Epitope Mapping by NMR of a Novel Anti-Aβ Antibody (STAB-MAb)

FP7-SP3-People-606950 POCI-01-0145-FEDER-007728 Project No 022161Alzheimer´s Disease (AD) is one of the most common neurodegenerative disorders worldwide. Excess of β-amyloid (Aβ), a peptide with a high propensity to misfold and self-aggregate, is believed to be the major contributor to the observed neuronal degeneration and cognitive decline in AD. Here, we characterize the epitope of a novel anti-Aβ monoclonal antibody, the STAB-MAb, which has previously demonstrated picomolar affinities for both monomers (KD = 80 pM) and fibrils (KD = 130 pM) of Aβ(1–42) and has shown therapeutic efficacy in preclinical mouse models of AD. Our findings reveal a widespread epitope that embraces several key Aβ residues that have been previously described as important in the Aβ fibrillation process. Of note, STAB-MAb exhibits a stronger affinity for the N-terminus of Aβ and stabilizes an α-helix conformation in the central to N-terminal region of the peptide, in addition to disrupting a characteristic salt-bridge of a hairpin structure present in fibrils. The NMR derived epitope supports the observed results from ThT-monitored fluorescence and electron microscopy experiments, in which STAB-MAb was shown to inhibit the formation of aggregates and promote disruption of pre-formed fibrils. In combination with the published in vitro and in vivo assays, our study highlights STAB-MAb as a rare and versatile antibody with analytical, diagnostic and therapeutic efficacy.publishersversionpublishe

Association of FTO and PPARG polymorphisms with obesity in Portuguese women

Purpose: We evaluated the association between risk of obesity in the Portuguese population and two obesity-related single-nucleotide gene polymorphisms: fat-mass and obesity-associated (FTO) rs9939609 and peroxisome proliferator-activated receptor gamma (PPARG) rs1801282. Patients and methods: A total of 194 Portuguese premenopausal female Caucasians aged between 18 and 50 years (95 with body mass index [BMI] ≥30 g/m2, 99 controls with BMI 18.5–24.9 kg/m2) participated in this study. The association of the single-nucleotide polymorphisms with obesity was determined by odds ratio calculation with 95% confidence intervals. Results: Significant differences in allelic expression of FTO rs9939609 (P0.05). Conclusion: For the first time, a study involving an adult Portuguese population shows that individuals harboring both risk alleles in the FTO gene locus are at higher risk for obesity, which is in agreement to what has been reported for other European populations

The Influence of Size

Funding Information: This work was financed by national funds from FCT – Fundação para a Ciência e a Tecnologia, I.P., in the scope of the following Projects: UIDP/04378/2020 and UIDB/04378/2020 of the Research Unit on Applied Molecular Biosciences – UCIBIO; LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy – i4HB; UIDB/50006/2020 and UIDP/50006/2020 of the Associate Laboratory for Green Chemistry – LAQV; and PTDC/NAN‐MAT/30589/2017 (NANOMODE). Dr. Rocío Jurado is acknowledged for initial work under the scope of the NANOMODE Project. Publisher Copyright: © 2022 The Authors. Particle & Particle Systems Characterization published by Wiley-VCH GmbH.Gold nanoparticles (AuNPs) are widely used in biomedical diagnostics due to their unique plasmonic properties, with larger AuNPs showing higher extinction coefficients for the plasmon band and, consequently, more intense colors than the more commonly used spherical 20 nm AuNPs. Other factors can be important in the performance of different-sized AuNPs, including surface area, colloidal stability, and curvature effects. Here, the properties of spherical 20 nm AuNPs and 35 nm AuNPs functionalized with a specific thiol-modified oligonucleotide–Au nanoprobes are compared when used on a colorimetric assay for the detection of a single nucleotide polymorphism related to lactose intolerance in humans. Successful functionalization of AuNPs is assessed by UV–vis spectroscopy, agarose gel electrophoresis, dynamic and electrophoretic light scattering, and nanoparticle tracking analysis. Statistical differences between Au-nanoprobe DNA target groups are calculated using analysis of variance and a post hoc Tukey's test. These results show that both 35 and 20 nm Au nanoprobes have similar detection limits using a 0.15 nmol dm−3 nanoprobe concentration compared to 2.5 nmol dm−3. Interestingly, the use of 35 nm Au nanoprobes allows a reduction of 80% and 48% in the amount of gold and oligonucleotide used in the assay, respectively.publishersversionpublishe

On the road of sequencing the genomes: Past, Present and Future

Desulfovibrio gigas is a model organism of sulfate-reducing bacteria of which energy metabolism and stress response have been extensively studied. The complete genomic context of this organism was however not yet available. This thesis is about the sequencing of the D. gigas genome and provides insights into the integrated network of energy conserving complexes and structures present in this bacterium. The work presented describes the technological processes used to sequence the genome. Chapter I is an overview of its most recent advances in bacterial genome sequencing, since the first genome published of Haemophilus influenzae till present; Chapter II is mostly an overview of the 4 different technologies that were necessary to finalize this genome sequencing project: shotgun sequencing through Sanger method and massive DNA sequencing from 3 different next generation sequencing (NGS) platforms: 454 (Roche);(...

