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Objective Fatigue, headache, problems sleeping and numerous other symptoms have been reported to be associated with long COVID. However, many of these symptoms coincide with symptoms reported by the general population, possibly exacerbated by restrictions/precautions experienced during the COVID-19 pandemic. This study examines the symptoms reported by individuals who tested positive for COVID-19 compared with those who tested negative.Design Observational study.Setting The study was conducted on adult residents in Alberta, Canada, from October 2021 to February 2023.Participants We evaluated self-reported symptoms in 7623 adults with positive COVID-19 tests and 1520 adults who tested negative, using surveys adapted from the internationally standardised International Severe Acute Respiratory and emerging Infection Consortium (ISARIC)-developed COVID-19 long-term follow-up tools. These individuals had an index COVID-19 test date between 1 March 2020 and 31 December 2022 and were over 28 days post-COVID-19 testing.Primary outcome measures The primary outcomes were to identify the symptoms associated with COVID-19 positivity and risk factors for reporting symptoms.Results Fatigue was the top reported symptom (42%) among COVID-19-positive respondents, while headache was the top reported symptom (32%) in respondents who tested negative. Compared with those who tested negative, COVID-19-positive individuals reported 1.5 times more symptoms and had higher odds of experiencing 31 out of the 40 listed symptoms during the postinfectious period. These symptoms included olfactory dysfunction, menstruation changes, cardiopulmonary and neurological symptoms. Female sex, middle age (41–55 years), Indigeneity, unemployment, hospital/intensive care unit (ICU) admission at the time of testing and pre-existing health conditions independently predicted a greater number and variety of symptoms.Conclusions Our results provide evidence that COVID-19 survivors continue to experience a significant number and variety of symptoms. These findings can help inform targeted strategies for the unequally affected population. It is important to offer appropriate management for symptom relief to those who have survived the acute COVID-19 illness.

Plain English summary Cancer patients and their family members have unique and lived experience with the condition. Therefore, collaborating with them is important in cancer science. We aimed to create such a collaboration in Newfoundland and Labrador, Canada. In 2021, we successfully formed our diverse collaborative group. Currently, our group includes 12 public representatives. We meet online and discuss matters important to members. We also design studies and events together. Our discussions have identified four topics that need further research and policy changes such as information needs and unique needs of caregivers and family members. Our activities expand over time. For example, lately we started to advocate for other cancer patients and families. In conclusion, we formed a successful cancer patient, family member, and researcher collaborative group. Our work informs the public, healthcare systems, and scientists on important cancer related matters.

IntroductionDigitalisation offers innovative solutions within maternity services; however, vulnerable groups risk being overlooked. University College London Hospital's (UCLH) successful implementation of a digital maternity app, MyCare, gives women access to test results, information about appointments, and enables communication with healthcare professionals (HCPs). Yet, little is known about access and engagement among vulnerable pregnant women.MethodologyResearch was conducted over a 3-month period (April–June 2022) in the Maternity Department at UCLH, UK. MyCare datasets were analysed, and anonymised surveys completed by vulnerable pregnant women and HCPs.ResultsLower rates of utilisation and engagement with MyCare were seen in vulnerable pregnant women especially among refugee/asylum seekers, those with mental health issues, and those facing domestic violence. Non-users were also more likely to be individuals from ethnic minority backgrounds, with a lower average social-deprivation-index decile, whose first language was not English, and with a significant history of non-attendance to appointments. Patient and HCP surveys highlighted various barriers to MyCare engagement, including a lack of motivation, limited language options, low e-literacy levels, and complex app interfaces.ConclusionThe use of a single digital tool, without a formulated pathway to identify and assist those not accessing or engaging with it, risks unequal care provision which may exacerbate health inequalities. This research advances the idea that digital exclusion is not necessarily a matter of access to technology, but an issue of a lack of engagement with these tools. Therefore, vulnerable women and HCPs must be integral to the implementation of digital strategies, to ensure no one is left behind.

Introduction Very little is known about how the COVID-19 pandemic has affected the health of residents and the healthcare system in Alberta, Canada. The purpose of this study is to establish an observational study to characterise the health of residents in Alberta, Canada, over time, covering a population that tested negative or positive for COVID-19 during the pandemic. The primary outcome is to characterise ‘long COVID-19’ and the health status of residents during the COVID-19 pandemic. Secondary outcomes include the estimation of the risk of and risk factors associated with adverse health outcomes and healthcare utilisation and burdens.Methods and analysis This is a population-level provincial observational study which will follow-up with Alberta residents who underwent testing for COVID-19 and completed surveys adapted from the ISARIC COVID-19 long-term follow-up survey. The survey data will be linked with medical records. Statistical analyses will be carried out to characterise ‘long COVID-19’ and the health status of residents during the pandemic. The outcomes of this study will inform strategies for primary care and rehabilitation services to prevent chronic consequences; contribute to healthcare management, interventional studies, rehabilitation and health management to reduce overall morbidity and improve long-term outcomes of COVID-19 and the COVID-19 pandemic and potentially guide a self-evaluation of a remote monitoring system to manage individuals’ health.Ethics and dissemination This study was reviewed and approved by the University of Alberta ethics committee (Study ID: Pro00112053 & Pro00113039) on 13 August 2021 and adheres to the Alberta Health Services research information management policy. Study results will be used to manage clinical care, published in peer-reviewed journals and presented at local, national and international conferences.Protocol version 6 June 2022EuroQol ID 161 015.

