Synthetic prions with novel strain-specified properties

Prions are infectious proteins that possess multiple self-propagating structures. The information for strains and structural specific barriers appears to be contained exclusively in the folding of the pathological isoform, PrP(Sc). Many recent studies determined that de novo prion strains could be generated in vitro from the structural conversion of recombinant (rec) prion protein (PrP) into amyloidal structures. Our aim was to elucidate the conformational diversity of pathological recPrP amyloids and their biological activities, as well as to gain novel insights in characterizing molecular events involved in mammalian prion conversion and propagation. To this end we generated infectious materials that possess different conformational structures. Our methodology for the prion conversion of recPrP required only purified rec full-length mouse (Mo) PrP and common chemicals. Neither infected brain extracts nor amplified PrP(Sc) were used. Following two different in vitro protocols recMoPrP converted to amyloid fibrils without any seeding factor. Mouse hypothalamic GT1 and neuroblastoma N2a cell lines were infected with these amyloid preparations as fast screening methodology to characterize the infectious materials. Remarkably, a large number of amyloid preparations were able to induce the conformational change of endogenous PrPC to harbor several distinctive proteinase-resistant PrP forms. One such preparation was characterized in vivo habouring a synthetic prion with novel strain specified neuropathological and biochemical properties

Cerebrospinal fluid beta-2-microglobulin in multiple sclerosis and AIDS dementia complex.

Cerebrospinal fluid (CSF) and serum concentrations of beta-2-microglobulin (beta-2-m) were evaluated in 19 patients with clinically definite multiple sclerosis (MS), in 21 with AIDS dementia complex (ADC), and in 20 subjects with other neurological diseases (OND). CSF beta-2-m and CSF/serum beta-2-m ratio were significantly higher in the patients with ADC than in the MS and OND patients. The CSF and serum levels of beta-2-m in MS patients were not significantly different from those of OND patients. These findings indicate that CSF beta-2-m and CSF/serum ratio may be a useful marker in the diagnosis of ADC. In MS patients the beta-2-m CSF determinations are of no value

Vision-Based Pose Estimation for Robot-Mediated Hand Telerehabilitation

Vision-based Pose Estimation (VPE) represents a non-invasive solution to allow a smooth and natural interaction between a human user and a robotic system, without requiring complex calibration procedures. Moreover, VPE interfaces are gaining momentum as they are highly intuitive, such that they can be used from untrained personnel (e.g., a generic caregiver) even in delicate tasks as rehabilitation exercises. In this paper, we present a novel master–slave setup for hand telerehabilitation with an intuitive and simple interface for remote control of a wearable hand exoskeleton, named HX. While performing rehabilitative exercises, the master unit evaluates the 3D position of a human operator’s hand joints in real-time using only a RGB-D camera, and commands remotely the slave exoskeleton. Within the slave unit, the exoskeleton replicates hand movements and an external grip sensor records interaction forces, that are fed back to the operator-therapist, allowing a direct real-time assessment of the rehabilitative task. Experimental data collected with an operator and six volunteers are provided to show the feasibility of the proposed system and its performances. The results demonstrate that, leveraging on our system, the operator was able to directly control volunteers’ hands movements

PMCA analysis of raw fibrils and cell lysates from infected N2a and GT1 cell lines.

<p>Seeding ability of amyloid #4 and #28 by means of PMCA using brain homogenates of CD1 mice as substrates for amplification (A). PMCA analysis of GT1 and N2a cell lysates (infected with preparations #4 and #28) and collected at passage six (P6) after the infection (B). Black arrow indicates PK-resistant PrP. Asterisk indicates unspecific binding. Western blots were performed using 6D11 monoclonal antibody to PrP (0.2 μg/mL, Covance). Blots were developed with the enhanced chemiluminescent system (ECL, Amersham Biosciences) and visualized using a G:BOX Chemi Syngene System.</p

Seeding assay of N2a and GT1 cell cultures with synthetic amyloid fibrils.

<p>Seeding of recMoPrP(23–231) amyloid preparations induced the conversion of endogenous PrP^C to mildly PK resistant forms (A) and accumulation (B) in mouse neuroblastoma N2a and mouse hypothalamic GT1 amyloid-infected cell lines analyzed six passages after the infection (P6). Western blotting shows the partial protease K (PK) resistance of N2a and GT1 amyloid fibril-infected cell lysates. Fibril-infected cell lysates (PK-lanes) were digested with PK at ratio 1:500 (w/w) (PK+ lanes). Western blots were performed using Fab D18 monoclonal antibody (1μg/mL) for GT1 infected cells and Clone-P (1μg/mL) for N2a infected cells. Blots were developed with the enhanced chemiluminescent system (ECL, Amersham Biosciences) and visualized on Hyperfilm (Amersham Biosciences) (A). Immunofluorescence imaging shows the accumulations of PrP in N2a and GT1 amyloid fibril-infected cell lines. The deposition and level of PrP (green) in amyloid fibril-infected cell lines after six passages were detected by Fab D18 monoclonal antibody (10 μg/mL final concentration). The nuclei (blue) were stained with DAPI. Scale bar is 20μm (B).</p

PK digestion assay of amyloid #4, #19, #28 and #32.

<p>Western blotting of PK digestion assay (amyloids #4, #19, #28, #32) showed partial protease K (PK) resistance of recMoPrP(23–231) (amyloids #4 and #28). RecMoPrP(23–231) amyloids (PK- lanes) were digested with PK at ratio 1:10 (w/w) (PK+ lanes) and 1:1 (w/w) (PK++ lanes). Western blots were performed using Fab D18 monoclonal antibody (1μg/mL). Blots were developed with the enhanced chemiluminescent system (ECL, Amersham Biosciences) and visualized on Hyperfilm (Amersham Biosciences)</p

PK digestion assay of GT1 and N2a cells collected at different passages after infection with amyloid fibrils.

<p>Western blotting of GT1 and N2a cell lines infected with PrP amyloid #4 was observed throughout, from first passage (P1) to sixth passage (P6) and after treatment with proteinase K at ratio 1:500 (w/w). Western blot was performed using Fab D18 monoclonal antibody (1μg/mL). Blots were developed with the enhanced chemiluminescent system (ECL, Amersham Biosciences) and visualized on Hyperfilm (Amersham Biosciences).</p