Impact of Aging and a High-Fat Diet on Adipose-Tissue-Derived Extracellular Vesicle miRNA Profiles in Mice.

BACKGROUND: Middle-aged adults have the highest obesity rates, leading to significant health complications in later years. Obesity triggers the release of altered molecules, including extracellular vesicles (EVs) from excess adipose tissue (AT), contributing to various health complications. In this study, we assessed the effects of age and a high-fat diet on AT-derived EV miRNA profiles to understand their potential roles in aging and obesity. METHOD: C57BL/6 male mice were subjected to a normal chow diet (NCD) or a high-fat diet (HFD) for either 10-12 weeks (young mice, n = 10) or 50-61 weeks (middle-aged mice, n = 12). After evaluating metabolic characteristics, peri-gonadal white AT was isolated and cultured to obtain EVs. AT-derived EV miRNAs were profiled using a NanoString miRNA panel (n = 599). RESULTS: Middle-aged mice exhibited obesity regardless of diet. Young mice fed an HFD showed similar metabolic traits to middle-aged mice. In the NCD group, 131 differentially expressed miRNAs (DE-miRNAs) emerged in middle-aged mice compared to young mice, including miR-21, miR-148a, and miR-29a, associated with cancer, neuro/psychological disorders, and reproductive diseases. In the HFD group, 55 DE-miRNAs were revealed in middle-aged mice compared to young mice. These miRNAs were associated with significantly suppressed IGF1R activity. CONCLUSION: This study demonstrates the potential significant impact of miRNAs of AT EVs on aging- and obesity-related diseases.

Extracellular vesicle miRNAs in breast milk of obese mothers.

Background: Breast milk has abundant extracellular vesicles (EVs) containing various biological molecules (cargo), including miRNAs. EVs are not degraded in the gastrointestinal system and circulation; thus, breast milk EVs (bEVs) are expected to interact with other organs in breastfed infants and modify the gene expression of recipient cells using miRNAs. Maternal pre-pregnancy BMI is a critical factor influencing the composition of breast milk. Thus, in mothers with obesity, miRNAs in bEVs can be altered, which might be associated with adverse health outcomes in infants. In this study, we examined 798 miRNAs to determine which miRNAs are altered in the bEVs of mothers with obesity and their potential impact on breastfed infants. Methods: We recruited healthy nursing mothers who were either of normal weight (BMI < 25) or with obesity (BMI ≥ 30) based on their pre-pregnancy BMI, and delivered a singleton baby in the prior 6 months. EVs were isolated from breast milk with ultracentrifugation. bEV characteristics were examined by flow cytometry and fluorescence imaging of EV markers. A total of 798 miRNAs were screened using a NanoString human miRNA panel to find differentially expressed miRNAs in bEVs of mothers with obesity compared to mothers of normal weight. Results: We included 65 nursing mothers: 47 of normal weight and 18 with obesity based on pre-pregnancy BMI. After bEV isolation, we confirmed the expression of various EV markers. Out of 37 EV markers, CD326 (EpCaM) was the most highly expressed in bEVs. The most abundant miRNAs in bEVs include miR-30b-5p, miR-4454, miR-494-3p, and let-7 miRNAs. Target genes of the top 10 miRNAs were associated with cancer, prolactin pathway, EGFR, ErbB, and FoxO signaling pathway. In bEVs of mothers with obesity, 19 miRNAs were differentially expressed (adjusted p < 0.05 cut-off), which include miR-575, miR-630, miR-642a-3p, and miR-652-5p. These miRNAs and their target genes were associated with neurological diseases and psychological disorders. Conclusion: In this study, we characterized bEVs and demonstrated altered miRNAs in bEVs of mothers with obesity and identified the pathways of their potential target genes. Our findings will provide insight for future studies investigating the role of bEVs in breastfed infants.

An annotated cDNA library of juvenile Euprymna scolopes with and without colonization by the symbiont Vibrio fischeri.

BACKGROUND: Biologists are becoming increasingly aware that the interaction of animals, including humans, with their coevolved bacterial partners is essential for health. This growing awareness has been a driving force for the development of models for the study of beneficial animal-bacterial interactions. In the squid-vibrio model, symbiotic Vibrio fischeri induce dramatic developmental changes in the light organ of host Euprymna scolopes over the first hours to days of their partnership. We report here the creation of a juvenile light-organ specific EST database. RESULTS: We generated eleven cDNA libraries from the light organ of E. scolopes at developmentally significant time points with and without colonization by V. fischeri. Single pass 3' sequencing efforts generated 42,564 expressed sequence tags (ESTs) of which 35,421 passed our quality criteria and were then clustered via the UIcluster program into 13,962 nonredundant sequences. The cDNA clones representing these nonredundant sequences were sequenced from the 5' end of the vector and 58% of these resulting sequences overlapped significantly with the associated 3' sequence to generate 8,067 contigs with an average sequence length of 1,065 bp. All sequences were annotated with BLASTX (E-value < -03) and Gene Ontology (GO). CONCLUSION: Both the number of ESTs generated from each library and GO categorizations are reflective of the activity state of the light organ during these early stages of symbiosis. Future analyses of the sequences identified in these libraries promise to provide valuable information not only about pathways involved in colonization and early development of the squid light organ, but also about pathways conserved in response to bacterial colonization across the animal kingdom.

1274 full-open reading frames of transcripts expressed in the developing mouse nervous system.

