Deciphering multi-way interactions in the human genome.

Chromatin architecture, a key regulator of gene expression, can be inferred using chromatin contact data from chromosome conformation capture, or Hi-C. However, classical Hi-C does not preserve multi-way contacts. Here we use long sequencing reads to map genome-wide multi-way contacts and investigate higher order chromatin organization in the human genome. We use hypergraph theory for data representation and analysis, and quantify higher order structures in neonatal fibroblasts, biopsied adult fibroblasts, and B lymphocytes. By integrating multi-way contacts with chromatin accessibility, gene expression, and transcription factor binding, we introduce a data-driven method to identify cell type-specific transcription clusters. We provide transcription factor-mediated functional building blocks for cell identity that serve as a global signature for cell types.

Evaluation of commercially available RNA amplification kits for RNA sequencing using very low input amounts of total RNA.

This article includes supplemental data. Please visit http://www.fasebj.org to obtain this information.Multiple recent publications on RNA sequencing (RNA-seq) have demonstrated the power of next-generation sequencing technologies in whole-transcriptome analysis. Vendor-specific protocols used for RNA library construction often require at least 100 ng total RNA. However, under certain conditions, much less RNA is available for library construction. In these cases, effective transcriptome profiling requires amplification of subnanogram amounts of RNA. Several commercial RNA amplification kits are available for amplification prior to library construction for next-generation sequencing, but these kits have not been comprehensively field evaluated for accuracy and performance of RNA-seq for picogram amounts of RNA. To address this, 4 types of amplification kits were tested with 3 different concentrations, from 5 ng to 50 pg, of a commercially available RNA. Kits were tested at multiple sites to assess reproducibility and ease of use. The human total reference RNA used was spiked with a control pool of RNA molecules in order to further evaluate quantitative recovery of input material. Additional control data sets were generated from libraries constructed following polyA selection or ribosomal depletion using established kits and protocols. cDNA was collected from the different sites, and libraries were synthesized at a single site using established protocols. Sequencing runs were carried out on the Illumina platform. Numerous metrics were compared among the kits and dilutions used. Overall, no single kit appeared to meet all the challenges of small input material. However, it is encouraging that excellent data can be recovered with even the 50 pg input total RNA.

Risk Factors Associated with Adjacent and Remote- Level Pathologic Vertebral Compression Fracture Following Balloon Kyphoplasty: 2-Year Follow-Up Comparison Versus Conservative Treatment.

Vertebral compression fractures are a significant source of morbidity and mortality among patients of all age groups. These fractures result in both acute and chronic pain. Patients who sustain such fractures are known to suffer from more comorbidities and have a higher mortality rate compared with healthy people in the same age group. In recent years, balloon kyphoplasty has become a popular method for treating vertebral compression fractures. However, as longer-term follow-up becomes available, the effects of cement augmentation on adjacent spinal segments require investigation. Here, we have performed a retrospective chart review of 258 consecutive patients with pathologic vertebral compression fractures secondary to osteoporosis, treated by either conservative measures or balloon kyphoplasty with polymethylmethacrylate cement augmentation. Multivariate analysis of patient comorbidities was performed to assess the risks associated with subsequent adjacent and remote compression fracture at a minimum of 2 years follow-up. A total of 258 patients had 361 vertebral compression fractures. A total of 121 patients were treated nonoperatively and 137 underwent balloon kyphoplasty with polymethylmethacrylate cement augmentation. The mean follow-up for both cohorts was 2.7 years (range, 2-6 years). The kyphoplasty cohort was significantly older than the nonoperative cohort (mean age, 78.5 versus 74.2 years; p = 0.02), had 24 more patients with diabetes mellitus (37 versus 13; p = 0.05), and had 34 more patients with a history of smoking (50 versus 16; p = 0.05). However, the kyphoplasty cohort had less patients with a history of non-steroidal anti-inflammatory drug (NSAID) use (45 versus 71; p = 0.07). There were no demographic differences between groups in patients with secondary fractures. Nonoperative treatment was identified as a statistically significant independent risk factor for subsequent vertebral compression fracture [odds ratio (OR), 2.28]. Univariate analysis identified age, diabetes mellitus, smoking, NSAID usage, and female gender as risk factors for subsequent vertebral compression fracture. When adjusted for multivariate analysis, no individual factor demonstrated increased risk for subsequent fracture. Patients diagnosed with vertebral compression fractures secondary to osteoporosis suffer from multiple medical comorbidities. No particular comorbidity was identified as solely attributable for increased risk of subsequent remote or adjacent compression fractures. Patients in this series treated with nonoperative (conservative) management had a 2.28 times greater risk for a subsequent vertebral compression fracture than patients treated with balloon kyphoplasty and polymethylmethacrylate cement augmentation.

