Mechanism of Allosteric Modulation of Nicotinamide Phosphoribosyltransferase to Elevate Cellular NAD.

In aging and disease, cellular nicotinamide adenine dinucleotide (NAD+) is depleted by catabolism to nicotinamide (NAM). NAD+ supplementation is being pursued to enhance human healthspan and lifespan. Activation of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting step in NAD+ biosynthesis, has the potential to increase the salvage of NAM. Novel NAMPT-positive allosteric modulators (N-PAMs) were discovered in addition to the demonstration of NAMPT activation by biogenic phenols. The mechanism of activation was revealed through the synthesis of novel chemical probes, new NAMPT co-crystal structures, and enzyme kinetics. Binding to a rear channel in NAMPT regulates NAM binding and turnover, with biochemical observations being replicated by NAD+ measurements in human cells. The mechanism of action of N-PAMs identifies, for the first time, the role of the rear channel in the regulation of NAMPT turnover coupled to productive and nonproductive NAM binding. The tight regulation of cellular NAMPT via feedback inhibition by NAM, NAD+, and adenosine 5'-triphosphate (ATP) is differentially regulated by N-PAMs and other activators, indicating that different classes of pharmacological activators may be engineered to restore or enhance NAD+ levels in affected tissues.

Microbial functional diversity covaries with permafrost thaw-induced environmental heterogeneity in tundra soil.

Permafrost soil in high latitude tundra is one of the largest terrestrial carbon (C) stocks and is highly sensitive to climate warming. Understanding microbial responses to warming-induced environmental changes is critical to evaluating their influences on soil biogeochemical cycles. In this study, a functional gene array (i.e., geochip 4.2) was used to analyze the functional capacities of soil microbial communities collected from a naturally degrading permafrost region in Central Alaska. Varied thaw history was reported to be the main driver of soil and plant differences across a gradient of minimally, moderately, and extensively thawed sites. Compared with the minimally thawed site, the number of detected functional gene probes across the 15-65 cm depth profile at the moderately and extensively thawed sites decreased by 25% and 5%, while the community functional gene ß-diversity increased by 34% and 45%, respectively, revealing decreased functional gene richness but increased community heterogeneity along the thaw progression. Particularly, the moderately thawed site contained microbial communities with the highest abundances of many genes involved in prokaryotic C degradation, ammonification, and nitrification processes, but lower abundances of fungal C decomposition and anaerobic-related genes. Significant correlations were observed between functional gene abundance and vascular plant primary productivity, suggesting that plant growth and species composition could be co-evolving traits together with microbial community composition. Altogether, this study reveals the complex responses of microbial functional potentials to thaw-related soil and plant changes and provides information on potential microbially mediated biogeochemical cycles in tundra ecosystems.

Biotic responses buffer warming-induced soil organic carbon loss in Arctic tundra.

Climate warming can result in both abiotic (e.g., permafrost thaw) and biotic (e.g., microbial functional genes) changes in Arctic tundra. Recent research has incorporated dynamic permafrost thaw in Earth system models (ESMs) and indicates that Arctic tundra could be a significant future carbon (C) source due to the enhanced decomposition of thawed deep soil C. However, warming-induced biotic changes may influence biologically related parameters and the consequent projections in ESMs. How model parameters associated with biotic responses will change under warming and to what extent these changes affect projected C budgets have not been carefully examined. In this study, we synthesized six data sets over 5 years from a soil warming experiment at the Eight Mile Lake, Alaska, into the Terrestrial ECOsystem (TECO) model with a probabilistic inversion approach. The TECO model used multiple soil layers to track dynamics of thawed soil under different treatments. Our results show that warming increased light use efficiency of vegetation photosynthesis but decreased baseline (i.e., environment-corrected) turnover rates of SOC in both the fast and slow pools in comparison with those under control. Moreover, the parameter changes generally amplified over time, suggesting processes of gradual physiological acclimation and functional gene shifts of both plants and microbes. The TECO model predicted that field warming from 2009 to 2013 resulted in cumulative C losses of 224 or 87 g/m2 , respectively, without or with changes in those parameters. Thus, warming-induced parameter changes reduced predicted soil C loss by 61%. Our study suggests that it is critical to incorporate biotic changes in ESMs to improve the model performance in predicting C dynamics in permafrost regions.

Wnt7b is required for epithelial progenitor growth and operates during epithelial-to-mesenchymal signaling in pancreatic development.