OBJECTIVES/GOALS: Older adults in rural communities are at an elevated risk of falling. Perturbation-Based Balance Training (PBT) is a highly effective fall prevention paradigm but its feasibility in the community is unknown. The purpose of this study was to determine the feasibility and acceptability of PBT to older adults and local clinicians in rural communities. METHODS/STUDY POPULATION: 19 older adults (60+ years) were recruited from communities in rural Montana to take part in a PBT program. The PBT program was implemented using a bespoke portable PBT treadmill developed by our research team and validated against commercial PBT treadmills. To increase ecological validity, the program was implemented by local clinicians. The PBT program lasted 6 weeks, with 1 session per week, participants received up to 35 trip perturbations per session. The feasibility and acceptability of PBT to participants and clinicians was assessed within the Theoretical Framework of Acceptability, prospectively and retrospectively via interviews and surveys, and concurrently via field notes. RESULTS/ANTICIPATED RESULTS: Data analysis is ongoing and will be complete by the time of presentation. Preliminary results suggest the portable PBT program was generally acceptable to older adults and local clinicians. Perceived effectiveness was generally positive and increased balance awareness and low burden are emerging as common themes. Local clinicians indicated the burden of implementing PBT was low, PBT fit within their views on fall prevention, and had a high level of perceived effectiveness. There appear to be a few limitations to feasibility, which are primarily related to the weight and transportation of the treadmill. We are currently working to refine the treadmill and lower its weight and are developing a grant with local clinicians and extension agents that would facilitate transportation to a broader network of communities. DISCUSSION/SIGNIFICANCE: Our preliminary findings suggest PBT is feasible in rural communities and accepted by older adults. Increased balance and fall awareness and low burden are emerging as important factors in the feasibility and acceptability of PBT. This study provides a foundation for future studies to translate other PBT modalities from the lab to the community.

Abstract Objective The advent of new technologies has made it possible to explore alternative ventilator manufacturing to meet the worldwide shortfall for mechanical ventilators especially in pandemics. We describe a method using rapid prototyping technologies to create an electro-mechanical ventilator in a cost effective, timely manner and provide results of testing using an in vitro–in vivo testing model. Results Rapid prototyping technologies (3D printing and 2D cutting) were used to create a modular ventilator. The artificial manual breathing unit (AMBU) bag connected to wall oxygen source using a flow meter was used as air reservoir. Controlled variables include respiratory rate, tidal volume and inspiratory: expiratory (I:E) ratio. In vitro testing and In vivo testing in the pig model demonstrated comparable mechanical efficiency of the test ventilator to that of standard ventilator but showed the material limits of 3D printed gears. Improved gear design resulted in better ventilator durability whilst reducing manufacturing time (

Most species of modern iguanas (Iguania, Iguanidae) dig burrows for dwelling and nesting, yet neither type of burrow has been interpreted as trace fossils in the geologic record. Here we describe and diagnose the first known fossil example of an iguana nesting burrow, preserved in the Grotto Beach Formation (Early Late Pleistocene, ~115 kya) on San Salvador Island, The Bahamas. The trace fossil, located directly below a protosol, is exposed in a vertical section of a cross-bedded oolitic eolianite. Abundant root traces, a probable land-crab burrow, and lack of ghost-crab burrows further indicate a vegetated inland dune as the paleoenvironmental setting. The trace fossil matches dimensions and overall forms of burrows made by modern iguanas, and internal structures indicate active backfilling consistent with modern iguana nesting burrows. The trace fossil is also located on an island with a modern native species of rock iguana (Cyclura riyeli riyeli), suggesting a presence of iguanas on San Salvador since the Late Pleistocene. This nesting burrow may provide a search image for more fossil iguana burrows in The Bahamas and other places with long-established iguana species and favorable geological conditions for preserving their burrows.

Uniting diverse stakeholders through communication, education or building a collaborative 'common vision' for biodiversity management is a recommended approach for enabling effective conservation in regions with multiple uses. However, socially focused strategies such as building a collaborative vision can require sharing scarce resources (time and financial resources) with the on-ground management actions needed to achieve conservation outcomes. Here we adapt current prioritisation tools to predict the likely return on the financial investment of building a stakeholder-led vision along with a portfolio of on-ground management strategies. Our approach brings together and analyses expert knowledge to estimate the cost-effectiveness of a common vision strategy and on-ground management strategies, before any investments in these strategies are made. We test our approach in an intensively-used Australian biodiversity hotspot with 179 threatened or at-risk species. Experts predicted that an effective stakeholder vision for the region would have a relatively low cost and would significantly increase the feasibility of on-ground management strategies. As a result, our analysis indicates that a common vision is likely to be a cost-effective investment, increasing the expected persistence of threatened species in the region by 9 to 52%, depending upon the strategies implemented. Our approach can provide the maximum budget that is worth investing in building a common vision or another socially focused strategy for building support for on-ground conservation actions. The approach can assist with decisions about whether and how to allocate scarce resources amongst social and ecological actions for biodiversity conservation in other regions worldwide.

