Aging exacerbates hypertension‐induced cerebral microhemorrhages in mice: role of resveratrol treatment in vasoprotection

Recent studies demonstrate that aging exacerbates hypertension‐induced cognitive decline, but the specific age‐related mechanisms remain elusive. Cerebral microhemorrhages (CMHs) are associated with rupture of small intracerebral vessels and are thought to progressively impair neuronal function. To determine whether aging exacerbates hypertension‐induced CMHs young (3 months) and aged (24 months) mice were treated with angiotensin II plus L‐NAME. We found that the same level of hypertension leads to significantly earlier onset and increased incidence of CMHs in aged mice than in young mice, as shown by neurological examination, gait analysis, and histological assessment of CMHs in serial brain sections. Hypertension‐induced cerebrovascular oxidative stress and redox‐sensitive activation of matrix metalloproteinases (MMPs) were increased in aging. Treatment of aged mice with resveratrol significantly attenuated hypertension‐induced oxidative stress, inhibited vascular MMP activation, significantly delayed the onset, and reduced the incidence of CMHs. Collectively, aging promotes CMHs in mice likely by exacerbating hypertension‐induced oxidative stress and MMP activation. Therapeutic strategies that reduce microvascular oxidative stress and MMP activation may be useful for the prevention of CMHs, protecting neurocognitive function in high‐risk elderly patients.     

Factors that drive the increasing use of FFPE tissue in basic and translational cancer research

The decision to use 10% neutral buffered formalin fixed, paraffin embedded (FFPE) archival pathology material may be dictated by the cancer research question or analytical technique, or may be governed by national ethical, legal and social implications (ELSI), biobank, and sample availability and access policy. Biobanked samples of common tumors are likely to be available, but not all samples will be annotated with treatment and outcomes data and this may limit their application. Tumors that are rare or very small exist mostly in FFPE pathology archives. Pathology departments worldwide contain millions of FFPE archival samples, but there are challenges to availability. Pathology departments lack resources for retrieving materials for research or for having pathologists select precise areas in paraffin blocks, a critical quality control step. When samples must be sourced from several pathology departments, different fixation and tissue processing approaches create variability in quality. Researchers must decide what sample quality and quality tolerance fit their specific purpose and whether sample enrichment is required. Recent publications report variable success with techniques modified to examine all common species of molecular targets in FFPE samples. Rigorous quality management may be particularly important in sample preparation for next generation sequencing and for optimizing the quality of extracted proteins for proteomics studies. Unpredictable failures, including unpublished ones, likely are related to pre-analytical factors, unstable molecular targets, biological and clinical sampling factors associated with specific tissue types or suboptimal quality management of pathology archives. Reproducible results depend on adherence to pre-analytical phase standards for molecular in vitro diagnostic analyses for DNA, RNA and in particular, extracted proteins. With continuing adaptations of techniques for application to FFPE, the potential to acquire much larger numbers of FFPE samples and the greater convenience of using FFPE in assays for precision medicine, the choice of material in the future will become increasingly biased toward FFPE samples from pathology archives. Recognition that FFPE samples may harbor greater variation in quality than frozen samples for several reasons, including variations in fixation and tissue processing, requires that FFPE results be validated provided a cohort of frozen tissue samples is available.     

Immunohistochemical expression of rabphilin-3A-like (Noc2) in normal and tumor tissues of human endocrine pancreas

Involvement of rabphilin-3A-like (RPH3AL), or Noc2, the potential effector of Ras-associated binding proteins Rab3A and Rab27A in the regulation of exocytotic processes in the endocrine pancreas has been demonstrated in experimental models. Noc2 expression together with other regulatory molecules of the exocytotic machinery in human tissues, however, has not been studied. We evaluated immunohistochemical expression of the key molecules of the exocytotic machinery, Noc2, Rab3A, Rab27A, and RIM2, together with the characteristic islet cell hormones, insulin and glucagon in normal and endocrine tumor tissues of human pancreas. Normal pancreatic islets were stained for all of these proteins and showed strong cytoplasmic localization. A similar pattern of strong cytoplasmic expression of these proteins was observed in the majority of endocrine tumors. By contrast, the exocrine portions of normal appearing pancreas completely lacked Rab27A staining and showed decreased expression of the proteins, Noc2, Rab3A, and RIM2. The staining pattern of Noc2 and Rab27A was similar to the staining pattern of glucagon-producing cells within the islets. The concomitant expression of Noc2 with these molecules suggests that Noc2 may serve as an effector for Rab3A and Rab27A and that it is involved in the regulation of exocytosis of the endocrine pancreas in humans.     

