integrated translation status with ribosomal density.

Dairy cattle suffer stress from management and production; contemporary farming tries to improve animal welfare and reduce stress. Therefore, the assessment of long-term hypothalamic-pituitary-adrenal function using non-invasive techniques is useful. The aims in this study were: to measure cortisol concentration in cow and calves hair by radioimmunoassay (RIA), to test cortisol accumulation in bovine hair after adrenocorticotropic hormone (ACTH) challenges, and determine the influence of hair color on cortisol concentrations. Fifteen Holstein heifers were allotted to 3 groups (n = 5 each): in control group (C), just the hair was sampled; in the saline solution group (SS), IV saline solution was administered on days 0, 7, and 14; and the ACTH group was challenged 3 times with ACTH (0.15 UI per kg of body weight) on days 0, 7, and 14. Serum samples from the SS and ACTH groups were obtained 0, 60 and 90 min post-injection. Serum cortisol concentration was greater 60 and 90 min after injection with ACTH. Hair was clipped on days 0, 14, 28, and 44. Hair cortisol was methanol extracted and measured by RIA. Hair cortisol was preserved for 11 mo. Hair cortisol concentrations in the ACTH group were greater than in the saline and control groups on days 14 and 28, but not on day 44. Concentrations were greater in calves than in cows and greater in white hair than in black hair. Cortisol accumulated in bovine hair after ACTH challenges, but the concentration was affected by both age and hair color. If hair color effects are taken into account, assessing cortisol concentration in hair is a potentially useful non-invasive method for assessing stress in cattle.

Neoplastic disease and its therapeutic options have a huge impact on the patient's quality of life from both the emotional and the working point of view. The project "Il Corpo Ritrovato" aims at creating an interdisciplinary network of physicians to improve the quality of life of the oncologic patient, focusing on such important aspects as dermocosmetological skin care but also on the evaluation of new therapeutic and diagnostic algorithms in order to make further progress in the field of prevention.

Pregnant mothers often report a special awareness of and bonding with their unborn child. Little is known about this relationship although it may offer potential for the assessment of the fetal condition. Recently we found evidence of short epochs of fetal-maternal heart rate synchronization under uncontrolled conditions with spontaneous maternal breathing. Here, we examine whether the occurrence of such epochs can be influenced by maternal respiratory arrhythmia induced by paced breathing at several different rates (10, 12, 15, and 20 cycles per minute). To test for such weak and nonstationary synchronizations among the fetal-maternal subsystems, we apply a multivariate synchronization analysis technique and test statistics based on twin surrogates. We find a clear increase in synchronization epochs mostly at high maternal respiratory rates in the original but not in the surrogate data. On the other hand, fewer epochs are found at low respiratory rates both in original and surrogate data. The results suggest that the fetal cardiac system seems to possess the capability to adjust its rate of activation in response to external--i.e., maternal--stimulation. Hence, the pregnant mothers' special awareness to the unborn child may also be reflected by fetal-maternal interaction of cardiac activity. Our approach opens up the chance to examine this interaction between independent but closely linked physiological systems.

