Melon and rostral muscle morphology of Gervais' beaked whale (Mesoplodon europaeus): Alternating patterns of bilateral asymmetry.

Toothed whales utilize specialized nasal structures such as the lipid-rich melon to produce sound and propagate it into the aquatic environment. Very little nasal morphology of mesoplodont beaked whales has been described in the literature, and the anatomy of the melon and associated musculature of Gervais' beaked whale (Mesoplodon europaeus) remains undescribed. Heads of three (n = 3) Gervais' beaked whales were examined in detail via dissection as well as computed tomography (CT) and magnetic resonance imaging (MRI). Two additional Gervais' beaked whale individuals (n = 2) were studied via archived CT and MRI scans. Representative transverse dissection sections of the melon were processed for polarized light imaging to verify the presence of tendons inserting into the melon tissue. Three-dimensional (3D) CT reconstructions of the melon, rostral muscles, and associated structures were performed to assess morphology and spatial relationships. In all individuals, the melon's main body demonstrated a bilaterally asymmetrical, curvilinear geometry. This curvilinear shape was defined by a pattern of alternating asymmetry in the medial rostral muscles that projected into the melon's tissue. In transverse polarized light imaging, a network of tendons originating from these asymmetrical rostral muscle projections was observed permeating the melon's lipid tissue. This curvilinear melon morphology and associated asymmetrical musculature suggest a means of lengthening the lipid pathway within a relatively short dimensional footprint. In addition, the species-specific arrangement of muscular projections suggests complex fine-tuning of the melon's geometry during echolocation. Further studies may lend additional insight into the function of this unusual melon morphology.

Pathology of short-term dorsal fin tag-attachments in tagged and re-stranded short-beaked common dolphins Delphinus delphis on Cape Cod, MA.

Odontocetes are difficult to study in the wild, making tagging and remote tracking a valuable practice. However, evaluations of host responses at tagging sites have been primarily limited to visual observations in the field. Here we explore the macro- and microscopic pathology of dorsal fin tag attachments in 13 stranded and released short-beaked common dolphins Delphinus delphis from Cape Cod, MA that later re-stranded and died or were euthanized 1-28 d post-tagging. Tags were attached to stranded dolphins' dorsal fins using 2 methods: core biopsy or piercing. Grossly, the piercing method resulted in epidermal compression into the dermis. One tag site had a necrotic border 28 d after application. Grossly, the biopsy method resulted in minimal to no tissue reaction. Two tag sites had granulation tissue accumulation 4 and 12 d after tagging. Histopathologic findings for all tag types and animals consisted of focal epithelial loss, dermal edema, perivascular edema, inflammation and hyperplasia, and inter- and extracellular edema in the adjacent epidermis. Minor expected pathological changes given the procedure were also observed: superficial epidermal necrosis in 3 cases, and superficial bacterial colonization in 2 cases. There was no evidence of sepsis and tagging was not related to cause of re-stranding or death in any case. These gross and histopathologic findings support previous observational conclusions in small delphinids that with appropriate sterile technique, the impacts of single pin dorsal fin tagging on the animal can be minimal and localized. Of the 2 methods, core biopsy may be a better tagging method.

Using Blood Gas Analysis and Capnography to Determine Oxygenation Status in Bottlenose Dolphins (Tursiops truncatus) Following the Deepwater Horizon Oil Spill.

