Visualization of fine-scale genomic structure by oligonucleotide-based high-resolution FISH

The discovery of complex structural variations that exist within individual genomes has prompted a need to visualize chromosomes at a higher resolution than previously possible. To address this concern, we established a robust, high-resolution fluorescence in situ hybridization (FISH) method that utilizes probes derived from high complexity libraries of long oligonucleotides (>150 mers) synthesized in massively parallel reactions. In silico selected oligonucleotides, targeted to only the most informative elements in 18 genomic regions of interest, eliminated the need for suppressive hybridization reagents. Because of the inherent flexibility in our probe design methods, we readily visualized regions as small as 6.7 kb with high specificity on human metaphase chromosomes, resulting in an overall success rate of 94%. Two-color FISH over a 479-kb duplication, initially reported as being identical in 2 individuals, revealed distinct 2-color patterns representing direct and inverted duplicons, demonstrating that visualization by high-resolution FISH provides further insight in the fine-scale complexity of genomic structures. The ability to design FISH probes for any sequenced genome along with the ease, reproducibility, and high level of accuracy of this technique suggests that it will be powerful for routine analysis of previously difficult genomic regions and structures.     

The effect of pioglitazone and resistance training on body composition in older men and women undergoing hypocaloric weight loss

Age‐related increases in ectopic fat accumulation are associated with greater risk for metabolic and cardiovascular diseases, and physical disability. Reducing skeletal muscle fat and preserving lean tissue are associated with improved physical function in older adults. PPARγ‐agonist treatment decreases abdominal visceral adipose tissue (VAT) and resistance training preserves lean tissue, but their effect on ectopic fat depots in nondiabetic overweight adults is unclear. We examined the influence of pioglitazone and resistance training on body composition in older (65–79 years) nondiabetic overweight/obese men (n = 48, BMI = 32.3 ± 3.8 kg/m²) and women (n = 40, BMI = 33.3 ± 4.9 kg/m²) during weight loss. All participants underwent a 16‐week hypocaloric weight‐loss program and were randomized to receive pioglitazone (30 mg/day) or no pioglitazone with or without resistance training, following a 2 × 2 factorial design. Regional body composition was measured at baseline and follow‐up using computed tomography (CT). Lean mass was measured using dual X‐ray absorptiometry. Men lost 6.6% and women lost 6.5% of initial body mass. The percent of fat loss varied across individual compartments. Men who were given pioglitazone lost more visceral abdominal fat than men who were not given pioglitazone (?1,160 vs. ?647 cm³, P = 0.007). Women who were given pioglitazone lost less thigh subcutaneous fat (?104 vs. ?298 cm³, P = 0.002). Pioglitazone did not affect any other outcomes. Resistance training diminished thigh muscle loss in men and women (resistance training vs. no resistance training men: ?43 vs. ?88 cm³, P = 0.005; women: ?34 vs. ?59 cm³, P = 0.04). In overweight/obese older men undergoing weight loss, pioglitazone increased visceral fat loss and resistance training reduced skeletal muscle loss. Additional studies are needed to clarify the observed gender differences and evaluate how these changes in body composition influence functional status.     

Astroglial inhibition of NF-κB does not ameliorate disease onset and progression in a mouse model for amyotrophic lateral sclerosis (ALS)

Motor neuron death in amyotrophic lateral sclerosis (ALS) is considered a "non-cell autonomous" process, with astrocytes playing a critical role in disease progression. Glial cells are activated early in transgenic mice expressing mutant SOD1, suggesting that neuroinflammation has a relevant role in the cascade of events that trigger the death of motor neurons. An inflammatory cascade including COX2 expression, secretion of cytokines and release of NO from astrocytes may descend from activation of a NF-κB-mediated pathway observed in astrocytes from ALS patients and in experimental models. We have attempted rescue of transgenic mutant SOD1 mice through the inhibition of the NF-κB pathway selectively in astrocytes. Here we show that despite efficient inhibition of this major pathway, double transgenic mice expressing the mutant SOD1(G93A) ubiquitously and the dominant negative form of IκBα (IκBαAA) in astrocytes under control of the GFAP promoter show no benefit in terms of onset and progression of disease. Our data indicate that motor neuron death in ALS cannot be prevented by inhibition of a single inflammatory pathway because alternative pathways are activated in the presence of a persistent toxic stimulus.     