Efficient Identification of Clinically Relevant Candida Yeast Species by Use of an Assay Combining Panfungal Loop-Mediated Isothermal DNA Amplification with Hybridization to Species-Specific Oligonucleotide Probes▿

The occurrence of invasive mycoses has progressively increased in recent years. Yeasts of the genus Candida remain the leading etiologic agents of those infections. Early identification of opportunistic yeasts may contribute significantly to improved disease management and the selection of appropriate antifungal therapy. We developed a rapid and reliable molecular identification system for clinically relevant yeasts that makes use of nonspecific primers to amplify a region of the 26S rRNA gene, followed by reverse hybridization of the digoxigenin-labeled products to a panel of species-specific oligonucleotide probes arranged on a nylon membrane macroarray format. DNA amplification was achieved by the recently developed loop-mediated isothermal DNA amplification technology, a promising option for the development of improved laboratory diagnostic kits. The newly developed method was successful in distinguishing among the major clinically relevant yeasts associated with bloodstream infections by using simple, rapid, and cost-effective procedures and equipment

Allele specific LAMP- gold nanoparticle for characterization of single nucleotide polymorphisms

Due to their relevance as disease biomarkers and for diagnostics, screening of single nucleotide polymorphism (SNPs) requires simple and straightforward strategies capable to provide results in medium throughput settings. Suitable approaches relying on isothermal amplification techniques have been evolving to substitute the cumbersome and highly specialized PCR amplification detection schemes. Nonetheless, identification of an individualâs genotype still requires sophisticated equipment and laborious methods.Here, we present a low-cost and reliable approach based on the allele specific loop-mediated isothermal amplification (AS-LAMP) coupled to ssDNA functionalized gold nanoparticle (Au-nanoprobe) colorimetric sequence discrimination. The Au-nanoprobe integration allows for the colorimetric detection of AS-LAMP amplification product that can be easily interpreted in less than 15 min. We targeted a clinical relevant SNP responsible for lactose intolerance (-13910C/T dbSNP rs#: 4988235) to demonstrate its proof of concept and full potential of this novel approach. Keywords: SNP, Isothermal amplification, Gold nanoparticles, Gold nanoprobes, Lactose intoleranc

Retrospective analysis of clinical yeast isolates in a hospital in the centre of Portugal: spectrum and revision of the identification procedures

We conducted a four-year (2003-2006) retrospective study of yeasts recovered in a hospital laboratory in the centre of Portugal to evaluate the epidemiology of yeast infections. Clinical isolates and data were gathered from 751 patients corresponding to 906 episodes of yeast infection. The isolates were first identified using classical and commercial methods, routinely employed at the hospital laboratory. We then re-identified the same isolates using RFLP of the ITS 5.8S rRNA gene and sequence of the D1/D2 domain of the 26S rRNA gene. Candida parapsilosis isolates were re-identified using the Ban I digestion of the SADH gene. C. albicans was the most frequently isolated of the yeasts found in the analysed specimens, with an overall incidence of 69.6% and then in deceasing order, C. glabrata, C. tropicalis, C. parapsilosis and C. krusei. C. parapsilosis was most frequently recovered from younger patients, decreasing with age, while C. glabrata occurrence increased with age. We found an increased number of cases of fungemia per 100,000 people per year, reaching a maximum of 4.4 during 2006

Molecular cloning and sequence analysis of the gene of the molybdenum‐containing aldehyde oxido‐reductase of Desulfovibrio gigas The deduced amino acid sequence shows similarity to xanthine dehydrogenase

In this report, we describe the isolation of a 4020‐bp genomic PstI fragment of Desulfovibrio gigas harboring the aldehyde oxido‐reductase gene. The aldehyde oxido‐reductase gene spans 2718 bp of genomic DNA and codes for a protein with 906 residues. The protein sequence shows an average 52% (± 1.5%) similarity to xanthine dehydrogenase from different organisms. The codon usage of the aldehyde oxidoreductase is almost identical to a calculated codon usage of the Desulfovibrio bacteria.publishersversionpublishe

Antibody-Functionalized Polymer Nanoparticle Leading to Memory Recovery in Alzheimer's Disease-like Transgenic Mouse Model

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