Following the Deepwater Horizon (DWH) oil spill in 2010, poor pulmonary health and reproductive failure in bottlenose dolphins (Tursiops truncatus) in the northern Gulf of Mexico were well-documented. One postulated etiology for the increased fetal distress syndrome and pneumonia found in affected perinatal dolphins was maternal hypoxia caused by lung disease. The objective of this study was to evaluate the utility of blood gas analysis and capnography in determining oxygenation status in bottlenose dolphins with and without pulmonary disease. Blood and breath samples were collected from 59 free-ranging dolphins in Barataria Bay, Louisiana (BB), during a capture-release health assessment program, and from 30 managed dolphins from the U.S. Navy Marine Mammal Program in San Diego, CA. The former was the oil-exposed cohort and the latter served as a control cohort with known health histories. Capnography and select blood gas parameters were compared based on the following factors: cohort, sex, age/length class, reproductive status, and severity of pulmonary disease. Animals with moderate-severe lung disease had higher bicarbonate concentrations (p = 0.005), pH (p < 0.001), TCO2 (p = 0.012), and more positive base excess (p = 0.001) than animals with normal-mild disease. Capnography (ETCO2) was found to have a weak positive correlation with blood PCO2 (p = 0.020), with a mean difference of 5.02 mmHg (p < 0.001). Based on these findings, indirect oxygenation measures, including TCO2, bicarbonate, and pH, show promise in establishing the oxygenation status in dolphins with and without pulmonary disease.

Use of Instagram to convey nutrition information to collegiate athletes.

Background: Nutrition professionals, who work with collegiate athletes, use Instagram to provide nutrition information. Although, limited focus is on the engagement of these athletes. The purpose of this descriptive study was to investigate collegiate athletes' engagement, as indicated by the total number of likes received on posts, based on the nutrition content posted on Instagram. Methods: Over a five-week period, 15 randomly selected accounts were examined daily for posts made within specific research-defined categories (n = 12). The number of posts and subsequent post likes were summed and tabulated per category. Results: The food feature post category received the highest number of likes (n = 1,130) and posts (n = 33), whereas the macronutrient post category received the fewest number of likes (n = 43) and posts (n = 2). A two-sample t-test assuming unequal variances was conducted to compare account engagement as defined by the mean number of post likes within the same conferences. Two conferences had differences in the number of posts (p < 0.05). Discussion: Posting on a weekly basis and within specific categories appeared to produce a higher degree of engagement on collegiate sports nutrition Instagram accounts. Future research is needed to determine the percentage of athletes following these Instagram accounts and the impact these nutrition posts influences dietary behaviors.

Teaching empathy to medical students.

BACKGROUND: Empathy is a core skill essential to patient-centred care. Yet a decline in empathy throughout undergraduate medical education is well recognised. The mainstay of existing teaching approaches to foster medical students' empathic ability has largely focused on the cognitive domain of empathy within the context of communication skills learning. However, there is growing evidence for educational initiatives that better evoke students' affective emotional responses. APPROACH: A student-led educational initiative was created to enhance medical students' understanding of empathy through undertaking different experiential approaches. Eight medical students were invited to participate in an empathy workshop that involved two experiential approaches: personal simulation, and patient narrative, selected given their expected focus on the affective domain. EVALUATION: A subsequent focus group discussion explored students' reactions, learning, and intended future change in practice. Discussions were coded and analysed for descriptive themes. Both initiatives were reported to generate students' empathic responses. Personal simulation evoked more affective domains whilst patient narratives additionally created cognitive empathic insight into the impact of the patient's condition. Students also reported intended changes to their future consulting practices. CONCLUSIONS: Although this is a small-scale exploration of medical students' experiences of empathy-related teaching initiatives, it offers insight into how each initiative could target different aspects of empathy. Data support the use of even brief experiential teaching to improve medical students' empathy during their undergraduate training. We provide recommendations for clinical educators who are considering designing similar initiatives within their undergraduate medical curricula.

Case report: Advice for schools on managing functional tic-like behaviours.

There has been an increase in the occurrence of sudden onset functional tic-like behaviours in adolescents during the COVID-19 pandemic, which has had a significant impact on the affected individual's ability to engage with education. The aim of this article is to generate discussion and inform practice within schools with regard to the management of functional tic-like behaviours. An advice sheet for schools has been produced based on clinical expertise and experience of consulting with schools around the management within education settings. Case examples are presented highlighting the importance and impact of these strategies. We also highlight the need for further evaluation of the effectiveness of the advice sheet in collaboration with schools and families.

Activated mast cells in skeletal muscle can be a potential mediator for cancer-associated cachexia.

