Structural, functional and molecular analysis of the effects of aging in the small intestine and colon of C57BL/6 J mice

ABSTRACT: BACKGROUND: By regulating digestion and absorption of nutrients and providing a barrier against the external environment the intestine provides a crucial contribution to the maintenance of health. To what extent aging-related changes in the intestinal system contribute to the functional decline associated with aging is still under debate. METHODS: Young (4 M) and old (21 M) male C57BL/6 J mice were fed a control low-fat (10E%) or a high-fat diet (45E%) for 2 weeks. During the intervention gross energy intake and energy excretion in the feces were measured. After sacrifice the small and large intestine were isolated and the small intestine was divided in three equal parts. Swiss rolls were prepared of each of the isolated segments for histological analysis and the luminal content was isolated to examine alterations in the microflora with 16S rRNA Q-PCR. Furthermore, mucosal scrapings were isolated from each segment to determine differential gene expression by microarray analysis and global DNA methylation by pyrosequencing. RESULTS: Digestible energy intake was similar between the two age groups on both the control and the high-fat diet. Microarray analysis on RNA from intestinal scrapings showed no marked changes in expression of genes involved in metabolic processes. Decreased expression of Cubilin was observed in the intestine of 21-month-old mice, which might contribute to aging-induced vitamin B12 deficiency. Furthermore, microarray data analysis revealed enhanced expression of a large number of genes involved in immune response and inflammation in the colon, but not in the small intestine of the 21-month-old mice. Aging-induced global hypomethylation was observed in the colon and the distal part of the small intestine, but not in the first two sections of the small intestine. CONCLUSION: In 21-month old mice the most pronounced effects of aging were observed in the colon, whereas very few changes were observed in the small intestine.     

Sociodemographic factors and clinical conditions associated to hospitalization in influenza A (H1N1) 2009 virus infected patients in Spain, 2009-2010

The emergence and pandemic spread of a new strain of influenza A (H1N1) virus in 2009 resulted in a serious alarm in clinical and public health services all over the world. One distinguishing feature of this new influenza pandemic was the different profile of hospitalized patients compared to those from traditional seasonal influenza infections. Our goal was to analyze sociodemographic and clinical factors associated to hospitalization following infection by influenza A(H1N1) virus. We report the results of a Spanish nationwide study with laboratory confirmed infection by the new pandemic virus in a case-control design based on hospitalized patients. The main risk factors for hospitalization of influenza A (H1N1) 2009 were determined to be obesity (BMI≥40, with an odds-ratio [OR] 14.27), hematological neoplasia (OR 10.71), chronic heart disease, COPD (OR 5.16) and neurological disease, among the clinical conditions, whereas low education level and some ethnic backgrounds (Gypsies and Amerinds) were the sociodemographic variables found associated to hospitalization. The presence of any clinical condition of moderate risk almost triples the risk of hospitalization (OR 2.88) and high risk conditions raise this value markedly (OR 6.43). The risk of hospitalization increased proportionally when for two (OR 2.08) or for three or more (OR 4.86) risk factors were simultaneously present in the same patient. These findings should be considered when a new influenza virus appears in the human population.     

Generation of transmitted/founder HIV-1 infectious molecular clones and characterization of their replication capacity in CD4 T lymphocytes and monocyte-derived macrophages

Genome sequences of transmitted/founder (T/F) HIV-1 have been inferred by analyzing single genome amplicons of acute infection plasma viral RNA in the context of a mathematical model of random virus evolution; however, few of these T/F sequences have been molecularly cloned and biologically characterized. Here, we describe the derivation and biological analysis of ten infectious molecular clones, each representing a T/F genome responsible for productive HIV-1 clade B clinical infection. Each of the T/F viruses primarily utilized the CCR5 coreceptor for entry and replicated efficiently in primary human CD4(+) T lymphocytes. This result supports the conclusion that single genome amplification-derived sequences from acute infection allow for the inference of T/F viral genomes that are consistently replication competent. Studies with monocyte-derived macrophages (MDM) demonstrated various levels of replication among the T/F viruses. Although all T/F viruses replicated in MDM, the overall replication efficiency was significantly lower compared to prototypic "highly macrophage-tropic" virus strains. This phenotype was transferable by expressing the env genes in an isogenic proviral DNA backbone, indicating that T/F virus macrophage tropism mapped to Env. Furthermore, significantly higher concentrations of soluble CD4 were required to inhibit T/F virus infection compared to prototypic macrophage-tropic virus strains. Our findings suggest that the acquisition of clinical HIV-1 subtype B infection occurs by mucosal exposure to virus that is not highly macrophage tropic and that the generation and initial biological characterization of 10 clade B T/F infectious molecular clones provides new opportunities to probe virus-host interactions involved in HIV-1 transmission.     

