Fungi and bacteria occupy distinct spatial niches within carious dentin.

The role of bacteria in the etiology of dental caries is long established, while the role of fungi has only recently gained more attention. The microbial invasion of dentin in advanced caries especially merits additional research. We evaluated the fungal and bacterial community composition and spatial distribution within carious dentin. Amplicon 16S rRNA gene sequencing together with quantitative PCR was used to profile bacterial and fungal species in caries-free children (n = 43) and 4 stages of caries progression from children with severe early childhood caries (n = 32). Additionally, healthy (n = 10) and carious (n = 10) primary teeth were decalcified, sectioned, and stained with Grocott's methenamine silver, periodic acid Schiff (PAS) and calcofluor white (CW) for fungi. Immunolocalization was also performed using antibodies against fungal ß-D-glucan, gram-positive bacterial lipoteichoic acid, gram-negative endotoxin, Streptococcus mutans, and Candida albicans. We also performed field emission scanning electron microscopy (FESEM) to visualize fungi and bacteria within carious dentinal tubules. Bacterial communities observed included a high abundance of S. mutans and the Veillonella parvula group, as expected. There was a higher ratio of fungi to bacteria in dentin-involved lesions compared to less severe lesions with frequent preponderance of C. albicans, C. dubliniensis, and in one case C. tropicalis. Grocott's silver, PAS, CW and immunohistochemistry (IHC) demonstrated the presence of fungi within carious dentinal tubules. Multiplex IHC revealed that fungi, gram-negative, and gram-positive bacteria primarily occupied separate dentinal tubules, with rare instances of colocalization. Similar findings were observed with multiplex immunofluorescence using anti-S. mutans and anti-C. albicans antibodies. Electron microscopy showed monomorphic bacterial and fungal biofilms within distinct dentin tubules. We demonstrate a previously unrecognized phenomenon in which fungi and bacteria occupy distinct spatial niches within carious dentin and seldom co-colonize. The potential significance of this phenomenon in caries progression warrants further exploration.

The Impact of Anti-Amyloid Immunotherapies on Stroke Care.

Anti-amyloid immunotherapies have recently emerged as treatments for Alzheimer's disease. While these therapies have demonstrated efficacy in clearing amyloid-ß and slowing cognitive decline, they have also been associated with amyloid-related imaging abnormalities (ARIA) which include both edema (ARIA-E) and hemorrhage (ARIA-H). Given that ARIA have been associated with significant morbidity in cases of antithrombotic or thrombolytic therapy, an understanding of mechanisms of and risk factors for ARIA is of critical importance for stroke care. We discuss the latest data regarding mechanisms of ARIA, including the role of underlying cerebral amyloid angiopathy, and implications for ischemic stroke prevention and management.

Altered localization of nucleoporin 98 in primary tauopathies.

Nucleoporin 98 is a nuclear pore complex component that is mislocalized in Alzheimer's disease and the alteration in nucleoporin 98 has been attributed to tau. In order to determine if nucleoporin 98 mislocalization is a general feature of tauopathies, we assessed the localization of nucleoporin 98 in neurons in primary tauopathies, including frontotemporal lobar degeneration-tau, corticobasal degeneration and progressive supranuclear palsy. Immunofluorescence staining was performed on frontal cortex and occipital cortex tissue from cases of primary tauopathies and controls without neurodegenerative disease using antibodies to identify nucleoporin 98, phospho-tau (Ser202, Thr205) monoclonal antibody and neuronal marker microtubule-associated protein 2. The stained tissue was imaged by fluorescence microscopy and the number of neurons with mislocalized nucleoporin 98 and phospho-tau (Ser202, Thr205) monoclonal antibody staining was quantified. In frontal cortex tissue, all primary tauopathies examined demonstrated significantly increased numbers of neurons with abnormal localization of nucleoporin 98 along the nuclear envelope compared with control tissue. Additionally, frontotemporal lobar degeneration-tau and corticobasal degeneration in the frontal cortex demonstrated significantly increased numbers of neurons with a cytoplasmic mislocalization of nucleoporin 98 compared with control tissue. The number of neurons with mislocalized nucleoporin 98 was significantly correlated with the number of neurons with phospho-tau (Ser202, Thr205) monoclonal antibody-positive tau staining. In the occipital cortex, which is relatively spared from pathological tau accumulations in these primary tauopathies, the localization of nucleoporin 98 was not significantly altered. This study demonstrates that nucleoporin 98 mislocalization is a feature of primary tauopathies and is associated with pathological tau accumulation. In the context of prior research demonstrating nucleoporin 98 mislocalization in Alzheimer's disease and an interaction between tau and nucleoporin 98, these results further support the hypothesis that pathological tau may contribute to nucleoporin 98 mislocalization. Given the critical role of the nuclear pore complex in nucleocytoplasmic transport, the identification of nucleoporin 98 mislocalization in primary tauopathies highlights a potential pathophysiological disruption in these disorders.

