Significance Analysis of Time Course Microarray Experiments

Characterizing the genome-wide dynamic regulation of gene expression is important and will be of much interest in the future. However, there is currently no established method for identifying differentially expressed genes in a time course study. Here we propose a significance method for analyzing time course microarray studies that can be applied to the typical types of comparisons and sampling schemes. This method is applied to two studies on humans. In one study, genes are identified that show differential expression over time in response to in vivo endotoxin administration. Using our method 7409 genes are called significant at a 1% FDR level, whereas several existing approaches fail to identify any genes. In another study, 417 genes are identified at a 10% FDR level that show expression changing with age in the kidney cortex. Here it is also shown that as many as 47% of the genes change with age in a manner more complex than simple exponential growth or decay. The methodology proposed here has been implemented in the freely distributed and open-source EDGE software package

Evaluation of Human Skin Reconstituted from Composite Grafts of Cultured Keratinocytes and Human Acellular Dermis Transplanted to Athymic Mice

This study evaluates the use of composite grafts of cultured human keratinocytes and de-epidermalized, acellular human dermis to close full-thickness wounds in athymic mice. Grafts were transplanted onto athymic mice and studied up to 8 wk. Graft take was excellent, with no instances of infection or graft loss. By 1 wk, the human keratinocytes had formed a stratified epidermis that was fused with mouse epithelium, and by 8 wk the grafts resembled human skin and could be freely moved over the mouse dorsum. Immunostaining for keratins 10 and 16 and for involucrin revealed an initial pattern of epithelial immaturity, which by 8 wk had normalized to that of mature unwounded epithelium. Mouse fibroblasts began to infiltrate the acellular dermis as early as 1 wk. By 8 wk fibroblasts had completely repopulated the dermis, and blood vessels were evident in the most superficial papillary projections, Dermal elements, such as rete ridges and elastin fibers, which were present in the starting dermis, persisted for the duration of the experiment. Grafts using keratinocytes from dark-skinned donors as opposed to light-skin donors had foci of pigmentation as early as 1 wk that progressed to homogenous pigmentation of the graft by 6 wk. These results indicate that melanocytes that persist in vitro are able to resume normal function in vivo. Our study demonstrates that composite grafts of cultured keratinocytes combined with acellular dermis are a useful approach for the closure of full-thickness wounds

The Quorum Sensing Volatile Molecule 2-Amino Acetophenon Modulates Host Immune Responses in a Manner that Promotes Life with Unwanted Guests

Increasing evidence indicates that bacterial quorum sensing (QS) signals are important mediators of immunomodulation. However, whether microbes utilize these immunomodulatory signals to maintain infection remain unclear. Here, we show that the Pseudomonas aeruginosa QS-regulated molecule 2-amino acetophenone (2-AA) modulates host immune responses in a manner that increases host ability to cope with this pathogen. Mice treated with 2-AA prior to infection had a 90% survival compared to 10% survival rate observed in the non-pretreated infected mice. Whilst 2-AA stimulation activates key innate immune response pathways involving mitogen-activated protein kinases (MAPKs), nuclear factor (NF)-$\kappa B$, and pro-inflammatory cytokines, it attenuates immune response activation upon pretreatment, most likely by upregulating anti-inflammatory cytokines. 2-AA host pretreatment is characterized by a transcriptionally regulated block of c-JUN N-terminal kinase (JNK) and NF-$\kappa B$ activation, with relatively preserved activation of extracellular regulated kinase (ERK) 1/2. These kinase changes lead to CCAAT/enhancer-binding protein-$\beta$ $(c/EBP\beta)$ activation and formation of the $c/EBP\beta-p65$ complex that prevents NF-$\kappa B$ activation. 2-AA's aptitude for dampening the inflammatory processes while increasing host survival and pathogen persistence concurs with its ability to signal bacteria to switch to a chronic infection mode. Our results reveal a QS immunomodulatory signal that promotes original aspects of interkingdom communication. We propose that this communication facilitates pathogen persistence, while enabling host tolerance to infection

