Real-time monitoring of protein conformational changes using a nano-mechanical sensor.

Proteins can switch between different conformations in response to stimuli, such as pH or temperature variations, or to the binding of ligands. Such plasticity and its kinetics can have a crucial functional role, and their characterization has taken center stage in protein research. As an example, Topoisomerases are particularly interesting enzymes capable of managing tangled and supercoiled double-stranded DNA, thus facilitating many physiological processes. In this work, we describe the use of a cantilever-based nanomotion sensor to characterize the dynamics of human topoisomerase II (Topo II) enzymes and their response to different kinds of ligands, such as ATP, which enhance the conformational dynamics. The sensitivity and time resolution of this sensor allow determining quantitatively the correlation between the ATP concentration and the rate of Topo II conformational changes. Furthermore, we show how to rationalize the experimental results in a comprehensive model that takes into account both the physics of the cantilever and the dynamics of the ATPase cycle of the enzyme, shedding light on the kinetics of the process. Finally, we study the effect of aclarubicin, an anticancer drug, demonstrating that it affects directly the Topo II molecule inhibiting its conformational changes. These results pave the way to a new way of studying the intrinsic dynamics of proteins and of protein complexes allowing new applications ranging from fundamental proteomics to drug discovery and development and possibly to clinical practice.

Using active learning for monitoring networks design: The example of wind power plants sites evaluation

Locating new wind farms is of crucial importance for energy policies of the next decade. To select the new location, an accurate picture of the wind fields is necessary. However, characterizing wind fields is a difficult task, since the phenomenon is highly nonlinear and related to complex topographical features. In this paper, we propose both a nonparametric model to estimate wind speed at different time instants and a procedure to discover underrepresented topographic conditions, where new measuring stations could be added. Compared to space filling techniques, this last approach privileges optimization of the output space, thus locating new potential measuring sites through the uncertainty of the model itself.

Diagnostic yield of short-term video-EEG monitoring for epilepsy and PNESs: a European assessment.

INTRODUCTION: Although long-term video-EEG monitoring (LVEM) is routinely used to investigate paroxysmal events, short-term video-EEG monitoring (SVEM) lasting <24 h is increasingly recognized as a cost-effective tool. Since, however, relatively few studies addressed the yield of SVEM among different diagnostic groups, we undertook the present study to investigate this aspect. METHODS: We retrospectively analyzed 226 consecutive SVEM recordings over 6 years. All patients were referred because routine EEGs were inconclusive. Patients were classified into 3 suspected diagnostic groups: (1) group with epileptic seizures, (2) group with psychogenic nonepileptic seizures (PNESs), and (3) group with other or undetermined diagnoses. We assessed recording lengths, interictal epileptiform discharges, epileptic seizures, PNESs, and the definitive diagnoses obtained after SVEM. RESULTS: The mean age was 34 (±18.7) years, and the median recording length was 18.6 h. Among the 226 patients, 127 referred for suspected epilepsy - 73 had a diagnosis of epilepsy, none had a diagnosis of PNESs, and 54 had other or undetermined diagnoses post-SVEM. Of the 24 patients with pre-SVEM suspected PNESs, 1 had epilepsy, 12 had PNESs, and 11 had other or undetermined diagnoses. Of the 75 patients with other diagnoses pre-SVEM, 17 had epilepsy, 11 had PNESs, and 47 had other or undetermined diagnoses. After SVEM, 15 patients had definite diagnoses other than epilepsy or PNESs, while in 96 patients, diagnosis remained unclear. Overall, a definitive diagnosis could be reached in 129/226 (57%) patients. CONCLUSIONS: This study demonstrates that in nearly 3/5 patients without a definitive diagnosis after routine EEG, SVEM allowed us to reach a diagnosis. This procedure should be encouraged in this setting, given its time-effectiveness compared with LVEM.

How reliable is the monitoring of permanent vegetation plots? A test with multiple observers

Questions: A multiple plot design was developed for permanent vegetation plots. How reliable are the different methods used in this design and which changes can we measure? Location: Alpine meadows (2430 m a.s.l.) in the Swiss Alps. Methods: Four inventories were obtained from 40 m(2) plots: four subplots (0.4 m(2)) with a list of species, two 10m transects with the point method (50 points on each), one subplot (4 m2) with a list of species and visual cover estimates as a percentage and the complete plot (40 m(2)) with a list of species and visual estimates in classes. This design was tested by five to seven experienced botanists in three plots. Results: Whatever the sampling size, only 45-63% of the species were seen by all the observers. However, the majority of the overlooked species had cover < 0.1%. Pairs of observers overlooked 10-20% less species than single observers. The point method was the best method for cover estimate, but it took much longer than visual cover estimates, and 100 points allowed for the monitoring of only a very limited number of species. The visual estimate as a percentage was more precise than classes. Working in pairs did not improve the estimates, but one botanist repeating the survey is more reliable than a succession of different observers. Conclusion: Lists of species are insufficient for monitoring. It is necessary to add cover estimates to allow for subsequent interpretations in spite of the overlooked species. The choice of the method depends on the available resources: the point method is time consuming but gives precise data for a limited number of species, while visual estimates are quick but allow for recording only large changes in cover. Constant pairs of observers improve the reliability of the records.

