Toll-like receptor adaptor signaling molecule MyD88 on intervertebral disk homeostasis: in vitro, ex vivo studies

MyD88 is an adapter protein that links toll-like receptors (TLRs) and Interleukin-1 receptors (IL-1Rs) with downstream signaling molecules. The MyD88 has been found to be an essential mediator in the development of osteoarthritis in articular cartilage. However, the role of the MyD88 pathway has yet to be elucidated in the intervertebral disk (IVD). Using in vitro techniques, we analyzed the effect of MyD88 pathway-specific inhibition on the potent inflammatory and catabolic mediator LPS and IL-1 in bovine and human nucleus pulposus (NP) cells by assessing matrix-degrading enzyme expression, including matrix metalloproteases (MMPs) and a disintegrin-like and metalloprotease with thrombospondin motifs (ADAMTS family). We also analyzed inhibition of MyD88 in the regulation of inducible nitric oxide synthase and TLR-2. Finally, we used an ex vivo organ culture model to assess the effects of MyD88 inhibitor (MyD88i) on catabolic factor-induced disk degeneration in mice lumbar disks. In bovine NP cells, MyD88i potently antagonizes LPS- or IL-1-mediated induction of cartilage-degrading enzyme production, including MMP-1, MMP-13, ADAMTS-4, and ADAMTS-5. MyD88i also attenuates the LPS- or IL-1-mediated induction of iNOS and TLR-2 gene expression. Our ex vivo findings reveal inhibition of MyD88 via counteraction of IL-1-mediated proteoglycan depletion. The findings from this study demonstrate the potent anti-inflammatory and anti-catabolic effects of inhibition of MyD88 pathway inhibition on IVD homeostasis, suggesting a potential therapeutic benefit of a MyD88i in degenerative disk disease in the future.     

Statistical discovery of site inter-dependencies in sub-molecular hierarchical protein structuring

Background

Much progress has been made in understanding the 3D structure of proteins using methods such as NMR and X-ray crystallography. The resulting 3D structures are extremely informative, but do not always reveal which sites and residues within the structure are of special importance. Recently, there are indications that multiple-residue, sub-domain structural relationships within the larger 3D consensus structure of a protein can be inferred from the analysis of the multiple sequence alignment data of a protein family. These intra-dependent clusters of associated sites are used to indicate hierarchical inter-residue relationships within the 3D structure. To reveal the patterns of associations among individual amino acids or sub-domain components within the structure, we apply a k-modes attribute (aligned site) clustering algorithm to the ubiquitin and transthyretin families in order to discover associations among groups of sites within the multiple sequence alignment. We then observe what these associations imply within the 3D structure of these two protein families.

Results

The k-modes site clustering algorithm we developed maximizes the intra-group interdependencies based on a normalized mutual information measure. The clusters formed correspond to sub-structural components or binding and interface locations. Applying this data-directed method to the ubiquitin and transthyretin protein family multiple sequence alignments as a test bed, we located numerous interesting associations of interdependent sites. These clusters were then arranged into cluster tree diagrams which revealed four structural sub-domains within the single domain structure of ubiquitin and a single large sub-domain within transthyretin associated with the interface among transthyretin monomers. In addition, several clusters of mutually interdependent sites were discovered for each protein family, each of which appear to play an important role in the molecular structure and/or function.

Conclusions

Our results demonstrate that the method we present here using a k- modes site clustering algorithm based on interdependency evaluation among sites obtained from a sequence alignment of homologous proteins can provide significant insights into the complex, hierarchical inter-residue structural relationships within the 3D structure of a protein family.

     

Diversity and succession of the intestinal bacterial community of the maturing broiler chicken

The diversity of bacterial floras in the ilea and ceca of chickens that were fed a vegetarian corn-soy broiler diet devoid of feed additives was examined by analysis of 1,230 partial 16S rRNA gene sequences. Nearly 70% of sequences from the ileum were related to those of Lactobacillus, with the majority of the rest being related to Clostridiaceae (11%), Streptococcus (6.5%), and Enterococcus (6.5%). In contrast, Clostridiaceae-related sequences (65%) were the most abundant group detected in the cecum, with the other most abundant sequences being related to Fusobacterium (14%), Lactobacillus (8%), and Bacteroides (5%). Statistical analysis comparing the compositions of the different 16S rRNA libraries revealed that population succession occurred during some sampling periods. The significant differences among cecal libraries at 3 and 7 days of age, at 14 to 28 days of age, and at 49 days of age indicated that successions occurred from a transient community to one of increasing complexity as the birds aged. Similarly, the ileum had a stable bacterial community structure for birds at 7 to 21 days of age and between 21 to 28 days of age, but there was a very unique community structure at 3 and 49 days of age. It was also revealed that the composition of the ileal and cecal libraries did not significantly differ when the birds were 3 days old, and in fact during the first 14 days of age, the cecal microflora was a subset of the ileal microflora. After this time, the ileum and cecum had significantly different library compositions, suggesting that each region developed its own unique bacterial community as the bird matured.     