Current assessment tools for sport-related concussion are limited by a reliance on subjective interpretation and patient symptom reporting. Robotic assessments may provide more objective and precise measures of neurological function than traditional clinical tests.To determine the reliability of assessments of sensory, motor and cognitive function conducted with the KINARM end-point robotic device in young adult elite athletes.Sixty-four randomly selected healthy, young adult elite athletes participated. Twenty-five individuals (25 M, mean age±SD, 20.2±2.1 years) participated in a within-season study, where three assessments were conducted within a single season (assessments labeled by session: S1, S2, S3). An additional 39 individuals (28M; 22.8±6.0 years) participated in a year-to-year study, where annual pre-season assessments were conducted for three consecutive seasons (assessments labeled by year: Y1, Y2, Y3). Forty-four parameters from five robotic tasks (Visually Guided Reaching, Position Matching, Object Hit, Object Hit and Avoid, and Trail Making B) and overall Task Scores describing performance on each task were quantified.Test-retest reliability was determined by intra-class correlation coefficients (ICCs) between the first and second, and second and third assessments. In the within-season study, ICCs were ≥0.50 for 68% of parameters between S1 and S2, 80% of parameters between S2 and S3, and for three of the five Task Scores both between S1 and S2, and S2 and S3. In the year-to-year study, ICCs were ≥0.50 for 64% of parameters between Y1 and Y2, 82% of parameters between Y2 and Y3, and for four of the five Task Scores both between Y1 and Y2, and Y2 and Y3.Overall, the results suggest moderate-to-good test-retest reliability for the majority of parameters measured by the KINARM robot in healthy young adult elite athletes. Future work will consider the potential use of this information for clinical assessment of concussion-related neurological deficits.

The efficient delivery of cellular cargo relies on the fusion of cargo-carrying vesicles with the correct membrane at the correct time. These spatiotemporal fusion events occur when SNARE proteins on the vesicle interact with cognate SNARE proteins on the target membrane. Regulatory Munc18 proteins are thought to contribute to SNARE interaction specificity through interaction with the SNARE protein Syntaxin. Neuronal Munc18a interacts with Syntaxin1 but not Syntaxin4, and adipocyte Munc18c interacts with Syntaxin4 but not Syntaxin1. Here we show that this accepted view of specificity needs revision. We find that Munc18c interacts with both Syntaxin4 and Syntaxin1, and appears to bind "non-cognate" Syntaxin1 a little more tightly than Syntaxin4. Munc18a binds Syntaxin1 and Syntaxin4, though it interacts with its cognate Syntaxin1 much more tightly. We also observed that when bound to non-cognate Munc18c, Syntaxin1 captures its neuronal SNARE partners SNAP25 and VAMP2, and Munc18c can bind to pre-formed neuronal SNARE ternary complex. These findings reveal that Munc18a and Munc18c bind Syntaxins differently. Munc18c relies principally on the Syntaxin N-peptide interaction for binding Syntaxin4 or Syntaxin1, whereas Munc18a can bind Syntaxin1 tightly whether or not the Syntaxin1 N-peptide is present. We conclude that Munc18a and Munc18c differ in their binding interactions with Syntaxins: Munc18a has two tight binding modes/sites for Syntaxins as defined previously but Munc18c has just one that requires the N-peptide. These results indicate that the interactions between Munc18 and Syntaxin proteins, and the consequences for in vivo function, are more complex than can be accounted for by binding specificity alone.

Climate-induced shifts in plant phenology may adversely affect animals that cannot or do not shift the timing of their reproductive cycle. The realized effect of potential trophic "mismatches" between a consumer and its food varies with the degree to which species rely on dietary income and stored capital. Large Arctic herbivores rely heavily on maternal capital to reproduce and give birth near the onset of the growing season but are they vulnerable to trophic mismatch? We evaluated the long-term changes in the temperatures and characteristics of the growing seasons (1970-2013), and compared growing conditions and dynamics of forage quality for caribou at peak parturition, peak lactation, and peak forage biomass, and plant senescence between two distinct time periods over 36 years (1977 and 2011-13). Despite advanced thaw dates (7-12 days earlier), increased growing season lengths (15-21 days longer), and consistent parturition dates, we found no decline in forage quality and therefore no evidence within this dataset for a trophic mismatch at peak parturition or peak lactation from 1977 to 2011-13. In Arctic ungulates that use stored capital for reproduction, reproductive demands are largely met by body stores deposited in the previous summer and autumn, which reduces potential adverse effects of any mismatch between food availability and timing of parturition. Climate-induced effects on forages growing in the summer and autumn ranges, however, do correspond with the demands of female caribou and their offspring to gain mass for the next reproductive cycle and winter. Therefore, we suggest the window of time to examine the match-mismatch framework in Arctic ungulates is not at parturition but in late summer-autumn, where the multiplier effects of small changes in forage quality are amplified by forage abundance, peak forage intake, and resultant mass gains in mother-offspring pairs.