Population Normalization with Ammonium in Wastewater-Based Epidemiology: Application to Illicit Drug Monitoring

Fluctuations in ammonium (NH4+), measured as NH4-N loads using an ion-selective electrode installed at the inlet of a sewage treatment plant, showed a distinctive pattern which was associated to weekly (i.e., commuters) and seasonal (i.e., holidays) fluctuations of the population. Moreover, population size estimates based on NH4-N loads were lower compared to census data. Diurnal profiles of benzoylecgonine (BE) and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH) were shown to be strongly correlated to NH4-N. Characteristic patterns, which reflect the prolonged nocturnal activity of people during the weekend, could be observed for BE, cocaine, and a major metabolite of MDMA (i.e., 4-hydroxy-3-methoxymethamphetamine). Additional 24 h composite samples were collected between February and September 2013. Per-capita loads (i.e., grams per day per 1000 inhabitants) were computed using census data and NH4-N measurements. Normalization with NH4-N did not modify the overall pattern, suggesting that the magnitude of fluctuations in the size of the population is negligible compared to those of illicit drug loads. Results show that fluctuations in the size of the population over longer periods of time or during major events can be monitored using NH4-N loads: either using raw NH4-N loads or population size estimates based on NH4-N loads, if information about site-specific NH4-N population equivalents is available.

Monitoring of human CD4 T cell responses in metastatic melanoma and arthritic patients

SUMMARY : Detailed knowledge of the different components of the immune system is required for the development of new immunotherapeutic strategies. CD4 T lymphocytes represent a highly heterogeneous group of cells characterized by various profiles of cytokine production and effector vs. regulatory functions. They are central players in orchestrating adaptive immune responses: unbalances between the different subtypes can lead either to aggressive autoimmune disorders or can favour the uncontrolled growth of malignancies. In this study we focused on the characterization of human CD4 T cells in advanced stage melanoma patients as well as in patients affected by various forms of autoimmune inflammatory spondyloarthropathies. In melanoma patients we report that a population of FOXP3 CD4 T cells, known as regulatory T cells, is overrepresented in peripheral blood, and even more in tumor-infitrated lymph nodes as well as at tumor sites, as compared to healthy donors. In tumor-infiltrated lymph nodes, but not in normal lymph nodes or in peripheral blood, FOXP3 CD4 T cells feature a highly differentiated phenotype (CD45RA-CCR7+/-), which suggests for a recent encounter with their cognate antigen. FOXP3 CD4 T cells have been described to be an important component of the several known immune escape mechanisms. We demonstrated that FOXP3 CD4 T cells isolated from melanoma patients exert an in vitro suppressive action on autologous CD4 T cells, thus possibly inhibiting an efficient anti-tumor response. Next, we aimed to analyse CD4 T cells at antigen-specific level. In advanced stage melanoma patients, we identified for the first time, using pMHCII multimers, circulating CD4 T cells specific for the melanoma antigen Melan-A, presented by HLA-DQB1 *0602. Interestingly, in a cohort of melanoma patients enrolled in an immunotherapy trails consisting of injection of a Melan-A derived peptide, we did not observe signif cant variations in the ex vivo frequencies of Melan-A specific CD4 T cells, but important differences in the quality of the specific CD4 T cells. In fact, up to 50% of the ex vivo Melan-A/DQ6 specific CD4 T cells displayed a regulatory phenotype and were hypoproliferative before vaccination, while more effector, cytokine-secreting Melan-A/DQ6 specific CD4 T cells were observed after immunization. These observations suggest that peptide vaccination may favourably modify the balance between regulatory and effector tumor-specific CD4 T cells. Finally, we identified another subset of CD4 T cells as possible mediator of pathology in a group of human autoimmune spondyloarthropathies, namely Th17 cells. These cells were recently described to play a critical role in the pathogenesis of some marine models of autommunity. We document an elevated presence of circulating Th17 cells in two members of seronegative spondyloarthropathies, e.g. psoriatic arthritis and ankylosing spondylitis, while we do not observe increased frequencies of Th17 cells in peripheral blood of rheumatoid arthritic patients. In addition, Th17 cells with a more advanced differentiation state (CD45RA-CCR7-CD27-) and polyfunctionality (concomitant secretion of IL-17, IL-2 and TNFα) were observed exclusively in patients with seronegative spondylarthropathies. Together, our observations emphasize the importance of CD4 T cells in various diseases and suggest that immunotherapeutic approaches considering CD4 T cells as targets should be evaluated in the future.