Developing Effective Alzheimer's Disease Therapies: Clinical Experience and Future Directions

Alzheimer's disease (AD) clinical trials, focused on disease modifying drugs and conducted in patients with mild to moderate AD, as well as prodromal (early) AD, have failed to reach efficacy endpoints in improving cognitive function in most cases to date or have been terminated due to adverse events. Drugs that have reached clinical stage were reviewed using web resources (such as clinicaltrials.gov, alzforum.org, company press releases, and peer reviewed literature) to identify late stage (Phase II and Phase III) efficacy clinical trials and summarize reasons for their failure. For each drug, only the latest clinical trials and ongoing trials that aimed at improving cognitive function were included in the analysis. Here we highlight the potential reasons that have hindered clinical success, including clinical trial design and choice of outcome measures, heterogeneity of patient populations, difficulties in diagnosing and staging the disease, drug design, mechanism of action, and toxicity related to the long-term use. We review and suggest approaches for AD clinical trial design aimed at improving our ability to identify novel therapies for this devastating disease

A Small Volatile Bacterial Molecule Triggers Mitochondrial Dysfunction in Murine Skeletal Muscle

Mitochondria integrate distinct signals that reflect specific threats to the host, including infection, tissue damage, and metabolic dysfunction; and play a key role in insulin resistance. We have found that the Pseudomonas aeruginosa quorum sensing infochemical, 2-amino acetophenone (2-AA), produced during acute and chronic infection in human tissues, including in the lungs of cystic fibrosis (CF) patients, acts as an interkingdom immunomodulatory signal that facilitates pathogen persistence, and host tolerance to infection. Transcriptome results have led to the hypothesis that 2-AA causes further harm to the host by triggering mitochondrial dysfunction in skeletal muscle. As normal skeletal muscle function is essential to survival, and is compromised in many chronic illnesses, including infections and CF-associated muscle wasting, we here determine the global effects of 2-AA on skeletal muscle using high-resolution magic-angle-spinning (HRMAS), proton (1H) nuclear magnetic resonance (NMR) metabolomics, in vivo 31P NMR, whole-genome expression analysis and functional studies. Our results show that 2-AA when injected into mice, induced a biological signature of insulin resistance as determined by 1H NMR analysis-, and dramatically altered insulin signaling, glucose transport, and mitochondrial function. Genes including Glut4, IRS1, PPAR-γ, PGC1 and Sirt1 were downregulated, whereas uncoupling protein UCP3 was up-regulated, in accordance with mitochondrial dysfunction. Although 2-AA did not alter high-energy phosphates or pH by in vivo 31P NMR analysis, it significantly reduced the rate of ATP synthesis. This affect was corroborated by results demonstrating down-regulation of the expression of genes involved in energy production and muscle function, and was further validated by muscle function studies. Together, these results further demonstrate that 2-AA, acts as a mediator of interkingdom modulation, and likely effects insulin resistance associated with a molecular signature of mitochondrial dysfunction in skeletal muscle. Reduced energy production and mitochondrial dysfunctional may further favor infection, and be an important step in the establishment of chronic and persistent infections

Role of Protein Farnesylation in Burn-Induced Metabolic Derangements and Insulin Resistance in Mouse Skeletal Muscle