The aim of the present study was to evaluate the signaling pathways involved in the follicle-stimulating hormone (FSH) regulated mitogenic activity. For this purpose, 18-d-old chick embryo testis cells were dissociated and cultured for 60 h on polycarbonate membranes. The culture medium was Dulbecco modified Eagle's medium with or without high pure human FSH (hFSH), human recombinant FSH, or different regulators of tyrosine kinase activity as herbimycin A and genistein, or serine/threonine kinases [cyclic adenosine monophosphate (cAMP)-dependent protein serine kinase and protein kinase C] as cAMP, phorbol myristate, and forskolin. In some experiments the regulators were added simultaneously with hFSH. The [3H]-thymidine incorporation was used as an indicator of DNA synthesis. In addition, fragments of chick embryo testis were cultured in the presence or absence of FSH or herbimycin A, and 5-bromo-2'-deoxy-uridine was added to identify the proliferating cell subpopulations. The effect of hFSH on [3H]-thymidine incorporation began at 24 h, and the increment was significant at 36 and 60 h of culture. The hFSH as well as human recombinant FSH significantly stimulated [3H]-thymidine incorporation to testicular cells. The 5-bromo-2'-deoxy-uridine technique showed a high signal in pericordonal and interstitial cells of the hFSH-treated groups, confirming the results obtained using [3H]-thymidine uptake. The treatment with the tyrosine kinase inhibitor herbimycin A increased the [3H]-thymidine uptake, but genistein did not. Regulators of PKA such as cAMP and forskolin, as well as PKC regulators and the phorbol ester phorbol myristate, did not influence cell proliferation. In summary, an inhibitor of tyrosine kinase, herbimycin A, induced per se an increment in chick embryo testis cell proliferation, a fact that strongly suggests that tyrosine kinase signaling pathway functions by inhibiting the proliferation of these cells. On the other hand, the cAMP-PKA pathway had no significant role during the embryonic stage of chick embryo testis. Our results also showed that the effect of FSH on chick embryo cell proliferation occurs mainly in pericordonal and interstitial testis cells.

In this paper, we present an approach to recover the dynamics from recurrences of a system and then generate (multivariate) twin surrogate (TS) trajectories. In contrast to other approaches, such as the linear-like surrogates, this technique produces surrogates which correspond to an independent copy of the underlying system, i.e. they induce a trajectory of the underlying system visiting the attractor in a different way. We show that these surrogates are well suited to test for complex synchronization, which makes it possible to systematically assess the reliability of synchronization analyses. We then apply the TS to study binocular fixational movements and find strong indications that the fixational movements of the left and right eye are phase synchronized. This result indicates that there might be only one centre in the brain that produces the fixational movements in both eyes or a close link between the two centres.

Prevalence of anthrax spores in feces of raptors was determined from samples collected in November-December 2000 and April-May 2001 in an agricultural region of Santa Fé province, Argentina. Feces were tested from 48 birds of six raptor species. One of 14 chimango caracaras (Milvago chimango) and one of eight road-side hawks (Buteo magnirostris) tested positive. The prevalence of Bacillus anthracis spores in feces for the six species was 4% (n=48). The prevalence was 7% (n=14) for chimango caracaras, 13% for road-side hawks (n=8), and 0% for the remaining species (Burrowing owl [Speotyto cunicularia] [n=17], Swainson's hawk [Buteo swainsoni] [n=3], Aplomado falcon [Falco femoralis] [n=2], and American kestrel [Falco sparverius] [n=4]). Grouped by their feeding habits, prevalence for scavenger species was not significantly different than for predators (7% vs. 3%, P>0.999). This study provides evidence that in central Argentina scavenger and non-scavenger raptors may have a role in the epidemiology of anthrax. Long-term studies to determine the extent of this potential involvement in the epidemiology of anthrax in central Argentina are required.

The development of the chick embryo gonads is influenced by gonadotropins [follicle-stimulating hormone (FSH), luteinizing hormone, human chorionic gonadotropin (hCG)]. We have previously shown that insulin enhanced the production of androgens in the testis of the newly hatched chicken and increased the proliferation of chick embryo testis cells. In the present paper, we have studied the effect of insulin on embryonic chick embryo ovarian cells and compared them with those of human FSH and hCG. The ovaries of 18-d-old chick embryos were dissociated and cultured for different periods in Dulbecco's modified Eagle's medium in the presence and absence of insulin, human FSH, hCG, and combinations of them. 3H-thymidine incorporation was used as an indicator of cell proliferation; steroids were measured by radioimmunoassay. Results showed that insulin enhanced the proliferation of ovarian cells in a dose- and time-dependent manner. Gonadotropins did not affect significantly the ovarian cell proliferation. Insulin did not change 17beta-estradiol production. The combination of insulin and FSH or insulin and hCG decreased the stimulation of estrogen secretion caused by the addition of the gonadotropins. In some experiments, ovarian cells were cultured with or without insulin, and subpopulations were identified. The results showed that insulin but not human FSH or hCG increased the proliferation of germinal cells after 60 h in culture. Insulin and human FSH did stimulate the other 2 subpopulations. In summary, present results suggest that insulin is an important hormone in the development of the chick embryo ovary.