Vesicular transport of cellular cargo requires targeted membrane fusion and formation of a SNARE protein complex that draws the two apposing fusing membranes together. Insulin-regulated delivery and fusion of glucose transporter-4 storage vesicles at the cell surface is dependent on two key proteins: the SNARE integral membrane protein Syntaxin4 (Sx4) and the soluble regulatory protein Munc18c. Many reported in vitro studies of Munc18c:Sx4 interactions and of SNARE complex formation have used soluble Sx4 constructs lacking the native transmembrane domain. As a consequence, the importance of the Sx4 C-terminal anchor remains poorly understood. Here we show that soluble C-terminally truncated Sx4 dissociates more rapidly from Munc18c than Sx4 where the C-terminal transmembrane domain is replaced with a T4-lysozyme fusion. We also show that Munc18c appears to inhibit SNARE complex formation when soluble C-terminally truncated Sx4 is used but does not inhibit SNARE complex formation when Sx4 is C-terminally anchored (by a C-terminal His-tag bound to resin, by a C-terminal T4L fusion or by the native C-terminal transmembrane domain in detergent micelles). We conclude that the C-terminus of Sx4 is critical for its interaction with Munc18c, and that the reported inhibitory role of Munc18c may be an artifact of experimental design. These results support the notion that a primary role of Munc18c is to support SNARE complex formation and membrane fusion.

Earlier research has revealed that the ndh loci have been pseudogenized, truncated, or deleted from most orchid plastomes sequenced to date, including in all available plastomes of the two most species-rich subfamilies, Orchidoideae and Epidendroideae. This study sought to resolve deeper-level phylogenetic relationships among major orchid groups and to refine the history of gene loss in the ndh loci across orchids. The complete plastomes of seven orchids, Oncidium sphacelatum (Epidendroideae), Masdevallia coccinea (Epidendroideae), Sobralia callosa (Epidendroideae), Sobralia aff. bouchei (Epidendroideae), Elleanthus sodiroi (Epidendroideae), Paphiopedilum armeniacum (Cypripedioideae), and Phragmipedium longifolium (Cypripedioideae) were sequenced and analyzed in conjunction with all other available orchid and monocot plastomes. Most ndh loci were found to be pseudogenized or lost in Oncidium, Paphiopedilum and Phragmipedium, but surprisingly, all ndh loci were found to retain full, intact reading frames in Sobralia, Elleanthus and Masdevallia. Character mapping suggests that the ndh genes were present in the common ancestor of orchids but have experienced independent, significant losses at least eight times across four subfamilies. In addition, ndhF gene loss was correlated with shifts in the position of the junction of the inverted repeat (IR) and small single-copy (SSC) regions. The Orchidaceae have unprecedented levels of homoplasy in ndh gene presence/absence, which may be correlated in part with the unusual life history of orchids. These results also suggest that ndhF plays a role in IR/SSC junction stability.

Microevolutionary adaptations and mechanisms of fungal pathogen resistance were explored in a melanic population of the Greater wax moth, Galleria mellonella. Under constant selective pressure from the insect pathogenic fungus Beauveria bassiana, 25(th) generation larvae exhibited significantly enhanced resistance, which was specific to this pathogen and not to another insect pathogenic fungus, Metarhizium anisopliae. Defense and stress management strategies of selected (resistant) and non-selected (susceptible) insect lines were compared to uncover mechanisms underpinning resistance, and the possible cost of those survival strategies. We hypothesize that the insects developed a transgenerationally primed resistance to the fungus B. bassiana, a costly trait that was achieved not by compromising life-history traits but rather by prioritizing and re-allocating pathogen-species-specific augmentations to integumental front-line defenses that are most likely to be encountered by invading fungi. Specifically during B. bassiana infection, systemic immune defenses are suppressed in favour of a more limited but targeted repertoire of enhanced responses in the cuticle and epidermis of the integument (e.g. expression of the fungal enzyme inhibitor IMPI, and cuticular phenoloxidase activity). A range of putative stress-management factors (e.g. antioxidants) is also activated during the specific response of selected insects to B. bassiana but not M. anisopliae. This too occurs primarily in the integument, and probably contributes to antifungal defense and/or helps ameliorate the damage inflicted by the fungus or the host's own immune responses.