Effects of orally administered tetracycline on the intestinal community structure of chickens and on tet determinant carriage by commensal bacteria and Campylobacter jejuni

There is a growing concern that antibiotic usage in animal production has selected for resistant food-borne bacteria. Since tetracyclines are common therapeutic antibiotics used in poultry production, we sought to evaluate the effects of oral administration on the resistance of poultry commensal bacteria and the intestinal bacterial community structure. The diversity indices calculated from terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA amplicons did not indicate significant changes in the cecal bacterial community in response to oxytetracycline. To evaluate its effects on cultivable commensals, Enterococcus spp., Escherichia coli, and Campylobacter spp. were isolated from the cecal droppings of broiler chickens. Enterococcus spp. and E. coli expressed tetracycline MICs of >8 microg/ml and harbored a variety of tet resistance determinants regardless of the tetracycline exposure history of the birds. The enterococcal isolates possessed tetM (61%), tetL (25.4%), and tetK (1.3%), as well as tetO (52.5%), the determinant known to confer a tetracycline resistance phenotype in Campylobacter jejuni. E. coli isolates harbored tetA (32.2%) or tetB (30.5%). Tetracycline MICs remained at <2 microg/ml for Campylobacter isolates before and after tetracycline treatment of the chickens, even though isolates expressing MICs of >16 mug/ml were commonly cultured from flocks that did not receive oxytetracycline. The results imply that complex ecological and genetic factors contribute to the prevalence of antibiotic resistance arising from resistance gene transfer in the production environment.     

Multistability maintains redox homeostasis in human cells

Cells metabolize nutrients through a complex metabolic and signaling network that governs redox homeostasis. At the core of this, redox regulatory network is a mutually inhibitory relationship between reduced glutathione and reactive oxygen species (ROS)—two opposing metabolites that are linked to upstream nutrient metabolic pathways (glucose, cysteine, and glutamine) and downstream feedback loops of signaling pathways (calcium and NADPH oxidase). We developed a nutrient‐redox model of human cells to understand system‐level properties of this network. Combining in silico modeling and ROS measurements in individual cells, we show that ROS dynamics follow a switch‐like, all‐or‐none response upon glucose deprivation at a threshold that is approximately two orders of magnitude lower than its physiological concentration. We also confirm that this ROS switch can be irreversible and exhibits hysteresis, a hallmark of bistability. Our findings evidence that bistability modulates redox homeostasis in human cells and provide a general framework for quantitative investigations of redox regulation in humans.     

Construction and analysis of a modular model of caspase activation in apoptosis

Background  

A key physiological mechanism employed by multicellular organisms is apoptosis, or programmed cell death. Apoptosis is triggered by the activation of caspases in response to both extracellular (extrinsic) and intracellular (intrinsic) signals. The extrinsic and intrinsic pathways are characterized by the formation of the death-inducing signaling complex (DISC) and the apoptosome, respectively; both the DISC and the apoptosome are oligomers with complex formation dynamics. Additionally, the extrinsic and intrinsic pathways are coupled through the mitochondrial apoptosis-induced channel via the Bcl-2 family of proteins.     