We examined the morphological, biochemical and molecular outcome of a nonspecific sulfhydryl reduction in cells, obtained by supplementation of N-acetyl-L-cysteine (NAC) in a 0.1-10 mM concentration range. In human normal primary keratinocytes and in colon and ovary carcinoma cells we obtained evidences for: (i) a dose-dependent inhibition of proliferation without toxicity or apoptosis; (ii) a transition from a proliferative mesenchymal morphology to cell-specific differentiated structures; (iii) a noticeable increase in cell-cell and cell-substratum junctions; (iv) a relocation of the oncogenic beta-catenin at the cell-cell junctions; (v) inhibition of microtubules aggregation; (vi) upregulation of differentiation-related genes including p53, heat shock protein 27 gene, N-myc downstream-regulated gene 1, E-cadherin, and downregulation of cyclooxygenase-2; (vii) inhibition of c-Src tyrosine kinase. In conclusion, a thiol reduction devoid of toxicity as that operated by NAC apparently leads to terminal differentiation of normal and cancer cells through a pleiade of converging mechanisms, many of which are targets of the recently developed differentiation therapy.

Background: Cancer prevention trials using different types of antioxidant supplements have been carried out at several occasions and one of the investigated compounds has been the antioxidant N-acetyl-L-cysteine (NAC). Studies at the cellular level have previously demonstrated that a single supplementation of NAC induces a ten-fold more rapid differentiation in normal primary human keratinocytes as well as a reversion of a colon carcinoma cell line from neoplastic proliferation to apical-basolateral differentiation. The investigated cells showed an early change in the organization of the cytoskeleton, several newly established adherens junctions with E-cadherin/beta-catenin complexes and increased focal adhesions, all features characterizing the differentiation process., Methods: In order to investigate the molecular mechanisms underlying the proliferation arrest and accelerated differentiation induced by NAC treatment of NHEK and Caco-2 cells in vitro, we performed global gene expression analysis of NAC treated cells in a time series (1, 12 and 24 hours post NAC treatment) using the Affymetrix GeneChip Human Genome U95Av2 chip, which contains approximately 12,000 previously characterized sequences. The treated samples were compared to the corresponding untreated culture at the same time point., Results: Microarray data analysis revealed an increasing number of differentially expressed transcripts over time upon NAC treatment. The early response (1 hour) was transient, while a constitutive trend was commonly found among genes differentially regulated at later time points (12 and 24 hours). Connections to the induction of differentiation and inhibition of growth were identified for a majority of up- and down-regulated genes. All of the observed transcriptional changes, except for seven genes, were unique to either cell line. Only one gene, ID-1, was mutually regulated at 1 hour post treatment and might represent a common mediator of early NAC action. The detection of several genes that previously have been identified as stimulated or repressed during the differentiation of NHEK and Caco-2 provided validation of results. In addition, real-time kinetic PCR analysis of selected genes also verified the differential regulation as identified by the microarray platform., Conclusion: NAC induces a limited and transient early response followed by a more consistent and extensively different expression at later time points in both the normal and cancer cell lines investigated. The responses are largely related to inhibition of proliferation and stimulation of differentiation in both cell types but are almost completely lineage specific. ID-1 is indicated as an early mediator of NAC action.