BACKGROUND: Eighty per cent of United States advanced cancer patients faces a worsened prognosis due to cancer-associated cachexia. Inflammation is one driver of muscle atrophy in cachexia, and skeletal muscle-resident immune cells could be a source of inflammation. This study explores the efficacy of cancer activated skeletal muscle-resident mast cells as a biomarker and mediator of cachexia. METHODS: Individual gene markers for immune cells were assessed in a publicly available colon carcinoma cohort of normal (n = 3), moderate cachexia (n = 3), and severe cachexia (n = 4) mice. Lewis lung carcinoma (LL/2) cells induced cachexia in C57BL/6 mice, and a combination of toluidine blue staining, immunofluorescence, quantitative polymerase chain reaction, and western blots measured innate immune cell expression in hind limb muscles. In vitro measurements included C2C12 myotube diameter before and after treatment with media from primary murine mast cells activated with LL/2 conditioned media. To assess translational potential in human samples, innate immune cell signatures were assessed for correlation with skeletal muscle atrophy and apoptosis, dietary excess, and cachexia signatures in normal skeletal muscle tissue. Gene set enrichment analysis was performed with innate immune cell signatures in publicly available cohorts for upper gastrointestinal (GI) cancer and pancreatic ductal adenocarcinoma (PDAC) patients (accession: GSE34111 and GSE130563, respectively). RESULTS: Individual innate immunity genes (TPSAB1 and CD68) showed significant increases in severe cachexia (weight loss > 15%) mice in a C26 cohort (GSE24112). Induction of cachexia in C57BL/6 mice with LL/2 subcutaneous injection significantly increased the number of activated skeletal muscle-resident degranulating mast cells. Murine mast cells activated with LL/2 conditioned media decreased C2C12 myotube diameter (P ≤ 0.05). Normal human skeletal muscle showed significant positive correlations between innate immune cell signatures and muscle apoptosis and atrophy, dietary excess, and cachexia signatures. The mast cell signature was up-regulated (positive normalized enrichment score and false discovery rate ≤ 0.1) in upper GI cachectic patients (n = 12) compared with control (n = 6), as well as in cachectic PDAC patients (n = 17) compared with control patients (n = 16). CONCLUSIONS: Activated skeletal muscle-resident mast cells are enriched in cachectic muscles, suggesting skeletal-muscle resident mast cells may serve as a biomarker and mediator for cachexia development to improve patient diagnosis and prognosis.

REVIEW: Assessing North Atlantic right whale health: threats, and development of tools critical for conservation of the species.

Whaling has decimated North Atlantic right whales Eubalaena glacialis (NARW) since the 11th century and southern right whales E. australis (SRW) since the 19th century. Today, NARWs are Critically Endangered and decreasing, whereas SRWs are recovering. We review NARW health assessment literature, NARW Consortium databases, and efforts and limitations to monitor individual and species health, survival, and fecundity. Photographs are used to track individual movement and external signs of health such as evidence of vessel and entanglement trauma. Post-mortem examinations establish cause of death and determine organ pathology. Photogrammetry is used to assess growth rates and body condition. Samples of blow, skin, blubber, baleen and feces quantify hormones that provide information on stress, reproduction, and nutrition, identify microbiome changes, and assess evidence of infection. We also discuss models of the population consequences of multiple stressors, including the connection between human activities (e.g. entanglement) and health. Lethal and sublethal vessel and entanglement trauma have been identified as major threats to the species. There is a clear and immediate need for expanding trauma reduction measures. Beyond these major concerns, further study is needed to evaluate the impact of other stressors, such as pathogens, microbiome changes, and algal and industrial toxins, on NARW reproductive success and health. Current and new health assessment tools should be developed and used to monitor the effectiveness of management measures and will help determine whether they are sufficient for a substantive species recovery.

Cyanobacterial neurotoxin BMAA and brain pathology in stranded dolphins.