Bacterial genome annotation.

Annotation of prokaryotic sequences can be separated into structural and functional annotation. Structural annotation is dependent on algorithmic interrogation of experimental evidence to discover the physical characteristics of a gene. This is done in an effort to construct accurate gene models, so understanding function or evolution of genes among organisms is not impeded. Functional annotation is dependent on sequence similarity to other known genes or proteins in an effort to assess the function of the gene. Combining structural and functional annotation across genomes in a comparative manner promotes higher levels of accurate annotation as well as an advanced understanding of genome evolution. As the availability of bacterial sequences increases and annotation methods improve, the value of comparative annotation will increase.

Genomics for key players in the N cycle from guinea pigs to the next frontier.

While sequencing methods were available in the late 1970s, it was not until the human genome project and a significant influx of funds for such research that this technology became high throughput. The fields of microbiology and microbial ecology, among many others, have been tremendously impacted over the years, to such an extent that the determination of complete microbial genome sequences is now commonplace. Given the lower costs of next-generation sequencing platforms, even small laboratories from around the world will be able to generate millions of base pairs of data, equivalent to entire genomes worth of sequence information. With this prospect just around the corner, it is timely to provide an overview of the genomics process: from sample preparation to some of the analytical methods used to gain functional knowledge from sequence information.

The roles of antimicrobial peptides in innate host defense.

Antimicrobial peptides (AMPs) are multi-functional peptides whose fundamental biological role in vivo has been proposed to be the elimination of pathogenic microorganisms, including Gram-positive and -negative bacteria, fungi, and viruses. Genes encoding these peptides are expressed in a variety of cells in the host, including circulating phagocytic cells and mucosal epithelial cells, demonstrating a wide range of utility in the innate immune system. Expression of these genes is tightly regulated; they are induced by pathogens and cytokines as part of the host defense response, and they can be suppressed by bacterial virulence factors and environmental factors which can lead to increased susceptibility to infection. New research has also cast light on alternative functionalities, including immunomodulatory activities, which are related to their unique structural characteristics. These peptides represent not only an important component of innate host defense against microbial colonization and a link between innate and adaptive immunity, but also form a foundation for the development of new therapeutic agents.

Computational analysis suggests beta-defensins are processed to mature peptides by signal peptidase.

Antimicrobial peptides (AMPs) are generally produced as precursor peptides containing a signal sequence, a pro-region and the mature peptide. A computational analysis of beta-defensin precursors predicts cleavage solely by signal peptidase to release the mature peptide, with no pro-region. This supports the extensive transcriptional control of beta-defensin expression compared with other AMP genes.

Activity of an antimicrobial peptide mimetic against planktonic and biofilm cultures of oral pathogens.

Antimicrobial peptides (AMPs) are naturally occurring, broad-spectrum antimicrobial agents that have recently been examined for their utility as therapeutic antibiotics. Unfortunately, they are expensive to produce and are often sensitive to protease digestion. To address this problem, we have examined the activity of a peptide mimetic whose design was based on the structure of magainin, exhibiting its amphiphilic structure. We demonstrate that this compound, meta-phenylene ethynylene (mPE), exhibits antimicrobial activity at nanomolar concentrations against a variety of bacterial and Candida species found in oral infections. Since Streptococcus mutans, an etiological agent of dental caries, colonizes the tooth surface and forms a biofilm, we quantified the activity of this compound against S. mutans growing under conditions that favor biofilm formation. Our results indicate that mPE can prevent the formation of a biofilm at nanomolar concentrations. Incubation with 5 nM mPE prevents further growth of the biofilm, and 100 nM mPE reduces viable bacteria in the biofilm by 3 logs. Structure-function analyses suggest that mPE inhibits the bioactivity of lipopolysaccharide and binds DNA at equimolar ratios, suggesting that it may act both as a membrane-active molecule, similar to magainin, and as an intracellular antibiotic, similar to other AMPs. We conclude that mPE and similar molecules display great potential for development as therapeutic antimicrobials.