High quality draft genome sequence of Segniliparus rugosus CDC 945(T)= (ATCC BAA-974(T))

Segniliparus rugosus represents one of two species in the genus Segniliparus, the sole genus in the family Segniliparaceae. A unique and interesting feature of this family is the presence of extremely long carbon-chain length mycolic acids bound in the cell wall. S. rugosus is also a medically important species because it is an opportunistic pathogen associated with mammalian lung disease. This report represents the second species in the genus to have its genome sequenced. The 3,567,567 bp long genome with 3,516 protein-coding and 49 RNA genes is part of the NIH Roadmap for Medical Research, Human Microbiome Project.     

Embryonic senescence and laminopathies in a progeroid zebrafish model

Background

Mutations that disrupt the conversion of prelamin A to mature lamin A cause the rare genetic disorder Hutchinson-Gilford progeria syndrome and a group of laminopathies. Our understanding of how A-type lamins function in vivo during early vertebrate development through aging remains limited, and would benefit from a suitable experimental model. The zebrafish has proven to be a tractable model organism for studying both development and aging at the molecular genetic level. Zebrafish show an array of senescence symptoms resembling those in humans, which can be targeted to specific aging pathways conserved in vertebrates. However, no zebrafish models bearing human premature senescence currently exist.

Principal Findings

We describe the induction of embryonic senescence and laminopathies in zebrafish harboring disturbed expressions of the lamin A gene (LMNA). Impairments in these fish arise in the skin, muscle and adipose tissue, and sometimes in the cartilage. Reduced function of lamin A/C by translational blocking of the LMNA gene induced apoptosis, cell-cycle arrest, and craniofacial abnormalities/cartilage defects. By contrast, induced cryptic splicing of LMNA, which generates the deletion of 8 amino acid residues lamin A (zlamin A-Δ8), showed embryonic senescence and S-phase accumulation/arrest. Interestingly, the abnormal muscle and lipodystrophic phenotypes were common in both cases. Hence, both decrease-of-function of lamin A/C and gain-of-function of aberrant lamin A protein induced laminopathies that are associated with mesenchymal cell lineages during zebrafish early development. Visualization of individual cells expressing zebrafish progerin (zProgerin/zlamin A-Δ37) fused to green fluorescent protein further revealed misshapen nuclear membrane. A farnesyltransferase inhibitor reduced these nuclear abnormalities and significantly prevented embryonic senescence and muscle fiber damage induced by zProgerin. Importantly, the adult Progerin fish survived and remained fertile with relatively mild phenotypes only, but had shortened lifespan with obvious distortion of body shape.

Conclusion

We generated new zebrafish models for a human premature aging disorder, and further demonstrated the utility for studying laminopathies. Premature aging could also be modeled in zebrafish embryos. This genetic model may thus provide a new platform for future drug screening as well as genetic analyses aimed at identifying modifier genes that influence not only progeria and laminopathies but also other age-associated human diseases common in vertebrates.     

Differential expression of microRNAs in tumors from chronically inflamed or genetic (APC(Min/+)) models of colon cancer

Background

Chronic inflammation associated with ulcerative colitis predisposes individuals to increased colon cancer risk. The aim of these studies was to identify microRNAs that are aberrantly regulated during inflammation and may participate in transformation of colonic epithelial cells in the inflammatory setting.