BackgroundThere are varying degrees of compatibility between malaria parasite-mosquito species, and understanding this compatibility may be crucial for developing effective transmission-blocking vaccines. This study investigates the compatibility of different biological forms of a malaria vector, Anopheles stephensi, to Plasmodium berghei ANKA strain.MethodsSeveral biologically different and allopatric forms of A. stephensi were studied. Three forms were isolated from different regions of southern Iran: the variety mysorensis, the intermediate form and the native type form, and an additional type form originated from India (Beech strain).The mosquitoes were experimentally infected with P. berghei to compare their susceptibility to parasitism. Anti-mosquito midgut antiserum was then raised in BALB/cs mice immunized against gut antigens from the most susceptible form of A. stephensi (Beech strain), and the efficacy of the antiserum was assessed in transmission-blocking assays conducted on the least susceptible mosquito biological form.ResultsThe susceptibility of different biological forms of A. stephensi mosquito to P. berghei was specifically inter-type varied. The Beech strain and the intermediate form were both highly susceptible to infection, with higher oocyst and sporozoite infection rates than intermediate and mysorensis forms. The oocyst infection, and particularly sporozite infection, was lowest in the mysorensis strain. Antiserum raised against midgut proteins of the Indian Beech type form blocked infection in this mosquito population, but it was ineffective at blocking both oocyst and sporozoite development in the permissive but geographically distant intermediate form mosquitoes. This suggests that a strong degree of incompatibility exists between the mosquito strains in terms of midgut protein(s) acting as putative ookinete receptors.ConclusionsThe incompatibility in the midgut protein profiles between two biological forms of A. stephensi demonstrates a well-differentiated population structure according to geographical origin. Therefore, the design of potential transmission-blocking strategies should incorporate a more thorough understanding of intra-species variations in host-parasite interactions.

Vesicle fusion is an indispensable cellular process required for eukaryotic cargo delivery. The Sec/Munc18 protein Munc18c is essential for insulin-regulated trafficking of glucose transporter4 (GLUT4) vesicles to the cell surface in muscle and adipose tissue. Previously, our biophysical and structural studies have used Munc18c expressed in SF9 insect cells. However to maximize efficiency, minimize cost and negate any possible effects of post-translational modifications of Munc18c, we investigated the use of Escherichia coli as an expression host for Munc18c. We were encouraged by previous reports describing Munc18c production in E. coli cultures for use in in vitro fusion assay, pulldown assays and immunoprecipitations. Our approach differs from the previously reported method in that it uses a codon-optimized gene, lower temperature expression and autoinduction media. Three N-terminal His-tagged constructs were engineered, two with a tobacco etch virus (TEV) or thrombin protease cleavage site to enable removal of the fusion tag. The optimized protocol generated 1-2 mg of purified Munc18c per L of culture at much reduced cost compared to Munc18c generated using insect cell culture. The purified recombinant Munc18c protein expressed in bacteria was monodisperse, monomeric, and functional. In summary, we developed methods that decrease the cost and time required to generate functional Munc18c compared with previous insect cell protocols, and which generates sufficient purified protein for structural and biophysical studies.

Evenks and Evens, Tungusic-speaking reindeer herders and hunter-gatherers, are spread over a wide area of northern Asia, whereas their linguistic relatives the Udegey, sedentary fishermen and hunter-gatherers, are settled to the south of the lower Amur River. The prehistory and relationships of these Tungusic peoples are as yet poorly investigated, especially with respect to their interactions with neighbouring populations. In this study, we analyse over 500 complete mtDNA genome sequences from nine different Evenk and even subgroups as well as their geographic neighbours from Siberia and their linguistic relatives the Udegey from the Amur-Ussuri region in order to investigate the prehistory of the Tungusic populations. These data are supplemented with analyses of Y-chromosomal haplogroups and STR haplotypes in the Evenks, Evens, and neighbouring Siberian populations. We demonstrate that whereas the North Tungusic Evenks and Evens show evidence of shared ancestry both in the maternal and in the paternal line, this signal has been attenuated by genetic drift and differential gene flow with neighbouring populations, with isolation by distance further shaping the maternal genepool of the Evens. The Udegey, in contrast, appear quite divergent from their linguistic relatives in the maternal line, with a mtDNA haplogroup composition characteristic of populations of the Amur-Ussuri region. Nevertheless, they show affinities with the Evenks, indicating that they might be the result of admixture between local Amur-Ussuri populations and Tungusic populations from the north.

Conflict arises in fisheries worldwide when piscivorous birds target fish species of commercial value. This paper presents a method for estimating size selectivity functions for piscivores and uses it to compare predation selectivities of Great Cormorants (Phalacrocorax carbo sinensis L. 1758) with that of gill-net fishing on a European perch (Perca fluviatilis L. 1758) population in the Curonian Lagoon, Lithuania. Fishers often regard cormorants as an unwanted "satellite species", but the degree of direct competition and overlap in size-specific selectivity between fishers and cormorants is unknown. This study showed negligible overlap in selectivity between Great Cormorants and legal-sized commercial nets. The selectivity estimation method has general application potential for use in conjunction with population dynamics models to assess fish population responses to size-selective fishing from a wide range of piscivorous predators.