Dolphin stranding events occur frequently in Florida and Massachusetts. Dolphins are an excellent sentinel species for toxin exposures in the marine environment. In this report we examine whether cyanobacterial neurotoxin, ß-methylamino-L-alanine (BMAA), is present in stranded dolphins. BMAA has been shown to bioaccumulate in the marine food web, including in the muscles and fins of sharks. Dietary exposure to BMAA is associated with the occurrence of neurofibrillary tangles and ß-amyloid plaques in nonhuman primates. The findings of protein-bound BMAA in brain tissues from patients with Alzheimer's disease has advanced the hypothesis that BMAA may be linked to dementia. Since dolphins are apex predators and consume prey containing high amounts of BMAA, we examined necropsy specimens to determine if dietary and environmental exposures may result in the accumulation of BMAA in the brains of dolphins. To test this hypothesis, we measured BMAA in a series of brains collected from dolphins stranded in Florida and Massachusetts using two orthogonal analytical methods: 1) high performance liquid chromatography, and 2) ultra-performance liquid chromatography with tandem mass spectrometry. We detected high levels of BMAA (20-748 µg/g) in the brains of 13 of 14 dolphins. To correlate neuropathological changes with toxin exposure, gross and microscopic examinations were performed on cortical brain regions responsible for acoustico-motor navigation. We observed increased numbers of ß-amyloid+ plaques and dystrophic neurites in the auditory cortex compared to the visual cortex and brainstem. The presence of BMAA and neuropathological changes in the stranded dolphin brain may help to further our understanding of cyanotoxin exposure and its potential impact on human health.

'Self-screening' for malnutrition with an electronic version of the Malnutrition Universal Screening Tool ('MUST') in hospital outpatients: concurrent validity, preference and ease of use.

Self-screening using an electronic version of the Malnutrition Universal Screening Tool ('MUST') has been developed but its implementation requires investigation. A total of 100 outpatients (mean age 50 (sd 16) years; 57 % male) self-screened with an electronic version of 'MUST' and were then screened by a healthcare professional (HCP) to assess concurrent validity. Ease of use, time to self-screen and prevalence of malnutrition were also assessed. A further twenty outpatients (mean age 54 (sd 15) years; 55 % male) examined preference between self- screening with paper and electronic versions of 'MUST'. For the three-category classification of 'MUST' (low, medium and high risk), agreement between electronic self-screening and HCP screening was 94 % (κ=0·74, se 0·092; P<0·001). For the two-category classification (low risk; medium+high risk) agreement was 96 % (κ=0·82, se 0·085; P<0·001), comparable with the previously reported paper-based self-screening. In all, 15 % of patients categorised themselves 'at risk' of malnutrition (5 % medium, 10 % high). Electronic self-screening took 3 min (sd 1·2 min), 40 % faster than previously reported for the paper-based version. Patients found the tool easy or very easy to understand (99 %) and complete (98 %). Patients that assessed both tools found the electronic tool easier to complete (65 %) and preferred it (55 %) to the paper version. Electronic self-screening using 'MUST' in a heterogeneous group of hospital outpatients is acceptable, user-friendly and has 'substantial to almost-perfect' agreement with HCP screening. The electronic format appears to be as agreeable and often the preferred format when compared with the validated paper-based 'MUST' self-screening tool.

Pharmacodynamic effects of C-domain-specific ACE inhibitors on the renin-angiotensin system in myocardial infarcted rats.