The present study evaluated the follicle-stimulating hormone (FSH) effect on cell proliferation and steroid production by chick embryo testis. Dissociated cells from 18-d-old embryos were cultured on polycarbonate membranes in defined media. In some experiments, cells were further separated by a metrizamide gradient, and 5 cellular subpopulations were recovered and cultured. [3H]thymidine was added to the culture media. When necessary, 17beta-estradiol, human FSH (hFSH), recombinant human FSH (rhFSH), or human chorionic gonadotropin (hCG) was added to the medium at the beginning of the culture. The total number of cells and the incorporation of [3H]thymidine increased when hFSH or rhFSH was added. No changes were produced by the addition of hCG or 17beta-estradiol. The dose-response curve to hFSH resulted in an ED50 of 0.25 IU/mL. The stimulatory effect of hFSH on total number of cells and on [3H]thymidine incorporation was significant at 36 h of culture and was maintained up to 60 h. Testosterone production increased with the addition of FSH or rhFSH, meanwhile estradiol production was below the limit of detection of RIA. The hFSH proliferative effect measured as [3H]thymidine incorporation was observed only in the F3, F4, and F5 fractions of the density gradient. Present results show that hFSH and rhFSH, but not hCG or estradiol, stimulate testis cell proliferation in a time- and dose-dependent manner. The combination of [3H]thymidine incorporation and testosterone production in fractions obtained from the metrizamide density gradients suggests that the cell fractions of the chick embryo testis show a differential response to FSH.

Aims: To compare the prevalence and incidence of respiratory symptoms and lung function values between hairdressing apprentices and office apprentices., Methods: A total of 322 hairdressing apprentices and 277 office apprentices (controls) were studied. Two cross sectional surveys were conducted in 1994 and 1996/97 with longitudinal follow up for a subgroup of apprentices (191 hairdressing apprentices and 189 office apprentices)., Results: In the initial phase, the prevalence of respiratory symptoms was significantly lower among hairdressing apprentices than among office apprentices. Lung function test results showed significantly higher values for hairdressing apprentices. Non-specific bronchial reactivity was similar in the two groups. In the final phase, results for respiratory symptoms were similar. The incidence of respiratory symptoms was not significantly different between hairdressing apprentices and office apprentices. Subjects who dropped out had lower values for FVC and FEV1 in the initial phase than those who completed the final phase. There was a significant deterioration of FEV1 and FEF25-75% in hairdressing apprentices compared to office apprentices. There was a link between atopy and the incidence of most of the respiratory symptoms (day/night cough, wheezing, dyspnoea, mucosal hyperresponsiveness) and between smoking and the incidence of bronchial hyperreactivity. There was no significant correlation between change in lung function tests and specific hairdressing activities reported at the end of the apprenticeship or with environmental working conditions in hairdressing salons., Conclusions: Although a healthy worker effect can be suspected, results showed a significant deterioration of baseline values of lung function tests in the hairdressing apprentice group. However, no clear link was shown between change in lung function tests and specific parameters of occupational activities.

We investigated whether insulin plays a role in the proliferation and androgen production of chick testis cells. Testes from 18-d-old chick embryos or newly hatched chickens were dissociated and precultured in the presence of fetal bovine serum (FBS) for 24 h. After this period, testis cells from 18-d-old chick embryos were cultured in serum-free medium for 1 h with 0, 10, 50, or 100 microg/mL of insulin and were then exposed to human chorionic gonadotropin (hCG) for 3 h. In addition, some cells were incubated for 18 h with only insulin or insulin plus hCG. Androgens were measured by radioimmunoassay in the spent media. To study the influence of insulin on testis cell proliferation, cells were exposed to insulin for 18 h. A pulse of 3H-thymidine was added thereafter. We found that 18-d-old embryonic testis cells responded to hCG, increasing androgen production. Incubation with insulin for 1 h did not affect basal androgen production but modified the subsequent response to hCG. The addition of insulin plus hCG for 18 h resulted in important downregulation of the hCG effect. In addition, insulin significantly increased the proliferation of embryonic testis cells. The cells from testes of newly hatched chickens were precultured as described for embryonic cells and then exposed to insulin for 1 h in a serum-free medium. This treatment significantly increased the basal androgen production. Insulin also significantly enhanced the response to hCG of the testis cells from newly hatched chickens. These results strongly suggest that insulin has a role in the activity and in the proliferation of cultured testis cells throughout the perinatal period.