Genomic Features and Phylogenetic Analysis of Antimicrobial-Resistant Salmonella Mbandaka ST413 Strains.

In recent years, Salmonella enterica subsp. enterica serovar Mbandaka (S. Mbandaka) has been increasingly isolated from laying hens and shell eggs around the world. Moreover, this serovar has been identified as the causative agent of several salmonellosis outbreaks in humans. Surprisingly, little is known about the characteristics of this emerging serovar, and therefore, we investigated antimicrobial resistance, virulence, and prophage genes of six selected Brazilian strains of Salmonella Mbandaka using Whole Genome Sequencing (WGS). Multi-locus sequence typing revealed that the tested strains belong to Sequence Type 413 (ST413), which has been linked to recent multi-country salmonellosis outbreaks in Europe. A total of nine resistance genes were detected, and the most frequent ones were aac(6')-Iaa, sul1, qacE, blaOXA-129, tet(B), and aadA1. A point mutation in ParC at the 57th position (threonine → serine) associated with quinolone resistance was present in all investigated genomes. A 112,960 bp IncHI2A plasmid was mapped in 4/6 strains. This plasmid harboured tetracycline (tetACDR) and mercury (mer) resistance genes, genes contributing to conjugative transfer, and genes involved in plasmid maintenance. Most strains (four/six) carried Salmonella genomic island 1 (SGI1). All S. Mbandaka genomes carried seven pathogenicity islands (SPIs) involved in intracellular survival and virulence: SPIs 1-5, 9, and C63PI. The virulence genes csgC, fimY, tcfA, sscA, (two/six), and ssaS (one/six) were absent in some of the genomes; conversely, fimA, prgH, and mgtC were present in all of them. Five Salmonella bacteriophage sequences (with homology to Escherichia phage phiV10, Enterobacteria phage Fels-2, Enterobacteria phage HK542, Enterobacteria phage ST64T, Salmonella phage SW9) were identified, with protein counts between 31 and 54, genome lengths of 24.7 bp and 47.7 bp, and average GC content of 51.25%. In the phylogenetic analysis, the genomes of strains isolated from poultry in Brazil clustered into well-supported clades with a heterogeneous distribution, primarily associated with strains isolated from humans and food. The phylogenetic relationship of Brazilian S. Mbandaka suggests the presence of strains with high epidemiological significance and the potential to be linked to foodborne outbreaks. Overall, our results show that isolated strains of S. Mbandaka are multidrug-resistant and encode a rather conserved virulence machinery, which is an epidemiological hallmark of Salmonella strains that have successfully disseminated both regionally and globally.

Probing the correlation of neuronal loss, neurofibrillary tangles, and cell death markers across the Alzheimer's disease Braak stages: a quantitative study in humans.

Clarifying the mechanisms connecting neurofibrillary tangle (NFT) neurotoxicity to neuronal dysfunction in humans is likely to be pivotal for developing effective treatments for Alzheimer's disease (AD). To model the temporal progression of AD in humans, we used a collection of brains with controls and individuals from each Braak stage to quantitatively investigate the correlation between intraneuronal caspase activation or macroautophagy markers, NFT burden, and neuronal loss, in the dorsal raphe nucleus and locus coeruleus, the earliest vulnerable areas to NFT accumulation. We fit linear regressions with each count as outcomes, with Braak score and age as the predictors. In progressive Braak stages, intraneuronal active caspase-6 positivity increases both alone and overlapping with NFTs. Likewise, the proportion of NFT-bearing neurons showing autophagosomes increases. Overall, caspases may be involved in upstream cascades in AD and are associated with higher NFTs. Macroautophagy changes correlate with increasing NFT burden from early AD stages.

Locus coeruleus volume and cell population changes during Alzheimer's disease progression: A stereological study in human postmortem brains with potential implication for early-stage biomarker discovery.

INTRODUCTION: Alzheimer's disease (AD) progression follows a specific spreading pattern, emphasizing the need to characterize those brain areas that degenerate first. The brainstem's locus coeruleus (LC) is the first area to develop neurofibrillary changes (neurofibrillary tangles [NFTs]). METHODS: The methods include unbiased stereological analyses in human brainstems to estimate LC volume and neuronal population in controls and individuals across all AD stages. RESULTS: As the Braak stage increases by 1 unit, the LC volume decreases by 8.4%. Neuronal loss started only midway through AD progression. Age-related changes spare the LC. DISCUSSION: The long gap between NFT accumulation and neuronal loss suggests that a second trigger may be necessary to induce neuronal death in AD. Imaging studies should determine whether LC volumetry can replicate the stage-wise atrophy observed here and how these changes are specific to AD. LC volumetry may develop into a screening biomarker for selecting high-yield candidates to undergo expensive and less accessible positron emission tomography scans and to monitor AD progression from presymptomatic stages.