INTRODUCTION: The renin-angiotensin system (RAS) is a dynamic network that plays a critical role in blood pressure regulation and fluid and electrolyte homeostasis. Modulators of the RAS, such as angiotensin-converting enzyme (ACE) inhibitors, are widely used to treat hypertension, heart failure and myocardial infarction. METHODS: The effect of ACE inhibitors (lisinopril and C-domain-selective LisW-S) on the constituent peptides of the RAS following myocardial infarction was examined in rats. Ten angiotensin peptides were analysed using a sensitive LC-MS/MS-based assay to examine both the circulating and equilibrium levels of these peptides. RESULTS: Administration of lisinopril or LisW-S caused a significant decrease in Ang 1-8/Ang 1-10 ratios as determined by circulating and equilibrium peptide level analysis. Furthermore, Ang 1-7 levels were elevated by both ACE inhibitors, but only lisinopril decreased the Ang 1-5/Ang 1-7 ratio. This indicates LisW-S C-domain specificity as Ang 1-5 is generated by hydrolysis of Ang 1-7 by the N-domain. Further corroboration of LisW-S C-domain specificity is that only lisinopril increased the circulating levels of the N-domain ACE substrate Ac-SDKP. CONCLUSION: LisW-S is able to effectively block ACE in vivo by C-domain-selective inhibition. The LC-MS/MS-based assay allows the evaluation of the pharmacologic impact of RAS inhibitors in different pathophysiological conditions.

Pharmacokinetic evaluation of lisinopril-tryptophan, a novel C-domain ACE inhibitor.

Angiotensin-converting enzyme (ACE, EC 3.4.15.1) is a metallopeptidase comprised of two homologous catalytic domains (N- and C-domains). The C-domain cleaves the vasoactive angiotensin II precursor, angiotensin I, more efficiently than the N-domain. Thus, C-domain-selective ACE inhibitors have been designed to investigate the pharmacological effects of blocking the C-terminal catalytic site of the enzyme and improve the side effect profile of current ACE inhibitors. Lisinopril-tryptophan (LisW-S), an analogue of the ACE inhibitor lisinopril, is highly selective for the C-domain. In this study, we have analysed the ex vivo domain selectivity and pharmacokinetic profile of LisW-S. The IC50 value of LisW-S was 38.5 nM in rat plasma using the fluorogenic substrate Abz-FRKP(Dnp)P-OH. For the pharmacokinetics analysis of LisW-S, a sensitive and selective LC-MS/MS method was developed and validated to determine the concentration of LisW-S in rat plasma. LisW-S was administered to Wistar rats at a dose of 1 mg/kg bodyweight intravenously, 5 mg/kg bodyweight orally. The Cmax obtained following oral administration of the drug was 0.082 µM and LisW-S had an apparent terminal elimination half-life of around 3.1 h. The pharmacokinetic data indicate that the oral bioavailability of LisW-S was approximately 5.4%. These data provide a basis for better understanding the absorption mechanism of LisW-S and evaluating its clinical application.

Long-term left ventricular remodelling in rat model of nonreperfused myocardial infarction: sequential MR imaging using a 3T clinical scanner.

Purpose. To evaluate whether 3T clinical MRI with a small-animal coil and gradient-echo (GE) sequence could be used to characterize long-term left ventricular remodelling (LVR) following nonreperfused myocardial infarction (MI) using semi-automatic segmentation software (SASS) in a rat model. Materials and Methods. 5 healthy rats were used to validate left ventricular mass (LVM) measured by MRI with postmortem values. 5 sham and 7 infarcted rats were scanned at 2 and 4 weeks after surgery to allow for functional and structural analysis of the heart. Measurements included ejection fraction (EF), end-diastolic volume (EDV), end-systolic volume (ESV), and LVM. Changes in different regions of the heart were quantified using wall thickness analyses. Results. LVM validation in healthy rats demonstrated high correlation between MR and postmortem values. Functional assessment at 4 weeks after MI revealed considerable reduction in EF, increases in ESV, EDV, and LVM, and contractile dysfunction in infarcted and noninfarcted regions. Conclusion. Clinical 3T MRI with a small animal coil and GE sequence generated images in a rat heart with adequate signal-to-noise ratio (SNR) for successful semiautomatic segmentation to accurately and rapidly evaluate long-term LVR after MI.

Malnutrition self-screening by using MUST in hospital outpatients: validity, reliability, and ease of use.

