CD8αα and -αβ isotypes are equally recruited to the immunological synapse through their ability to bind to MHC class I

Bimolecular fluorescence complementation was used to engineer CD8 molecules so that CD8αα and CD8αβ dimers can be independently visualized on the surface of a T cell during antigen recognition. Using this approach, we show that CD8αα is recruited to the immunological synapse almost as well as CD8αβ, but because the kinase Lck associates preferentially with CD8αβ in lipid rafts, CD8αα is the weaker co-receptor. During recognition of the strong CD8αα ligand H2-TL, CD8αα is preferentially recruited. Thus, recruitment of the two CD8 species correlates with their relative binding to the available ligands, rather than with the co-receptor functions of the CD8 species.     

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

Muscle strains are one of the most common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and physical exam alone, however the clinical presentation can vary greatly depending on the extent of injury, the patient''s pain tolerance, etc. In patients with muscle injury or muscle disease, assessment of muscle damage is typically limited to clinical signs, such as tenderness, strength, range of motion, and more recently, imaging studies. Biological markers, such as serum creatine kinase levels, are typically elevated with muscle injury, but their levels do not always correlate with the loss of force production. This is even true of histological findings from animals, which provide a "direct measure" of damage, but do not account for all the loss of function. Some have argued that the most comprehensive measure of the overall health of the muscle in contractile force. Because muscle injury is a random event that occurs under a variety of biomechanical conditions, it is difficult to study. Here, we describe an in vivo animal model to measure torque and to produce a reliable muscle injury. We also describe our model for measurement of force from an isolated muscle in situ. Furthermore, we describe our small animal MRI procedure.Download video file.^{(50M, mov)}     

Measurement of the membrane potential in small cells using patch clamp methods

The resting membrane potential, E(m), of mammalian cells is a fundamental physiological parameter. Even small changes in E(m) can modulate excitability, contractility and rates of cell migration. At present accurate, reproducible measurements of E(m) and determination of its ionic basis remain significant challenges when patch clamp methods are applied to small cells. In this study, a mathematical model has been developed which incorporates many of the main biophysical principles which govern recordings of the resting potential of 'small cells'. Such a prototypical cell (approx. capacitance, 6 pF; input resistance 5 GΩ) is representative of neonatal cardiac myocytes, and other cells in the cardiovascular system (endothelium, fibroblasts) and small cells in other tissues, e.g. bone (osteoclasts) articular joints (chondrocytes) and the pancreas (β cells). Two common experimental conditions have been examined: (1) when the background K(+) conductance is linear; and (2) when this K(+) conductance is highly nonlinear and shows pronounced inward rectification. In the case of a linear K(+) conductance, the presence of a "leakage" current through the seal resistance between the cell membrane and the patch pipette always depolarizes E(m). Our calculations confirm that accurate characterization of E(m) is possible when the seal resistance is at least 5 times larger than the input resistance of the targeted cell. Measurement of E(m) under conditions in which the main background current includes a markedly nonlinear K(+) conductance (due to inward rectification) yields complex and somewhat counter-intuitive findings. In fact, there are at least two possible stable values of resting membrane potential for a cell when the nonlinear, inwardly rectifying K(+) conductance interacts with the seal current. This type of bistable behavior has been reported in a variety of small mammalian cells, including those from the heart, endothelium, smooth muscle and bone. Our theoretical treatment of these two common experimental situations provides useful mechanistic insights, and suggests practical methods by which these significant limitations, and their impact, can be minimized.     

Identification and validation of novel cerebrospinal fluid biomarkers for staging early Alzheimer's disease

Background

Ideally, disease modifying therapies for Alzheimer disease (AD) will be applied during the ‘preclinical’ stage (pathology present with cognition intact) before severe neuronal damage occurs, or upon recognizing very mild cognitive impairment. Developing and judiciously administering such therapies will require biomarker panels to identify early AD pathology, classify disease stage, monitor pathological progression, and predict cognitive decline. To discover such biomarkers, we measured AD-associated changes in the cerebrospinal fluid (CSF) proteome.