Annual draft systems are the principal method used by teams in major sporting leagues to recruit amateur players. These draft systems frequently take one of three forms: a lottery style draft, a weighted draft, or a reverse-order draft. Reverse-order drafts can create incentives for teams to deliberately under-perform, or tank, due to the perceived gain from obtaining quality players at higher draft picks. This paper uses a dynamic simulation model that captures the key components of a win-maximising sporting league, including the amateur player draft, draft choice error, player productivity, and between-team competition, to explore how competitive balance and incentives to under-perform vary according to league characteristics. We find reverse-order drafts can lead to some teams cycling between success and failure and to other teams being stuck in mid-ranking positions for extended periods of time. We also find that an incentive for teams to tank exists, but that this incentive decreases (i) as uncertainty in the ability to determine quality players in the draft increases, (ii) as the number of teams in the league reduces, (iii) as team size decreases, and (iv) as the number of teams adopting a tanking strategy increases. Simulation models can be used to explore complex stochastic dynamic systems such as sports leagues, where managers face difficult decisions regarding the structure of their league and the desire to maintain competitive balance.

Lhx3 is a LIM-homeodomain (LIM-HD) transcription factor that regulates neural cell subtype specification and pituitary development in vertebrates, and mutations in this protein cause combined pituitary hormone deficiency syndrome (CPHDS). The recently published structures of Lhx3 in complex with each of two key protein partners, Isl1 and Ldb1, provide an opportunity to understand the effect of mutations and posttranslational modifications on key protein-protein interactions. Here, we use small-angle X-ray scattering of an Ldb1-Lhx3 complex to confirm that in solution the protein is well represented by our previously determined NMR structure as an ensemble of conformers each comprising two well-defined halves (each made up of LIM domain from Lhx3 and the corresponding binding motif in Ldb1) with some flexibility between the two halves. NMR analysis of an Lhx3 mutant that causes CPHDS, Lhx3(Y114C), shows that the mutation does not alter the zinc-ligation properties of Lhx3, but appears to cause a structural rearrangement of the hydrophobic core of the LIM2 domain of Lhx3 that destabilises the domain and/or reduces the affinity of Lhx3 for both Ldb1 and Isl1. Thus the mutation would affect the formation of Lhx3-containing transcription factor complexes, particularly in the pituitary gland where these complexes are required for the production of multiple pituitary cell types and hormones.

A subset of victims of ocular sulfur mustard (SM) exposure develops an irreversible, idiotypic keratitis with associated secondary pathologies, collectively referred to as mustard gas keratopathy (MGK). MGK involves a progressive corneal degeneration resulting in chronic ocular discomfort and impaired vision for which clinical interventions have typically had poor outcomes. Using a rabbit corneal vapor exposure model, we previously demonstrated a clinical progression with acute and chronic sequelae similar to that observed in human casualties. However, a better understanding of the temporal changes that occur during the biphasic SM injury is crucial to mechanistic understanding and therapeutic development. Here we evaluate the histopathologic, biochemical and ultrastructural expressions of pathogenesis of the chronic SM injury over eight weeks. We confirm that MGK onset exhibits a biphasic trajectory involving corneal surface regeneration over the first two weeks, followed by the rapid development and progressive degeneration of corneal structure. Preclinical markers of corneal dysfunction were identified, including destabilization of the basal corneal epithelium, basement membrane zone abnormalities and stromal deformation. Clinical sequelae of MGK appeared abruptly three weeks after exposure, and included profound anterior edema, recurring corneal erosions, basement membrane disorganization, basal cell necrosis and stromal degeneration. Unlike resolved corneas, MGK corneas exhibited frustrated corneal wound repair, with significantly elevated histopathology scores. Increased lacrimation, disruption of the basement membrane and accumulation of pro-inflammatory mediators in the aqueous humor provide several mechanisms for corneal degeneration. These data suggest that the chronic injury is fundamentally distinct from the acute lesion, involving injury mechanisms that operate on different time scales and in different corneal tissues. Corneal edema appears to be the principal pathology of MGK, in part resulting from persistent necrosis of the basal corneal epithelium and deterioration of the basement membrane. The findings also provide a potential explanation as to why administration of anti-inflammatories transiently delays, but does not prevent, the development of MGK sequelae.