BACKGROUND: Although nutritional screening with a tool such as the Malnutrition Universal Screening Tool (MUST) is recommended for outpatients, staff are under pressure to undertake a variety of other tasks. Little attention has been paid to the validity of patient self-screening with MUST. OBJECTIVE: This study in 205 outpatients with a mean (±SD) age of 55 ± 17 y (56% male) assessed the practicalities of self-screening, its agreement with screening undertaken by a trained health care professional (HCP), and its test-retest reliability. DESIGN: After the participants provided consent, screening was undertaken by the patients themselves and then by a trained HCP who was unaware of the self-screening results. All patients completed an ease-of-use questionnaire. Test-retest reliability of self-screening was established in a subset of 60 patients. RESULTS: A total of 19.6% of patients categorized themselves as "at risk" of malnutrition (9.8% medium, 9.8% high). For the 3-category classification of MUST (low, medium, high), agreement between self-screening and HCP screening was 90% (κ = 0.70; SE = 0.058, P < 0.001). For the 2-category classification (low risk, medium + high risk), agreement was 93% (κ = 0.78, SE = 0.057, P < 0.001). Disagreements were not systematically under- or overcategorized. Test-retest reliability was almost perfect (κ = 0.94, P < 0.001). Most patients (71%) completed self-screening in <5 min. Patients found the tool easy or very easy to understand (96%) and complete (98%), with 94% reporting that they were happy to screen themselves. CONCLUSION: Self-screening involving MUST in outpatients is acceptable to patients, user-friendly, reliable, and associated with good agreement with HCP screening. This trial was registered at clinicaltrials.gov as NCT00714324.

Apparent ploidy effects on silencing are post-transcriptional at HML and telomeres in Saccharomyces cerevisiae.

The repression of genes in regions of heterochromatin is known as transcriptional silencing. It occurs in a wide range of organisms and can have importance in adaptation to the environment, developmental changes and disease. The model organism Saccharomyces cerevisiae has been used for many years to study transcriptional silencing, but until recently no study has been made in relation to ploidy. The aim of this work was to compare transcriptional silencing in haploids and diploids at both telomeres and the hidden mating-type (HM) loci. Transcriptional silencing was assayed, by growth on 5-fluoroorotic acid (5-FOA) media or by flow cytometry, on strains where a telomere or HM locus was marked. RNA levels were measured by quantitative RT-PCR to confirm that effects were transcriptional. 5-FOA assays and flow cytometry were consistent with transcriptional silencing at telomeres and at HML being reduced as ploidy increases which agreed with conclusions in previous publications. However, QRT-PCR revealed that transcriptional silencing was unaffected by ploidy and thus protein levels were increasing independently of RNA levels. At telomere XI left (XI-L), changes in protein level were strongly influenced by mating-type, whereas at HML mating-type had much less influence. The post-transcriptional effects seen in this study, illustrate the often ignored need to measure RNA levels when assaying transcriptional silencing in Saccharomyces cerevisiae.

Identification of a major rif transcript common to gametocytes and sporozoites of Plasmodium falciparum.

BACKGROUND: The Plasmodium falciparum parasite is transmitted in its sexual gametocyte stage from man to mosquito and as asexual sporozoites from mosquito to man. Developing gametocytes sequester preferentially in the bone marrow, but mature stage gametocytes are released to the bloodstream. Sexual stage parasite surface proteins are of interest as candidate target antigens for transmission blocking vaccines. METHODS: In this study, the transcript profiles of rif and var genes, known to encode surface antigens in asexual blood stage parasites, were investigated at different stages of 3D7/NF54 gametocytogenesis and in sporozoites. RESULTS: Gametocytes exhibited a rif transcript profile unlinked to the rif and var transcript profile of the asexual progenitors. At stage V, mature gametocytes produced high levels of a single rif gene, PF13_0006, which also dominated the rif transcript profile of sporozoites. All var genes appeared to be silenced in sporozoites. CONCLUSIONS: The most prominent variant surface antigen transcribed in both gametocytes and sporozoites of 3D7/NF54 is a single variant of the RIFIN protein family. This discovery may lead to the identification of the parasites binding ligands responsible for the adhesion during sexual stages and potentially to novel vaccine candidates.

Mortality trends of stranded marine mammals on Cape Cod and southeastern Massachusetts, USA, 2000 to 2006.