Objective: Metabolic derangements, including insulin resistance and hyperlactatemia, are a major complication of major trauma (e.g., burn injury) and affect the prognosis of burn patients. Protein farnesylation, a posttranslational lipid modification of cysteine residues, has been emerging as a potential component of inflammatory response in sepsis. However, farnesylation has not yet been studied in major trauma. To study a role of farnesylation in burn-induced metabolic aberration, we examined the effects of farnesyltransferase (FTase) inhibitor, FTI-277, on burn-induced insulin resistance and metabolic alterations in mouse skeletal muscle. Methods: A full thickness burn (30% total body surface area) was produced under anesthesia in male C57BL/6 mice at 8 weeks of age. After the mice were treated with FTI-277 (5 mg/kg/day, IP) or vehicle for 3 days, muscle insulin signaling, metabolic alterations and inflammatory gene expression were evaluated. Results: Burn increased FTase expression and farnesylated proteins in mouse muscle compared with sham-burn at 3 days after burn. Simultaneously, insulin-stimulated phosphorylation of insulin receptor (IR), insulin receptor substrate (IRS)-1, Akt and GSK-3β was decreased. Protein expression of PTP-1B (a negative regulator of IR-IRS-1 signaling), PTEN (a negative regulator of Akt-mediated signaling), protein degradation and lactate release by muscle, and plasma lactate levels were increased by burn. Burn-induced impaired insulin signaling and metabolic dysfunction were associated with increased inflammatory gene expression. These burn-induced alterations were reversed or ameliorated by FTI-277. Conclusions: Our data demonstrate that burn increased FTase expression and protein farnesylation along with insulin resistance, metabolic alterations and inflammatory response in mouse skeletal muscle, all of which were prevented by FTI-277 treatment. These results indicate that increased protein farnesylation plays a pivotal role in burn-induced metabolic dysfunction and inflammatory response. Our study identifies FTase as a novel potential molecular target to reverse or ameliorate metabolic derangements in burn patients

Spontaneous Neutrophil Migration Patterns during Sepsis after Major Burns

Finely tuned to respond quickly to infections, neutrophils have amazing abilities to migrate fast and efficiently towards sites of infection and inflammation. Although neutrophils ability to migrate is perturbed in patients after major burns, no correlations have yet been demonstrated between altered migration and higher rate of infections and sepsis in these patients when compared to healthy individuals. To probe if such correlations exist, we designed microfluidic devices to quantify the neutrophil migration phenotype with high precision. Inside these devices, moving neutrophils are confined in channels smaller than the neutrophils and forced to make directional decisions at bifurcations and around posts. We employed these devices to quantify neutrophil migration across 18 independent parameters in 74 blood samples from 13 patients with major burns and 3 healthy subjects. Blinded, retrospective analysis of clinical data and neutrophil migration parameters revealed that neutrophils isolated from blood samples collected during sepsis migrate spontaneously inside the microfluidic channels. The spontaneous neutrophil migration is a unique phenotype, typical for patients with major burns during sepsis and often observed one or two days before the diagnosis of sepsis is confirmed. The spontaneous neutrophil migration phenotype is rare in patients with major burns in the absence of sepsis, and is not encountered in healthy individuals. Our findings warrant further studies of neutrophils and their utility for early diagnosing and monitoring sepsis in patients after major burns

Inhibitors of Pathogen Intercellular Signals as Selective Anti-Infective Compounds

Long-term antibiotic use generates pan-resistant super pathogens. Anti-infective compounds that selectively disrupt virulence pathways without affecting cell viability may be used to efficiently combat infections caused by these pathogens. A candidate target pathway is quorum sensing (QS), which many bacterial pathogens use to coordinately regulate virulence determinants. The Pseudomonas aeruginosa MvfR-dependent QS regulatory pathway controls the expression of key virulence genes; and is activated via the extracellular signals 4-hydroxy-2-heptylquinoline (HHQ) and 3,4-dihydroxy-2-heptylquinoline (PQS), whose syntheses depend on anthranilic acid (AA), the primary precursor of 4-hydroxy-2-alkylquinolines (HAQs). Here, we identified halogenated AA analogs that specifically inhibited HAQ biosynthesis and disrupted MvfR-dependent gene expression. These compounds restricted P. aeruginosa systemic dissemination and mortality in mice, without perturbing bacterial viability, and inhibited osmoprotection, a widespread bacterial function. These compounds provide a starting point for the design and development of selective anti-infectives that restrict human P. aeruginosa pathogenesis, and possibly other clinically significant pathogens