Wnt signaling is a well conserved pathway critical for growth, patterning and differentiation of multiple tissues and organs. Previous studies on Wnt signaling in the pancreas have been based predominantly on downstream pathway effector genes such as ß-catenin. We here provide evidence that the canonical-pathway member Wnt7b is a physiological regulator of pancreatic progenitor cell growth. Genetic deletion of Wnt7b in the developing pancreas leads to pancreatic hypoplasia due to reduced proliferation of pancreatic progenitor cells during the phase of pancreas development marked by rapid progenitor cell growth. While the differentiation potential of pancreatic progenitor cells is unaffected by Wnt7b deletion, through a gain-of-function analysis, we find that early pancreatic progenitor cells are highly sensitive to Wnt7b expression, but later lose such competence. By modulating the level and the temporal windows of Wnt7b expression we demonstrate a significant impact on organ growth and morphogenesis particularly during the early branching stages of the organ, which negatively affects generation of the pro-endocrine (Ngn3(+)/Nkx6.1(+)), and pro-acinar (Ptf1A(+)) fields. Consequently, Wnt7b gain-of-function results in failed morphogenesis and almost complete abrogation of the differentiation of endocrine and acinar cells, leading to cystic epithelial metaplasia expressing ductal markers including Sox9, Hnf6 and Hnf1ß. While Wnt7b is expressed exclusively in the developing pancreatic epithelium, adjacent mesenchymal cells in the organ display a direct trophic response to elevated Wnt7b and increase expression of Lef1, cFos and desmin. Of note, in contrast to the pancreatic epithelium, the pancreatic mesenchyme remains competent to respond to Wnt7b ligand, at later stages in development. We conclude that Wnt7b helps coordinate pancreatic development through autocrine, as well as paracrine mechanisms, and as such represents a novel bi-modal morphogen ligand.

Hydrogen-fed biofilm reactors reducing selenate and sulfate: Community structure and capture of elemental selenium within the biofilm.

Remediation of selenate (SeO4 (2-) ) contamination through microbial reduction is often challenging due to the presence of sulfate (SO4 (2-) ), which can lead to competition for the electron donor and the co-production of toxic H2 S. Microbial reduction of SeO4 (2-) in the presence of SO4 (2-) was studied in two hydrogen-based membrane biofilm reactors (MBfRs). One MBfR was initiated with SO4 (2-) -reducing conditions and gradually shifted to SeO4 (2-) reduction. The second MBfR was developed with a SeO4 (2-) -reducing biofilm, followed by SO4 (2-) introduction. Biofilms within both MBfRs achieved greater than 90% SeO4 (2-) reduction, even though the SeO4 (2-) concentration ranged from 1,000-11,000 µg/L, more than 20-200 times the maximum contaminant level for drinking water (50 µg/L). Biofilm microbial community composition, assessed by 16S rRNA gene-based amplicon pyrosequencing, was distinct between the two MBfRs and was framed by alterations in SeO4 (2-) loading. Specifically, high SeO4 (2-) loading resulted in communities mainly composed of denitrifying bacteria (e.g., Denitratisoma and Dechloromonas). In contrast, low loading led to mostly sulfate-reducing bacteria (i.e., Desulfovibrio) and sulfur-oxidizing bacteria (i.e., Sulfuricurvum and Sulfurovum). SeO4 (2-) was reduced to elemental selenium (Se°), which was visualized within the biofilm as crystalloid aggregates, with its fate corresponding to that of biofilm solids. In conclusion, microbial biofilm communities initiated under either SeO4 (2-) or SO4 (2-) -reducing conditions attained high SeO4 (2-) removal rates even though their microbial community composition was quite distinct. Biotechnol. Bioeng. 2016;113: 1736-1744. © 2016 Wiley Periodicals, Inc.

Nicotinamide Phosphoribosyltransferase Positive Allosteric Modulators Attenuate Neuronal Oxidative Stress.

Evidence supports boosting nicotinamide adenine dinucleotide (NAD+) to counteract oxidative stress in aging and neurodegenerative disease. One approach is to enhance the activity of nicotinamide phosphoribosyltransferase (NAMPT). Novel NAMPT positive allosteric modulators (N-PAMs) were identified. A cocrystal structure confirmed N-PAM binding to the NAMPT rear channel. Early hit-to-lead efforts led to a 1.88-fold maximum increase in the level of NAD+ in human THP-1 cells. Select N-PAMs were assessed for mitigation of reactive oxygen species (ROS) in HT-22 neuronal cells subject to inflammatory stress using tumor necrosis factor alpha (TNFα). N-PAMs that increased NAD+ more effectively in THP-1 cells attenuated TNFα-induced ROS more effectively in HT-22 cells. The most efficacious N-PAM completely attenuated ROS elevation in glutamate-stressed HT-22 cells, a model of neuronal excitotoxicity. This work demonstrates for the first time that N-PAMs are capable of mitigating elevated ROS in neurons stressed with TNFα and glutamate and provides support for further N-PAM optimization for treatment of neurodegenerative diseases.

Synthesis, Optimization, and Structure-Activity Relationships of Nicotinamide Phosphoribosyltransferase (NAMPT) Positive Allosteric Modulators (N-PAMs).

