Digital microfluidic devices for isothermal circular strand displacement polymerization of microRNAs

Nucleic acid amplification is a key step in nucleic acid detection assays. The use of digital microfluidic devices to miniaturize amplification protocols reduces the required sample volume and the analysis times and offers new possibilities for the process automation and integration in one single device. Unlike polymerase chain reaction (PCR) amplification methods (requiring repeated cycles of three or two temperature-dependent steps during amplification), low temperature isothermal amplification methods have no need for thermal cycling thus requiring simplified microfluidic device features. Here, the combined use of digital microfluidics and molecular beacon (MB)-assisted isothermal circular strand displacement polymerization (ICSDP) technique is described for detection of miR-210, the most consistently and predominantly upregulated hypoxia-inducible microRNA. The ability of the droplet ICSDP isothermal method to discriminate between full-matched, single mismatched and unrelated sequences has been demonstrated. The detection of a range of nM/pM miR-210 solutions compartmentalized in nanoliter-sized droplets was performed to demonstrate the method capabilities for detecting as low as 10-18 moles of microRNA target sequences compartmentalized in 20 nL droplets. The method capability to operate with biological samples was tested by detecting miR-210 from transfected K562 cells. When combined with digital microfluidics ICSDP offers a convenient environment for fast, simple and effective DNA and microRNA detection. In addition, droplet ICSDP isothermal procedure simplifies the integration of nucleic acids detection protocols in microfluidic devices. Potentials offered by the method have been demonstrated by designing specific experiments using a total of 200 nL of solutions carrying a total of as low as 6 x 107 molecules. In addition, the method offers possibilities for specifically detecting as low as 3.3 10-18 moles of microRNA target sequences compartmentalized in 20 nL droplets. The new approach represents a promising tool for the development of simpler and more convenient lab-on-chip devices for miRNAs detection

Genetic risk factors and candidate biomarkers for Alzheimer’s disease

Alzheimer's disease is a multifactorial and progressive neurodegenerative disease, extremely diffused and with an increasing prevalence worldwide. There is an urgent need for biomarkers to diagnose AD early in its course. Furthermore, accurate biomarkers would be able to determine the clinical efficacy of novel neuroprotective strategies. Although the heritability of late-onset AD is high, our knowledge of the underlying putative susceptibility genes remains incomplete and the only unequivocally established late-onset AD gene is APOE. Nevertheless a number of susceptibility loci seems to influence the pathogenesis of AD, and variations in numerous genes have been considered to be important in the risk for AD. Many advances have been made in identifying biochemical indices of brain dysfunction, measured in body fluids such as cerebrospinal fluid and plasma, with different methodological approaches. Although these biomarkers are promising, none of them can predict AD with 100% confidence to date. This review will elaborate on the available selection of genetic and biochemical biomarkers for AD, with a particular reference to those linked to inflammation and oxidative stress

Toward Sustainable Development Trajectories? Estimating Urban Footprints from High-Resolution Copernicus Layers in Athens, Greece

Land imperviousness reflects settlement growth and urban sprawl. Grounded on a comparative approach, a set of multidimensional statistical techniques were adopted here to quantify the evolution of land imperviousness from Copernicus High-Resolution Layers (HRLs) in a representative case study of Southern Europe (Athens, Greece). A two-way data matrix reporting the percent share of the surface land exposed to different sealing levels (101 classes ranging continuously from 0% to 100%) in the total municipal area was computed for two years (2006 and 2018) individually for 115 municipalities in metropolitan Athens. This matrix represented the information base needed to derive place-specific urban footprints and a comprehensive (global) profile of land imperviousness. Results of a Detrended Correspondence Analysis (DCA) delineated a metropolitan structure still organized along the density gradient, moving from dense settlements in central locations with dominant land classes sealed for more than 90% of their surface area to completely pervious land (0%) typical of rural locations. While the density gradient became less steep between 2006 and 2018, it continued to aliment a socioeconomic polarization in urban and rural districts with distinctive profiles of land imperviousness. Intermediate locations had more mixed imperviousness profiles as a result of urban sprawl. Differential profiles reflect place-specific urban footprints with distinctive land take rates

Areas Activated in Patient GN for HFF (unknown faces wrongly recognized as familiar > unknown faces correctly recognized as unfamiliar).

<p>Areas Activated in Patient GN for HFF (unknown faces wrongly recognized as familiar > unknown faces correctly recognized as unfamiliar).</p

Sulcal Depth of Patient’s GN Brain.

<p>(A). Three-dimensional reconstruction of the sulcal depth in GN’s brain, from surface and shallow depth values (green/yellow) to deep values (orange/red). Note the deepening and widening of the temporal sulci in the left compared to right hemisphere as outlined by expanded red areas in lateral sulcus and in the STS.</p

Anatomical MRI and SPECT of Patient’s GN Brain.

<p>(A) T1-weighted MRI transversal and coronal images showing atrophy in the left temporal lobe (white circles). (B) SPECT transversal and coronal images showing hypoperfusion in the left tempo-parietal region (white circles).</p

Patient GN’s Neuropsychological Assessment.

<p>^aAll scores are corrected for age and education.</p><p>^bMMSE = Mini Mental State Evaluation.</p><p>^cFAB = Frontal Assessment Battery.</p><p>* = Score under normal limits.</p><p>Patient GN’s Neuropsychological Assessment.</p

Brain areas significantly activated by the presentation of faces (known and unknown faces pooled together > baseline activity).

<p>(A) Activations in the left and right FFA. (B) Activations in the left and right OFA. (C) Activation in the right pSTS. (D) Activation in the PCUN/pCING.</p

Recognition of Face Familiarity in Patient GN and in the Twelve Age-, Gender- and Education-matched Controls.

<p>Recognition of Face Familiarity in Patient GN and in the Twelve Age-, Gender- and Education-matched Controls.</p

Sulcal Depth Values and Signal Detection Response Parameters.

<p>(A) Sulcal depth values for the lateral sulcus, STS and the anterior part of the ITS in the left (LH) and right hemisphere (RH) of patient GN and of the twelve age-, gender, and education-matched controls. (B) Signal detection parameters in patient GN (red lines) and averaged parameters in the twelve controls (black lines). High discrimination sensitivity between familiar and unfamiliar faces (<i>d</i>_<i>a</i>) is graphically represented by reduced overlapping between signal distributions (familiarity) (continuous lines) and noise distributions (unfamiliarity) (dashed lines). Response bias (<i>c</i>_<i>a</i>) is represented by the vertical lines, with negative values indicating a loose response criterion (i.e., a tendency to favour familiarity responses).</p