The mechanism of inactivation of human enzyme N-acylethanolamine-hydrolyzing acid amidase (hNAAA), with selected inhibitors identified in a novel fluorescent based assay developed for characterization of both reversible and irreversible inhibitors, was investigated kinetically and using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). 1-Isothiocyanatopentadecane (AM9023) was found to be a potent, selective and reversible hNAAA inhibitor, while two others, 5-((biphenyl-4-yl)methyl)-N,N-dimethyl-2H-tetrazole-2-carboxamide (AM6701) and N-Benzyloxycarbonyl-L-serine β-lactone (N-Cbz-serine β-lactone), inhibited hNAAA in a covalent and irreversible manner. MS analysis of the hNAAA/covalent inhibitor complexes identified modification only of the N-terminal cysteine (Cys126) of the β-subunit, confirming a suggested mechanism of hNAAA inactivation by the β-lactone containing inhibitors. These experiments provide direct evidence of the key role of Cys126 in hNAAA inactivation by different classes of covalent inhibitors, confirming the essential role of cysteine for catalysis and inhibition in this cysteine N-terminal nucleophile hydrolase enzyme. They also provide a methodology for the rapid screening and characterization of large libraries of compounds as potential inhibitors of NAAA, and subsequent characterization or their mechanism through MALDI-TOF MS based bottom up-proteomics.

ARHGAP22 is a RhoGAP protein comprising an N-terminal PH domain, a RhoGAP domain and a C-terminal coiled-coil domain. It has recently been identified as an Akt substrate that binds 14-3-3 proteins in response to treatment with growth factors involved in cell migration. We used a range of biophysical techniques to investigate the weak interaction between 14-3-3 and a truncated form of ARHGAP22 lacking the coiled-coil domain. This weak interaction could be stabilized by chemical cross-linking which we used to show that: a monomer of ARHGAP22 binds a dimer of 14-3-3; the ARHGAP22 PH domain is required for the 14-3-3 interaction; the RhoGAP domain is unlikely to participate in the interaction; Ser16 is the more important of two predicted 14-3-3 binding sites; and, phosphorylation of Ser16 may not be necessary for 14-3-3 interaction under the conditions we used. Small angle X-ray scattering and cross-link information were used to generate solution structures of the isolated proteins and of the cross-linked ARHGAP22:14-3-3 complex, showing that no major rearrangement occurs in either protein upon binding, and supporting a role for the PH domain and N-terminal peptide of ARHGAP22 in the 14-3-3 interaction. Small-angle X-ray scattering measurements of mixtures of ARHGAP22 and 14-3-3 were used to establish that the affinity of the interaction is ∼30 µM.

The tumor necrosis factor receptor (TNFR) superfamily mediates signals critical for regulation of the immune system. One family member, CD40, is important for the efficient activation of antibody-producing B cells and other antigen-presenting cells. The molecules and mechanisms that mediate CD40 signaling are only partially characterized. Proteins known to interact with the cytoplasmic domain of CD40 include members of the TNF receptor-associated factor (TRAF) family, which regulate signaling and serve as links to other signaling molecules. To identify additional proteins important for CD40 signaling, we used a combined stimulation/immunoprecipitation procedure to isolate CD40 signaling complexes from B cells and characterized the associated proteins by mass spectrometry. In addition to known CD40-interacting proteins, we detected SMAC/DIABLO, HTRA2/Omi, and HOIP/RNF31/PAUL/ZIBRA. We found that these previously unknown CD40-interacting partners were recruited in a TRAF2-dependent manner. HOIP is a ubiquitin ligase capable of mediating NF-kappaB activation through the ubiquitin-dependent activation of IKKgamma. We found that a mutant HOIP molecule engineered to lack ubiquitin ligase activity inhibited the CD40-mediated activation of NF-kappaB. Together, our results demonstrate a powerful approach for the identification of signaling molecules associated with cell surface receptors and indicate an important role for the ubiquitin ligase activity of HOIP in proximal CD40 signaling.

Historic DNA data have the potential to identify phenotypic information otherwise invisible in the historical, archaeological and palaeontological record. In order to determine whether a single nucleotide polymorphism typing protocol based on single based extension (SNaPshot™) could produce reliable phenotypic data from historic samples, we genotyped three coat colour markers for a sample of historic Thoroughbred horses for which both phenotypic and correct genotypic information were known from pedigree information in the General Stud Book. Experimental results were consistent with the pedigrees in all cases. Thus we demonstrate that historic DNA techniques can produce reliable phenotypic information from museum specimens.

BACKGROUND: To utilize the power of high-throughput sequencers, target enrichment methods have been developed. The majority of these require reagents and equipment that are only available from commercial vendors and are not suitable for the targets that are a few kilobases in length. METHODOLOGY/PRINCIPAL FINDINGS: We describe a novel and economical method in which custom made long-range PCR products are used to capture complete human mitochondrial genomes from complex DNA mixtures. We use the method to capture 46 complete mitochondrial genomes in parallel and we sequence them on a single lane of an Illumina GA(II) instrument. CONCLUSIONS/SIGNIFICANCE: This method is economical and simple and particularly suitable for targets that can be amplified by PCR and do not contain highly repetitive sequences such as mtDNA. It has applications in population genetics and forensics, as well as studies of ancient DNA.