Cytoplasmic Mislocalization of RNA Polymerase II Subunit RPB1 in Alzheimer Disease Is Linked to Pathologic Tau.

Abnormal protein accumulation and mislocalization is a general hallmark of Alzheimer disease. Recent data suggest nucleocytoplasmic transport may be compromised by tau in Alzheimer disease. In this context, we have examined the RNA polymerase II subunit RPB1, which is the catalytic subunit that plays a critical role in transcription. Using immunofluorescence staining in control and Alzheimer disease hippocampal tissue, we show that 2 phosphoisoforms of RPB1 mislocalize from the nucleus to the cytoplasm of neurons in Alzheimer disease. The number of neurons with this cytoplasmic mislocalization is correlated with the burden of pathologic tau (AT8-immunopositive neurons). In order to test whether there is a causal relationship between pathologic tau and cytoplasmic RPB1 accumulation, we used the rTg4510 mouse model, which expresses a regulatable pathologic human tau species harboring the P301L mutation. Using immunofluorescence staining on brain tissue from young (2.5-month-old) and aged (8.5- to 10-month-old) rTg4510 mice, we found a tau- and age-dependent increase in cytoplasmic mislocalization of Rpb1. In summary, this study provides evidence that tau induces mislocalization of RPB1 in Alzheimer disease, and since RPB1 is essential for transcription, this raises the possibility that RPB1 mislocalization could lead to fundamental alterations in neuronal health.

Alternative polyadenylation and miR-34 family members regulate tau expression.

Tau pathologically aggregates in Alzheimer's disease, and evidence suggests that reducing tau expression may be safe and beneficial for the prevention or treatment of this disease. We sought to examine the role of the 3'-untranslated region (3'-UTR) of human tau mRNA in regulating tau expression. Tau expresses two 3'-UTR isoforms, long and short, as a result of alternative polyadenylation. Using luciferase reporter constructs, we found that expression from these isoforms is differentially controlled in human neuroblastoma cell lines M17D and SH-SY5Y. Several microRNAs were computationally identified as candidates that might bind the long, but not short, tau 3'-UTR isoform. A hit from a screen of candidates, miR-34a, was subsequently shown to repress the expression of endogenous tau protein in M17D cells. Conversely, inhibition of endogenously expressed miR-34 family members leads to increased endogenous tau expression. In addition, through an unbiased screen of fragments of the human tau 3'-UTR using a luciferase reporter assay, we identified several other regions in the long tau 3'-UTR isoform that contain regulatory cis-elements. Improved understanding of the regulation of tau expression by its 3'-UTR may ultimately lead to the development of novel therapeutic strategies for the treatment of Alzheimer's disease and other tauopathies. mRNA 3'-untranslated regions (3'-UTR) often regulate transcript stability or translation. Despite the centrality of the tau protein in Alzheimer's and other neurodegenerative diseases, the human tau 3'-UTR has been little studied. This report identifies regions of the tau 3'-UTR that influence expression and shows that microRNA (miR)-34a targets this 3'-UTR to lower expression, which is considered an important therapeutic goal.