Methods and Findings

CSF samples from individuals with mild AD (Clinical Dementia Rating [CDR] 1) (n = 24) and cognitively normal controls (CDR 0) (n = 24) were subjected to two-dimensional difference-in-gel electrophoresis. Within 119 differentially-abundant gel features, mass spectrometry (LC-MS/MS) identified 47 proteins. For validation, eleven proteins were re-evaluated by enzyme-linked immunosorbent assays (ELISA). Six of these assays (NrCAM, YKL-40, chromogranin A, carnosinase I, transthyretin, cystatin C) distinguished CDR 1 and CDR 0 groups and were subsequently applied (with tau, p-tau181 and Aβ42 ELISAs) to a larger independent cohort (n = 292) that included individuals with very mild dementia (CDR 0.5). Receiver-operating characteristic curve analyses using stepwise logistic regression yielded optimal biomarker combinations to distinguish CDR 0 from CDR>0 (tau, YKL-40, NrCAM) and CDR 1 from CDR<1 (tau, chromogranin A, carnosinase I) with areas under the curve of 0.90 (0.85–0.94 95% confidence interval [CI]) and 0.88 (0.81–0.94 CI), respectively.

Conclusions

Four novel CSF biomarkers for AD (NrCAM, YKL-40, chromogranin A, carnosinase I) can improve the diagnostic accuracy of Aβ42 and tau. Together, these six markers describe six clinicopathological stages from cognitive normalcy to mild dementia, including stages defined by increased risk of cognitive decline. Such a panel might improve clinical trial efficiency by guiding subject enrollment and monitoring disease progression. Further studies will be required to validate this panel and evaluate its potential for distinguishing AD from other dementing conditions.     

Geographic variation in cardiovascular inflammation among healthy women in the Women's Health Study

Background

Geographic variation in traditional cardiovascular disease (CVD) risk factors has been observed among women in the US. It is not known whether state-level variation in cardiovascular inflammation exists or could be explained by traditional clinical risk factors and behavioral lifestyle factors.

Methods and Results

We used multilevel linear regression to estimate state-level variation in inflammatory biomarker patterns adjusted for clinical and lifestyle characteristics among 26,029 women free of CVD. Participants derived from the Women''s Health Study, a national cohort of healthy middle-aged and older women. Inflammatory biomarker patterns (plasma levels of high-sensitivity C-reactive protein (hsCRP), soluble intercellular adhesion molecule-1 (sICAM-1), and fibrinogen) were compared to state-level patterns of traditional CVD risk factors and global risk scores. We found that all three inflammatory biomarkers exhibited significant state-level variation including hsCRP (lowest vs. highest state median 1.3 mg/L vs. 2.7 mg/L, unadjusted random effect estimate 1^st to 99^th percentile range for log hsCRP 0.52, p<.001), sICAM-1 (325 ng/ml vs. 366ng/ml, unadjusted random effect estimate 1^st to 99^th percentile range 0.44, p<.001), and fibrinogen (322 mg/dL vs. 367 mg/dL, unadjusted random effect estimate 1^st to 99^th percentile range 0.41, p = .001). Neither demographic, clinical or lifestyle characteristics explained away state-level effects in biomarker patterns. Southern and Appalachian states (Arkansas, West Virginia) had the highest inflammatory biomarker values. Regional geographic patterns of traditional CVD risk factors and risk scores did not completely overlap with biomarkers of inflammation.

Conclusions

There is state-level geographic variation in inflammatory biomarkers among otherwise healthy women that cannot be completely attributed to traditional clinical risk factors or lifestyle characteristics. Future research should aim to identify additional factors that may explain geographic variation in biomarkers of inflammation among healthy women.     

MicroRNA alterations and associated aberrant DNA methylation patterns across multiple sample types in oral squamous cell carcinoma

Background

MicroRNA (miRNA) expression is broadly altered in cancer, but few studies have investigated miRNA deregulation in oral squamous cell carcinoma (OSCC). Epigenetic mechanisms are involved in the regulation of >30 miRNA genes in a range of tissues, and we aimed to investigate this further in OSCC.