As part of the trans-National Institutes of Health (NIH) Mouse Brain Molecular Anatomy Project (BMAP), and in close coordination with the NIH Mammalian Gene Collection Program (MGC), we initiated a large-scale project to clone, identify, and sequence the complete open reading frame (ORF) of transcripts expressed in the developing mouse nervous system. Here we report the analysis of the ORF sequence of 1274 cDNAs, obtained from 47 full-length-enriched cDNA libraries, constructed by using a novel approach, herein described. cDNA libraries were derived from size-fractionated cytoplasmic mRNA isolated from brain and eye tissues obtained at several embryonic stages and postnatal days. Altogether, including the full-ORF MGC sequences derived from these libraries by the MGC sequencing team, NIH_BMAP full-ORF sequences correspond to approximately 20% of all transcripts currently represented in mouse MGC. We show that NIH_BMAP clones comprise 68% of mouse MGC cDNAs > or =5 kb, and 54% of those > or =4 kb, as of March 15, 2004. Importantly, we identified transcripts, among the 1274 full-ORF sequences, that are exclusively or predominantly expressed in brain and eye tissues, many of which encode yet uncharacterized proteins.

Development of evaluation procedures for local exhaust ventilation for United States postal service mail-processing equipment.

Researchers from the National Institute for Occupational Safety and Health (NIOSH) have conducted several evaluations of local exhaust ventilation (LEV) systems for the United States Postal Service (USPS) since autumn 2001 when (a) terrorist(s) employed the mail system for acts of bioterrorism. As a part of the USPS 2002 Emergency Preparedness Plan, the development and installation of LEV onto USPS mail-processing equipment can reduce future exposures to operators from potentially hazardous contaminants, such as anthrax, which might be emitted during the processing of mail. This article describes how NIOSH field testing led to the development of recommended testing procedures for evaluations of LEV capture efficiency for mail-processing equipment, including tracer gas measurements, smoke release observations, air velocity measurements, and decay-rate testing under access hoods.

High-throughput gene discovery in the rat.

The rat is an important animal model for human diseases and is widely used in physiology. In this article we present a new strategy for gene discovery based on the production of ESTs from serially subtracted and normalized cDNA libraries, and we describe its application for the development of a comprehensive nonredundant collection of rat ESTs. Our new strategy appears to yield substantially more EST clusters per ESTs sequenced than do previous approaches that did not use serial subtraction. However, multiple rounds of library subtraction resulted in high frequencies of otherwise rare internally primed cDNAs, defining the limits of this powerful approach. To date, we have generated >200,000 3' ESTs from >100 cDNA libraries representing a wide range of tissues and developmental stages of the laboratory rat. Most importantly, we have contributed to approximately 50,000 rat UniGene clusters. We have identified, arrayed, and derived 5' ESTs from >30,000 unique rat cDNA clones. Complete information, including radiation hybrid mapping data, is also maintained locally at http://genome.uiowa.edu/clcg.html. All of the sequences described in this article have been submitted to the dbEST division of the NCBI.

EST-based gene discovery in pig: virtual expression patterns and comparative mapping to human.

A molecular understanding of porcine reproduction is of biological interest and economic importance. Our Midwest Consortium has produced cDNA libraries containing the majority of genes expressed in major female reproductive tissues, and we have deposited into public databases 21,499 expressed sequence tag (EST) gene sequences from the 3' end of clones from these libraries. These sequences represent 10,574 different genes, based on sequence comparison among these data, and comparison with existing porcine ESTs and genes indicate as many as 4652 of these EST clusters are novel. In silico analysis identified sequences that are expressed in specific pig tissues or organs and confirmed the broad expression in pig for many genes ubiquitously expressed in human tissues. Furthermore, we have developed computer software to identify sequence similarity of these pig genes with their human counterparts, and to extract the mapping information of these human homologues from genome databases. We demonstrate the utility of this software for comparative mapping by localizing 61 genes on the porcine physical map for Chromosomes (Chrs) 5, 10, and 14.

Control of wake-induced exposure using an interrupted oscillating jet.

A problem may arise in ventilation design when the contaminant source is located in the worker's wake, where turbulence and vortex formation can carry the contaminant into the breathing zone even though the source is downwind. It was found previously that forced directional variations in the flow can reduce or eliminate the vortex formation that causes these local reversals. Reported here is a simple realization of this concept, in which an oscillating jet of air was directed at a mannequin in an otherwise steady flow of air. A 50th percentile male mannequin was placed in a nearly uniform flow of approximately 0.18 m/sec (36 ft/min). A low-velocity tracer gas source (isobutylene) was held in the standing mannequin's hands with the upper arms vertical and the elbows at 90 degrees. Four ventilation scenarios were compared by concentration measurements in the breathing zone, using photoionization detectors: (A) uniform flow; (B) addition of a steady jet with initial velocity 5.1 m/sec (1.0 x 10(3) ft/min) directed at the mannequin's back, parallel to the main flow; (C) making the jet oscillate to 45 degrees on either side of the centerline with a period of 13 sec; and (D) introducing a blockage at the centerline so the oscillating jet never blew directly at the worker. At the 97.5% confidence level the interrupted oscillating jet (case D) achieved at least 99% exposure reduction compared with the uniform flow by itself (case A), at least 93% compared with the steady jet (case B), and at least 45% exposure reduction compared with the unblocked oscillating jet (case C).