Effects of feed additives on the development on the ileal bacterial community of the broiler chicken

Intensifying concerns about the use of antimicrobials in meat and poultry production has enhanced interest in the application of prebiotics, probiotics and enzymes to enhance growth and prevent disease in food animals. Growth-promoting antibiotics enhance growth of animals by reducing the load of bacteria in the intestine, by reducing colonization by intestinal pathogens or by enhancing the growth and/or metabolism of beneficial bacteria in the intestine. Recently, molecular ecology, utilizing DNA-sequence heterogeneity of the 16S rRNA gene, has revealed a surprising diversity of uncharacterized bacteria inhabiting this ecosystem. We used this approach to determine the effect of growth-promoting antibiotics on the development and composition of the ileal bacterial community. Pairwise comparisons, correspondence analysis and community diversity indices revealed significant differences among the treatments (bacitracin/virginiamycin or monensin) and controls. Antibiotics reduced the diversity of the ileal bacterial community and induced communities rich in Clostridia throughout the life of the broiler chicken. These results indicate that some bacterial species, such as lactobacilli, were suppressed and also suggest that many intestinal Clostridia may be non-pathogenic. Future studies should focus on characterizing the important bacterial species needed to stabilize the intestinal microbiota and identifying those commensals that stimulate and enhance development of intestinal function.     

Ranking and compacting binding segments of protein families using aligned pattern clusters

Background

Discovering sequence patterns with variation can unveil functions of a protein family that are important for drug discovery. Exploring protein families using existing methods such as multiple sequence alignment is computationally expensive, thus pattern search, called motif finding in Bioinformatics, is used. However, at present, combinatorial algorithms result in large sets of solutions, and probabilistic models require a richer representation of the amino acid associations. To overcome these shortcomings, we present a method for ranking and compacting these solutions in a new representation referred to as Aligned Pattern Clusters (APCs). To tackle the problem of a large solution set, our method reveals a reduced set of candidate solutions without losing any information. To address the problem of representation, our method captures the amino acid associations and conservations of the aligned patterns. Our algorithm renders a set of APCs in which a set of patterns is discovered, pruned, aligned, and synthesized from the input sequences of a protein family.

Results

Our algorithm identifies the binding or other functional segments and their embedded residues which are important drug targets from the cytochrome c and the ubiquitin protein families taken from Unitprot. The results are independently confirmed by pFam's multiple sequence alignment. For cytochrome c protein the number of resulting patterns with variations are reduced by 76.62% from the number of original patterns without variations. Furthermore, all of the top four candidate APCs correspond to the binding segments with one of each of their conserved amino acid as the binding residue. The discovered proximal APCs agree with pFam and PROSITE results. Surprisingly, the distal binding site discovered by our algorithm is not discovered by pFam nor PROSITE, but confirmed by the three-dimensional cytochrome c structure. When applied to the ubiquitin protein family, our results agree with pFam and reveals six of the seven Lysine binding residues as conserved aligned columns with entropy redundancy measure of 1.0.

Conclusion

The discovery, ranking, reduction, and representation of a set of patterns is important to avert time-consuming and expensive simulations and experimentations during proteomic study and drug discovery.

     

Hepatitis C (HCV) therapy for HCV mono-infected and HIV-HCV co-infected individuals living in Nepal

BackgroundDespite direct-acting antivirals (DAA), aims to “eradicate” viral hepatitis by 2030 remain unlikely. In Nepal, an expert consortium was established to treat HCV through Nepal earthquakes aftermath offering a model for HCV treatment expansion in a resource-poor setting.Methodology/Principal findingsIn 2015, we established a network of hepatologists, laboratory experts, and community-based leaders at 6 Opioid Substitution Treatment (OST) sites from 4 cities in Nepal screening 838 patients for a treatment cohort of 600 individuals with HCV infection and past or current drug use. During phase 1, patients were treated with interferon-based regimens (n = 46). During phase 2, 135 patients with optimal predictors (HIV controlled, without cirrhosis, low baseline HCV viral load) were treated with DAA-based regimens. During phase 3, IFN-free DAA treatment was expanded, regardless of HCV disease severity, HIV viremia or drug use. Sustained virologic response (SVR) was assessed at 12 weeks.Median age was 37 years and 95.5% were males. HCV genotype was 3 (53.2%) or 1a (40.7%) and 32% had cirrhosis; 42.5% were HIV-HCV coinfected. The intention-to-treat (ITT) SVR rates in phase 2 and 3 were 97% and 81%, respectively. The overall per-protocol and ITT SVR rates were 97% and 85%, respectively. By multivariable analysis, treatment at the Kathmandu site was protective and substance use, treatment during phase 3 were associated with failure to achieve SVR.Conclusions/SignificanceVery high SVR rates may be achieved in a difficult-to-treat, low-income population whatever the patient’s profile and disease severity. The excellent treatment outcomes observed in this real-life community study should prompt further HCV treatment initiatives in Nepal.