Expression of NgR1-Antagonizing Proteins Decreases with Aging and Cognitive Decline in Rat Hippocampus

The myelin-associated inhibitor/Nogo-66 receptor 1 (NgR1) pathway directly functions in negative modulation of structural and electrophysiological synaptic plasticity. A previous study has established an important role of NgR1 pathway signaling in cognitive function, and we have demonstrated that multiple components of this pathway, including ligands, NgR1 co-receptors, and RhoA, are upregulated at the protein level specifically in cognitively impaired, but not age-matched cognitively intact aged rats. Recent studies have identified two novel endogenous NgR1 antagonists, LOTUS and LGI1, and an alternative co-receptor, ADAM22, which act to suppress NgR1 pathway signaling. To determine whether these endogenous NgR1-inhibiting proteins may play a compensatory role in age-related cognitive impairment by counteracting overexpression of NgR1 agonists and co-receptors, we quantified the expression of LOTUS, LGI1, and ADAM22 in hippocampal CA1, CA3 and DG subregions dissected from mature adult and aged rats cognitively phenotyped for spatial learning and memory by Morris water maze testing. We have found that endogenous inhibitors of NgR1 pathway action decrease significantly with aging and cognitive decline and that lower expression levels correlate with declining cognitive ability, particularly in CA1 and CA3. These data suggest that decreased expression of NgR1-antagonizing proteins may exert a combinatorial effect with increased NgR1 signaling pathway components to result in abnormally strong suppression of synaptic plasticity in age-related cognitive impairment.     

Modulation of microbial predator-prey dynamics by phosphorus availability: growth patterns and survival strategies of bacterial phylogenetic clades

We simultaneously studied the impact of top-down (protistan grazing) and bottom-up (phosphorus availability) factors on the numbers and biomasses of bacteria from various phylogenetic lineages, and on their growth and activity parameters in the oligo-mesotrophic Piburger See, Austria. Enhanced grazing resulted in decreased proportions of bacteria with high nucleic acid content (high-NA bacteria) and lower detection rates by FISH. There was a change in the composition of the bacterial assemblage, whereby Betaproteobacteria were heavily grazed while Alphaproteobacteria and Cytophaga-Flavobacterium-Bacteroides were less affected by predators. Changes in bacterial assemblage composition were also apparent in the treatments enriched with phosphorus, and even more pronounced in the incubations in dialysis tubes (allowing relatively free nutrient exchange). Here, Betaproteobacteria became dominant and appeared to act as successful opportunistic competitors for nutrients. In contrast, Actinobacteria did not respond to surplus phosphorus by population growth, and, moreover, maintained their small size, which resulted in a very low biomass contribution. In addition, significant relationships between high-NA bacteria and several bacterial phylogenetic clades were found, indicating an enhanced activity status. By combining several single-cell methods, new insight is gained into the competitive abilities of freshwater bacteria from a variety of phylogenetic lineages under contrasting sets of bottom-up and top-down constraints.     

Evaluation of Fluorophores to Label SNAP-Tag Fused Proteins for Multicolor Single-Molecule Tracking Microscopy in Live Cells

Single-molecule tracking has become a widely used technique for studying protein dynamics and their organization in the complex environment of the cell. In particular, the spatiotemporal distribution of membrane receptors is an active field of study due to its putative role in the regulation of signal transduction. The SNAP-tag is an intrinsically monovalent and highly specific genetic tag for attaching a fluorescent label to a protein of interest. Little information is currently available on the choice of optimal fluorescent dyes for single-molecule microscopy utilizing the SNAP-tag labeling system. We surveyed 6 green and 16 red excitable dyes for their suitability in single-molecule microscopy of SNAP-tag fusion proteins in live cells. We determined the nonspecific binding levels and photostability of these dye conjugates when bound to a SNAP-tag fused membrane protein in live cells. We found that only a limited subset of the dyes tested is suitable for single-molecule tracking microscopy. The results show that a careful choice of the dye to conjugate to the SNAP-substrate to label SNAP-tag fusion proteins is very important, as many dyes suffer from either rapid photobleaching or high nonspecific staining. These characteristics appear to be unpredictable, which motivated the need to perform the systematic survey presented here. We have developed a protocol for evaluating the best dyes, and for the conditions that we evaluated, we find that Dy 549 and CF 640 are the best choices tested for single-molecule tracking. Using an optimal dye pair, we also demonstrate the possibility of dual-color single-molecule imaging of SNAP-tag fusion proteins. This survey provides an overview of the photophysical and imaging properties of a range of SNAP-tag fluorescent substrates, enabling the selection of optimal dyes and conditions for single-molecule imaging of SNAP-tagged fusion proteins in eukaryotic cell lines.     

Multidimensional characterization and differentiation of neurons in the anteroventral cochlear nucleus

Multiple parallel auditory pathways ascend from the cochlear nucleus. It is generally accepted that the origin of these pathways are distinct groups of neurons differing in their anatomical and physiological properties. In extracellular in vivo recordings these neurons are typically classified on the basis of their peri-stimulus time histogram. In the present study we reconsider the question of classification of neurons in the anteroventral cochlear nucleus (AVCN) by taking a wider range of response properties into account. The study aims at a better understanding of the AVCN's functional organization and its significance as the source of different ascending auditory pathways. The analyses were based on 223 neurons recorded in the AVCN of the Mongolian gerbil. The range of analysed parameters encompassed spontaneous activity, frequency coding, sound level coding, as well as temporal coding. In order to categorize the unit sample without any presumptions as to the relevance of certain response parameters, hierarchical cluster analysis and additional principal component analysis were employed which both allow a classification on the basis of a multitude of parameters simultaneously. Even with the presently considered wider range of parameters, high number of neurons and more advanced analytical methods, no clear boundaries emerged which would separate the neurons based on their physiology. At the current resolution of the analysis, we therefore conclude that the AVCN units more likely constitute a multi-dimensional continuum with different physiological characteristics manifested at different poles. However, more complex stimuli could be useful to uncover physiological differences in future studies.