To understand the cause of death of 405 marine mammals stranded on Cape Cod and southeastern Massachusetts between 2000 and 2006, a system for coding final diagnosis was developed and categorized as (1) disease, (2) human interaction, (3) mass-stranded with no significant findings, (4) single-stranded with no significant findings, (5) rock and/or sand ingestion, (6) predatory attack, (7) failure to thrive or dependent calf or pup, or (8) other. The cause of death for 91 animals could not be determined. For the 314 animals that could be assigned a cause of death, gross and histological pathology results and ancillary testing indicated that disease was the leading cause of mortality in the region, affecting 116/314 (37%) of cases. Human interaction, including harassment, entanglement, and vessel collision, fatally affected 31/314 (10%) of all animals. Human interaction accounted for 13/29 (45%) of all determined gray seal Halichoerus grypus mortalities. Mass strandings were most likely to occur in northeastern Cape Cod Bay; 97/106 (92%) of mass stranded animals necropsied presented with no significant pathological findings. Mass strandings were the leading cause of death in 3 of the 4 small cetacean species: 46/67 (69%) of Atlantic white-sided dolphin Lagenorhynchus acutus, 15/21 (71%) of long-finned pilot whale Globicephala melas, and 33/54 (61%) of short-beaked common dolphin Delphinus delphis. These baseline data are critical for understanding marine mammal population health and mortality trends, which in turn have significant conservation and management implications. They not only afford a better retrospective analysis of strandings, but ultimately have application for improving current and future response to live animal stranding.

Repressive and non-repressive chromatin at native telomeres in Saccharomyces cerevisiae.

BACKGROUND: In Saccharomyces cerevisiae genes that are located close to a telomere can become transcriptionally repressed by an epigenetic process known as telomere position effect. There is large variation in the level of the telomere position effect among telomeres, with many native ends exhibiting little repression. RESULTS: Chromatin analysis, using microccocal nuclease and indirect end labelling, reveals distinct patterns for ends with different silencing states. Differences were observed in the promoter accessibility of a subtelomeric reporter gene and a characteristic array of phased nucleosomes was observed on the centromere proximal side of core X at a repressive end. The silent information regulator proteins 2 - 4, the yKu heterodimer and the subtelomeric core X element are all required for the maintenance of the chromatin structure of repressive ends. However, gene deletions of particular histone modification proteins can eliminate the silencing without the disruption of this chromatin structure. CONCLUSION: Our data identifies chromatin features that correlate with the silencing state and indicate that an array of phased nucleosomes is not sufficient for full repression.

Segregating YKU80 and TLC1 alleles underlying natural variation in telomere properties in wild yeast.

In yeast, as in humans, telomere length varies among individuals and is controlled by multiple loci. In a quest to define the extent of variation in telomere length, we screened 112 wild-type Saccharomyces sensu stricto isolates. We found extensive telomere length variation in S. paradoxus isolates. This phenotype correlated with their geographic origin: European strains were observed to have extremely short telomeres (<150 bp), whereas American isolates had telomeres approximately three times as long (>400 bp). Insertions of a URA3 gene near telomeres allowed accurate analysis of individual telomere lengths and telomere position effect (TPE). Crossing the American and European strains resulted in F1 spores with a continuum of telomere lengths consistent with what would be predicted if many quantitative trait loci (QTLs) were involved in length maintenance. Variation in TPE is similarly quantitative but only weakly correlated with telomere length. Genotyping F1 segregants indicated several QTLs associated with telomere length and silencing variation. These QTLs include likely candidate genes but also map to regions where there are no known genes involved in telomeric properties. We detected transgressive segregation for both phenotypes. We validated by reciprocal hemizygosity that YKU80 and TLC1 are telomere-length QTLs in the two S. paradoxus subpopulations. Furthermore, we propose that sequence divergence within the Ku heterodimer generates negative epistasis within one of the allelic combinations (American-YKU70 and European-YKU80) resulting in very short telomeres.