Methods

TaqMan® qRT-PCR arrays and individual assays were used to profile miRNA expression in a panel of 25 tumors with matched adjacent tissues from patients with OSCC, and 8 control paired oral stroma and epithelium from healthy volunteers. Associated DNA methylation changes of candidate epigenetically deregulated miRNA genes were measured in the same samples using the MassArray® mass spectrometry platform. MiRNA expression and DNA methylation changes were also investigated in FACS sorted CD44^high oral cancer stem cells from primary tumor samples (CSCs), and in oral rinse and saliva from 15 OSCC patients and 7 healthy volunteers.

Results

MiRNA expression patterns were consistent in healthy oral epithelium and stroma, but broadly altered in both tumor and adjacent tissue from OSCC patients. MiR-375 is repressed and miR-127 activated in OSCC, and we confirm previous reports of miR-137 hypermethylation in oral cancer. The miR-200 s/miR-205 were epigenetically activated in tumors vs normal tissues, but repressed in the absence of DNA hypermethylation specifically in CD44^high oral CSCs. Aberrant miR-375 and miR-200a expression and miR-200c-141 methylation could be detected in and distinguish OSCC patient oral rinse and saliva from healthy volunteers, suggesting a potential clinical application for OSCC specific miRNA signatures in oral fluids.

Conclusions

MiRNA expression and DNA methylation changes are a common event in OSCC, and we suggest miR-375, miR-127, miR-137, the miR-200 family and miR-205 as promising candidates for future investigations. Although overall activated in OSCC, miR-200/miR-205 suppression in oral CSCs indicate that cell specific silencing of these miRNAs may drive tumor expansion and progression.     

Diet restriction and life history trade-offs in short- and long-lived species of Daphnia

The life-extending effects of diet restriction are well documented. One evolutionary model that accounts for this widespread conservation is the resource allocation model, where the selected individuals are those that can delay reproduction during periods of resource limitation. In this study, we use closely related species of a model organism, Daphnia, with widely divergent lifespans to address the relationship between diet restriction and longevity and assess whether the relationships are owing to trade-offs between reproductive and somatic investment. Specifically, we conducted a common garden experiment and constructed reaction norms for lifespan, fecundity, and body size as a function of food concentration. Our study provides evidence that the short-lived species in our study, D. pulex, shows the classically observed relationship of enhanced lifespan in response to reduced diet intake, but does not divert resources to somatic maintenance at the expense of reproduction during chronic diet restriction. In contrast, we find no evidence that the long-lived species in our study, D. pulicaria, gains any life-extending effects through diet restriction. Combined, our results provide evidence that the resource allocation model is not sufficient to explain the evolution of diet-mediated lifespan plasticity.     

Man and the last great wilderness: human impact on the deep sea

The deep sea, the largest ecosystem on Earth and one of the least studied, harbours high biodiversity and provides a wealth of resources. Although humans have used the oceans for millennia, technological developments now allow exploitation of fisheries resources, hydrocarbons and minerals below 2000 m depth. The remoteness of the deep seafloor has promoted the disposal of residues and litter. Ocean acidification and climate change now bring a new dimension of global effects. Thus the challenges facing the deep sea are large and accelerating, providing a new imperative for the science community, industry and national and international organizations to work together to develop successful exploitation management and conservation of the deep-sea ecosystem. This paper provides scientific expert judgement and a semi-quantitative analysis of past, present and future impacts of human-related activities on global deep-sea habitats within three categories: disposal, exploitation and climate change. The analysis is the result of a Census of Marine Life--SYNDEEP workshop (September 2008). A detailed review of known impacts and their effects is provided. The analysis shows how, in recent decades, the most significant anthropogenic activities that affect the deep sea have evolved from mainly disposal (past) to exploitation (present). We predict that from now and into the future, increases in atmospheric CO(2) and facets and consequences of climate change will have the most impact on deep-sea habitats and their fauna. Synergies between different anthropogenic pressures and associated effects are discussed, indicating that most synergies are related to increased atmospheric CO(2) and climate change effects. We identify deep-sea ecosystems we believe are at higher risk from human impacts in the near future: benthic communities on sedimentary upper slopes, cold-water corals, canyon benthic communities and seamount pelagic and benthic communities. We finalise this review with a short discussion on protection and management methods.