Methodology/Principal Findings

We have use quantitative PCR arrays to compare microRNA (miRNA) expression in tumors and control colonic epithelial cells isolated from distal colons of chronically inflamed mice and APC^Min/+ mice. Rank order statistics was utilized to identify differentially regulated miRNAs in tumors that arose due to chronic inflammation and/or to germline APC mutation. Eight high priority miRNAs were identified: miR-215, miR-137, miR-708, miR-31, and miR-135b were differentially expressed in APC tumors and miR-215, miR-133a, miR-467d, miR-218, miR-708, miR-31, and miR-135b in colitis-associated tumors. Four of these (miR-215, miR-708, miR-31, and miR-135b) were common to both tumors types, and dysregulation of these miRNAs was confirmed in an independent sample set. Target prediction and pathway analysis suggests that these microRNAs, in the aggregate, regulate signaling pathways related to MAPK, PI3K, WNT, and TGF-β, all of which are known to be involved in transformation.

Conclusions/Significance

We conclude that these four miRNAs are dysregulated at some very early stage in transformation of colonic epithelial cells. This response is not dependent on the mechanism of initiation of transformation (inflammation versus germline mutation), suggesting that the miRNAs that we have identified are likely to regulate critical signaling pathways that are central to early events in transformation of colonic epithelial cells.     

Bacteria in sputum of stable severe asthma and increased airway wall thickness

Background

Patients with chronic asthma have thicker intrapulmonary airways measured on high resolution computed tomography (HRCT). We determined whether the presence of lower airway bacteria was associated with increased airway wall thickness.

Methods

In 56 patients with stable severe asthma, sputum specimens obtained either spontaneously or after induction with hypertonic saline were cultured for bacteria and thoracic HRCT scans obtained. Wall thickness (W_T) and area (W_A) expressed as a ratio of airway diameter (D) and total area, respectively, were measured at five levels.

Results

Positive bacterial cultures were obtained in 29 patients, with H. influenzae, P. aeruginosa and S. aureus being the commonest strains. Logistic regression analysis showed that this was associated with the duration of asthma and the exacerbations during the past year. In airways > 2 mm, there was no significant difference in W_A (67.5 ± 5.4 vs 66.4 ± 5.4) and W_T/D (21.6 ± 2.7 vs 21.3 ± 2.4) between the culture negative versus positive groups. Similarly, in airways (≤ 2 mm), there were no significant differences in these parameters. The ratio of √wall area to P_i was negatively correlated with FEV₁% predicted (p < 0.05).

Conclusions

Bacterial colonization of the lower airways is common in patients with chronic severe asthma and is linked to the duration of asthma and having had exacerbations in the past year, but not with an increase in airway wall thickness.     

Evolutionary Genomics of Transposable Elements in Saccharomyces cerevisiae

Saccharomyces cerevisiae is one of the premier model systems for studying the genomics and evolution of transposable elements. The availability of the S. cerevisiae genome led to unprecedented insights into its five known transposable element families (the LTR retrotransposons Ty1-Ty5) in the years shortly after its completion. However, subsequent advances in bioinformatics tools for analysing transposable elements and the recent availability of genome sequences for multiple strains and species of yeast motivates new investigations into Ty evolution in S. cerevisiae. Here we provide a comprehensive phylogenetic and population genetic analysis of all Ty families in S. cerevisiae based on a systematic re-annotation of Ty elements in the S288c reference genome. We show that previous annotation efforts have underestimated the total copy number of Ty elements for all known families. In addition, we identify a new family of Ty3-like elements related to the S. paradoxus Ty3p which is composed entirely of degenerate solo LTRs. Phylogenetic analyses of LTR sequences identified three families with short-branch, recently active clades nested among long branch, inactive insertions (Ty1, Ty3, Ty4), one family with essentially all recently active elements (Ty2) and two families with only inactive elements (Ty3p and Ty5). Population genomic data from 38 additional strains of S. cerevisiae show that the majority of Ty insertions in the S288c reference genome are fixed in the species, with insertions in active clades being predominantly polymorphic and insertions in inactive clades being predominantly fixed. Finally, we use comparative genomic data to provide evidence that the Ty2 and Ty3p families have arisen in the S. cerevisiae genome by horizontal transfer. Our results demonstrate that the genome of a single individual contains important information about the state of TE population dynamics within a species and suggest that horizontal transfer may play an important role in shaping the genomic diversity of transposable elements in unicellular eukaryotes.