Novel information on the epitope of an inverse agonist monoclonal antibody provides insight into the structure of the TSH receptor

The TSH receptor (TSHR) comprises an extracellular leucine-rich domain (LRD) linked by a hinge region to the transmembrane domain (TMD). Insight into the orientation of these components to each other is required for understanding how ligands activate the receptor. We previously identified residue E251 at the LRD-hinge junction as contributing to coupling TSH binding with receptor activation. However, a single residue cannot stabilize the LRD-hinge unit. Therefore, based on the LRD crystal structure we selected for study four other potential LRD-hinge interface charged residues. Alanine substitutions of individual residues K244, E247, K250 and R255 (as well as previously known E251A) did not affect TSH binding or function. However, the cumulative mutation of these residues in varying permutations, primarily K250A and R255A when associated with E251A, partially uncoupled TSH binding and function. These data suggest that these three residues, spatially very close to each other at the LRD base, interact with the hinge region. Unexpectedly and most important, monoclonal antibody CS-17, a TSHR inverse agonist whose epitope straddles the LRD-hinge, was found to interact with residues K244 and E247 at the base of the convex LRD surface. These observations, together with the functional data, exclude residues K244 and E247 from the TSHR LRD-hinge interface. Further, for CS-17 accessibility to K244 and E247, the concave surface of the TSHR LRD must be tilted forwards towards the hinge region and plasma membrane. Overall, these data provide insight into the mechanism by which ligands either activate the TSHR or suppress its constitutive activity.     

Caveolin-1-enhanced motility and focal adhesion turnover require tyrosine-14 but not accumulation to the rear in metastatic cancer cells

Caveolin-1 is known to promote cell migration, and increased caveolin-1 expression is associated with tumor progression and metastasis. In fibroblasts, caveolin-1 polarization and phosphorylation of tyrosine-14 are essential to promote migration. However, the role of caveolin-1 in migration of metastatic cells remains poorly defined. Here, caveolin-1 participation in metastatic cell migration was evaluated by shRNA targeting of endogenous caveolin-1 in MDA-MB-231 human breast cancer cells and ectopic expression in B16-F10 mouse melanoma cells. Depletion of caveolin-1 in MDA-MB-231 cells reduced, while expression in B16-F10 cells promoted migration, polarization and focal adhesion turnover in a sequence of events that involved phosphorylation of tyrosine-14 and Rac-1 activation. In B16-F10 cells, expression of a non-phosphorylatable tyrosine-14 to phenylalanine mutant failed to recapitulate the effects observed with wild-type caveolin-1. Alternatively, treatment of MDA-MB-231 cells with the Src family kinase inhibitor PP2 reduced caveolin-1 phosphorylation on tyrosine-14 and cell migration. Surprisingly, unlike for fibroblasts, caveolin-1 polarization and re-localization to the trailing edge were not observed in migrating metastatic cells. Thus, expression and phosphorylation, but not polarization of caveolin-1 favor the highly mobile phenotype of metastatic cells.     

Changes in structure and composition of evergreen forests on an altitudinal gradient in the Venezuelan Guayana shield

There have been several ecological studies in forests of the Guayana Shield, but so far none had examined the changes in structure and composition of evergreen forests with altitude. This study describes and analyzes the structure, species composition and soil characteristics of forest stands at different altitudinal zones in Southeastern Venezuelan Guayana, in order to explain the patterns and the main factors that determine the structure and composition of evergreen forests along the altitudinal gradient. Inventories of 3 948 big (>10cm DBH) and 1 328 small (5-10cm DBH) woody stems were carried out in eleven plots, ranging from 0.1 to 1.0ha, along a 188km long transect with elevations between 290 and 1 395masl. It has been found that 1) hemiepihytes become more dominant and lianas reduce their dominance with increasing altitude and 2) the forest structure in the study area is size-dependent. Five families and 12 genera represented only 9% of the total number of families and genera, respectively, recorded troughout the gradient, but the two groups of taxa comprised more than 50% of the Importance Value (the sum of the relative density and the relative dominance) of all measured stems. Moreover, the results suggest that low species richness seems to be associated with the dominance of one or few species. Stand-level wood density (WD) of trees decreased significantly with increasing elevation. WD is an indicator of trees'life history strategy. Its decline suggests a change in the functional composition of the forest with increasing altitude. The Canonical Correspondence Analysis (CCA) indicated a distinction of the studied forests on the basis of their altitudinal levels and geographic location, and revealed different ecological responses by the forests, to environmental variables along the altitudinal gradient. The variation in species composition, in terms of basal area among stands, was controlled primarily by elevation and secondarily by rainfall and soil conditions. There are other interacting factors not considered in this study like disturbance regime, biological interactions, productivity, and dispersal history, which could affect the structure and composition of the forests in the altitudinal gradient. In conclusion, it appears that the structural and floristic variability observed in the studied transect is produced by a combination of different climates and randomly expressed local processes interacting across a complex physical landscape.     

Olfactory sensitivity for six amino acids: a comparative study in CD-1 mice and spider monkeys

Using a conditioning paradigm, the olfactory sensitivity of five CD-1 mice for the l- and d-forms of cysteine, methionine, and proline was investigated. With all six stimuli, the animals discriminated concentrations ≤0.1 ppm (parts per million) from the odorless solvent, and with three of the six stimuli the best-scoring animals were even able to detect concentrations <0.1 ppb (parts per billion). Three spider monkeys tested in parallel were found to detect the same six stimuli at concentrations <1 ppm, and with four of the six stimuli the best-scoring animals detected concentrations ≤1 ppb. Both CD-1 mice and spider monkeys displayed a higher olfactory sensitivity with the l- and d-forms of cysteine and methionine than with the prolines, suggesting an important role of the sulfur-containing functional groups for detectability. Accordingly, the across-odorant patterns of detection thresholds obtained with mice and spider monkeys showed a significant positive correlation. A comparison of the detection thresholds between the two species tested here and those obtained in human subjects suggests that neither the number of functional olfactory receptor genes nor the absolute or the relative size of the olfactory bulbs reliably predicts a species’ olfactory sensitivity for amino acids.