We investigated the perceived quality of teleradiology services offered at a rural hospital in comparison with radiology provided by three other methods: 'circuit riding', an on-site radiologist, and a hybrid arrangement. The research design was a case study, with interviews of administrators, technologists and physicians at the rural hospital, followed by a structured survey of all staff physicians. Responses were analysed both qualitatively and quantitatively. Both interviews and the survey indicated that teleradiology was perceived to be as accurate as on-site film interpretation. All other aspects of the service--efficiency, reports, communications and the overall contribution to patient care--were judged to be poorer than on-site radiology. We conclude that the provision of acceptable teleradiology requires considerable attention to all aspects of the radiology service, with attention to differences in institutional culture and mission, and to communication. [ABSTRACT FROM AUTHOR]

5-Aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR), an analog of AMP is widely used as an activator of AMP-kinase (AMPK), a protein that regulates the responses of the cell to energy change. Recently, we showed that AICAR-induced AMPK activation inhibits the growth of retinoblastoma cells in vitro by decreasing cyclins and by inducing apoptosis and S-phase arrest. In this study, we investigated the effects of AMPK activator AICAR on the growth of retinoblastoma in vivo. Intraperitoneal injection of AICAR resulted in 48% growth inhibition of Y79 retinoblastoma cell tumors in mice. Tumors isolated from mice treated with AICAR had decreased expression of Ki67 and increased apoptotic cells (TUNEL positive) compared with the control. In addition, AICAR treatment suppressed significantly tumor vessel density and macrophage infiltration. We also showed that AICAR administration resulted in AMPK activation and mTOR pathway inhibition. Paradoxically observed down-regulation of p21, which indicates that p21 may have a novel function of an oncogene in retinoblastoma tumor. Our results indicate that AICAR treatment inhibited the growth of retinoblastoma tumor in vivo via AMPK/mTORC1 pathway and by apoptogenic, anti-proliferative, anti-angiogenesis mechanism. AICAR is a promising novel non-chemotherapeutic drug that may be effective as an adjuvant in treating Retinoblastoma.

Background: Visual loss in glaucoma is associated with pathological changes in retinal ganglion cell (RGC) axons and a slow decline in the RGC population. Age and elevated intraocular pressure (IOP) are the main risk factors for glaucomatous loss of vision. Several studies have implicated the proinflammatory cytokine tumor necrosis factor- α (TNF-α) as a link between elevated IOP and RGC death, but the cellular source of TNF-α and its causative role in RGC death remain uncertain. Here, using a rat model of glaucoma, we investigated the source of elevated TNF- α and examined whether Etanercept, a TNF-α blocker that is in common clinical use for other indications, is protective against RGC death. Methodology/Principal Findings: Episcleral vein cauterization (EVC) caused intraocular pressure (IOP) to be elevated for at least 28 days. IOP elevation resulted in a dramatic increase in TNF-α levels within a few days, axonal degeneration, and a 38% loss of RGCs by 4 weeks. Immunostaining coupled with confocal microscopy showed that OHT induced robust induction of TNF-α in Iba-1-positive microglia around the optic nerve head (ONH). Despite persistent elevation of IOP, Etanercept reduced microglial activation, TNF-α levels, axon degeneration in the optic nerve, and the loss of RGCs. Conclusions/Significance: Ocular hypertension (OHT) triggers an inflammatory response characterized by the appearance of activated microglia around the ONH that express TNF-α. Blocking TNF-α activity with a clinically approved agent inhibits this microglial response and prevents axonal degeneration and loss of RGCs. These findings suggest a new treatment strategy for glaucoma using TNF- α antagonists or suppressors of inflammation.

Purpose: The integrin αvβ3 is differentially expressed on neovascular endothelial cells. We investigated whether a novel intravenously injectable αvβ3 integrin-ligand coupled nanoparticle (NP) can target choroidal neovascular membranes (CNV) for imaging and targeted gene therapy. Methods: CNV lesions were induced in rats using laser photocoagulation. The utility of NP for in vivo imaging and gene delivery was evaluated by coupling the NP with a green fluorescing protein plasmid (NP-GFPg). Rhodamine labeling (Rd-NP) was used to localize NP in choroidal flatmounts. Rd-NP-GFPg particles were injected intravenously on weeks 1, 2, or 3. In the treatment arm, rats received NP containing a dominant negative Raf mutant gene (NP-ATPμ-Raf) on days 1, 3, and 5. The change in CNV size and leakage, and TUNEL positive cells were quantified. Results: GFP plasmid expression was seen in vivo up to 3 days after injection of Rd-NP-GFPg. Choroidal flatmounts confirmed the localization of the NP and the expression of GFP plasmid in the CNV. Treating the CNV with NP-ATPμ-Raf decreased the CNV size by 42% (P<0.001). OCT analysis revealed that the reduction of CNV size started on day 5 and reached statistical significance by day 7. Fluorescein angiography grading showed significantly less leakage in the treated CNV (P<0.001). There were significantly more apoptotic (TUNEL-positive) nuclei in the treated CNV. Conclusion: Systemic administration of αvβ3 targeted NP can be used to label the abnormal blood vessels of CNV for imaging. Targeted gene delivery with NP-ATPμ-Raf leads to a reduction in size and leakage of the CNV by induction of apoptosis in the CNV.