GRN and MAPT Mutations in 2 Frontotemporal Dementia Research Centers in Brazil.

BACKGROUND: Mutations in GRN (progranulin) and MAPT (microtubule-associated protein tau) are among the most frequent causes of monogenic frontotemporal dementia (FTD), but data on the frequency of these mutations in regions such as Latin America are still lacking. OBJECTIVE: We aimed to investigate the frequencies of GRN and MAPT mutations in FTD cohorts from 2 Brazilian dementia research centers, the University of Sao Paulo and the Federal University of Minas Gerais medical schools. METHODS: We included 76 probands diagnosed with behavioral-variant FTD (n=55), semantic-variant Primary Progressive Aphasia (PPA) (n=11), or nonfluent-variant PPA (n=10). Twenty-five percent of the cohort had at least 1 relative affected with FTD. RESULTS: Mutations in GRN were identified in 7 probands, and in MAPT, in 2 probands. We identified 3 novel GRN mutations (p.Q130X, p.317Afs*12, and p.K259Afs*23) in patients diagnosed with nonfluent-variant PPA or behavioral-variant FTD. Plasma progranulin levels were measured and a cutoff value of 70 ng/mL was found, with 100% sensitivity and specificity to detect null GRN mutations. CONCLUSIONS: The frequency of GRN mutations was 9.6% and that of MAPT mutations was 7.1%. Among familial cases of FTD, the frequency of GRN mutations was 31.5% and that of MAPT mutations was 10.5%.

Laminin-111 derived peptides AG73 and C16 regulate invadopodia activity of a human adenoid cystic carcinoma cell line.

Adenoid cystic carcinoma is a frequently occurring malignant salivary gland neoplasm with high level of recurrence and distant metastasis long time after treatment. Metastatic tumor cells that actively migrate and invade surrounding tissues rely on invadopodia to degrade extracellular matrix (ECM) barriers. Invadopodia are actin-rich membrane protrusions that localize enzymes required for ECM degradation. Breakdown of ECM macromolecules releases fragments and bioactive peptides. We have already demonstrated that laminin-111 and its derived peptides regulate migration, invasion and protease activity of adenocarcinoma cells. Here we addressed the role of laminin-111 peptides AG73 and C16 in invadopodia activity of cells (CAC2) derived from human adenoid cystic carcinoma. CAC2 cells were treated by AG73 and C16, and subjected to fluorescent gelatin substrate degradation assay. In this assay invadopodia activity areas appear as black dots in a fluorescent background. Both peptides significantly increased invadopodia formation and activity compared to controls. We analyzed putative receptors and signaling pathways related to peptide effects. ß1 integrin silencing by siRNA decreased AG73- and C16-induced invadopodia. Furthermore inhibition of Rac1 and ERK signaling pathways decreased both C16- and AG73-related invadopodia activities. We propose that laminin-111 peptides AG73 and C16 increase invadopodia activity in CAC2 cells through ß1 integrin. Rac1 and ERK1/2 signaling pathways would transduce signals generated by both peptides.

Role of MMP9 on invadopodia formation in cells from adenoid cystic carcinoma. Study by laser scanning confocal microscopy.

Migration, invasion and protease activity are essential for tumor progression and metastasis. Metastatic cells rely on invadopodia to degrade and invade extracellular matrix (ECM). Invadopodia are membrane protrusions with enzymes required for ECM degradation. These protrusions contain cortactin and membrane type 1 matrix metalloproteinase (MT1-MMP) superimposed to areas of digested matrix. Here we characterized invadopodia in a cell line (CAC2) derived from human adenoid cystic carcinoma. We carried out fluorescent-substrate degradation assay to assess in situ protease activity of CAC2 cells. Digestion spots in fluorescent substrate appear as black areas in green background. Cells were cultured on Matrigel-gelatin-FITC and fixed after 1 h and 3 h. CAC2 cells were double labeled to actin and cortactin. Cells were also double stained to actin and MT1-MMP. Samples were studied by laser scanning confocal microscopy. In all time points CAC2 cells showed actin, cortactin, and MT1-MMP colocalized with digestion spots in fluorescent substrate. We searched for other proteases involved in invadopodia activity. We have previously demonstrated that MMP9 influences adenoid cystic carcinoma behavior. This prompted us to investigate role played by MMP9 on invadopodia formation. CAC2 cells had MMP9 silenced by siRNA. After 1 h in fluorescent substrate, cells with silenced MMP9 showed clear decrease in matrix digestion compared with controls. No differences were found in cells with silenced MMP9 grown for 3 h on fluorescent substrate. Our results showed that CAC2 cells exhibit functional invadopodia containing cortactin and MT1-MMP. Furthermore, MMP9 would be required in the initial steps of invadopodia formation.