Depletion of nicotinamide adenine dinucleotide (NAD+) is associated with aging and disease, spurring the study of dietary supplements to replenish NAD+. The catabolism of NAD+ to nicotinamide (NAM) requires the salvage of NAM to replenish cellular NAD+, which relies on the rate-limiting enzyme nicotinamide phosphoribosyltransferase (NAMPT). Pharmacological activation of NAMPT provides an alternative to dietary supplements. Screening for activators of NAMPT identified small molecule NAMPT positive allosteric modulators (N-PAMs). N-PAMs bind to the rear channel of NAMPT increasing enzyme activity and alleviating feedback inhibition by NAM and NAD+. Synthesis of over 70 N-PAMs provided an excellent correlation between rear channel binding affinity and potency for enzyme activation, confirming the mechanism of allosteric activation via binding to the rear channel. The mechanism accounts for higher binding affinity leading to loss of efficacy. Enzyme activation translated directly to elevation of NAD+ measured in cells. Optimization led to an orally bioavailable N-PAM.

mdx(5cv) mice manifest more severe muscle dysfunction and diaphragm force deficits than do mdx Mice.

Duchenne muscular dystrophy (DMD) is characterized by progressive skeletal muscle dysfunction leading to premature death by the third decade of life. The mdx mouse, the most widely used animal model of DMD, has been extremely useful to study disease mechanisms and to screen new therapeutics. However, unlike patients with DMD, mdx mice have a very mild motor function deficit, posing significant limitations for its use as a platform to assess the impact of treatments on motor function. It has been suggested that an mdx variant, the mdx(5cv) mouse, might be more severely affected. Here, we compared the motor activity, histopathology, and individual muscle force measurements of mdx and mdx(5cv) mice. Our study revealed that mdx(5cv) mice showed more severe exercise-induced fatigue, Rotarod performance deficits, and gait anomalies than mdx mice and that these deficits began at a younger age. Muscle force studies showed more severe strength deficits in the diaphragm of mdx(5cv) mice compared to mdx mice, but similar force generation in the extensor digitorum longus. Muscle histology was similar between the two strains. Differences in genetic background (genetic modifiers) probably account for these functional differences between mdx strains. Overall, our findings indicate that the mdx and mdx(5cv) mouse models of DMD are not interchangeable and identify the mdx(5cv) mouse as a valuable platform for preclinical studies that require assessment of muscle function in live animals.

DNA-Stable Isotope Probing Shotgun Metagenomics Reveals the Resilience of Active Microbial Communities to Biochar Amendment in Oxisol Soil.

The functions and interactions of individual microbial populations and their genes in agricultural soils amended with biochar remain elusive but are crucial for a deeper understanding of nutrient cycling and carbon (C) sequestration. In this study, we coupled DNA stable isotope probing (SIP) with shotgun metagenomics in order to target the active community in microcosms which contained soil collected from biochar-amended and control plots under napiergrass cultivation. Our analyses revealed that the active community was composed of high-abundant and low-abundant populations, including Actinobacteria, Proteobacteria, Gemmatimonadetes, and Acidobacteria. Although biochar did not significantly shift the active taxonomic and functional communities, we found that the narG (nitrate reductase) gene was significantly more abundant in the control metagenomes. Interestingly, putative denitrifier genomes generally encoded one gene or a partial denitrification pathway, suggesting denitrification is typically carried out by an assembly of different populations within this Oxisol soil. Altogether, these findings indicate that the impact of biochar on the active soil microbial community are transient in nature. As such, the addition of biochar to soils appears to be a promising strategy for the long-term C sequestration in agricultural soils, does not impart lasting effects on the microbial functional community, and thus mitigates un-intended microbial community shifts that may lead to fertilizer loss through increased N cycling.

Diazotroph Diversity and Nitrogen Fixation in Summer Active Perennial Grasses in a Mediterranean Region Agricultural Soil.

Summer-growing perennial grasses such as Panicum coloratum L. cv. Bambatsi (Bambatsi panic), Chloris gayana Kunth cv. Katambora (Rhodes grass) and Digitaria eriantha Steud. cv. Premier (Premier digit grass) growing in the poor fertility sandy soils in the Mediterranean regions of southern Australia and western Australia mainly depend upon soil N and biological N inputs through diazotrophic (free living or associative) N fixation. We investigated the community composition and diversity (nifH-amplicon sequencing), abundance (qPCR) and functional capacity (15N incubation assay) of the endophytic diazotrophic community in the below and above ground plant parts of field grown and unfertilized grasses. Results showed a diverse and abundant diazotrophic community inside plant both above and below-ground and there was a distinct diazotrophic assemblage in the different plant parts in all the three grasses. There was a limited difference in the diversity between leaves, stems and roots except that Panicum grass roots harbored greater species richness. Nitrogen fixation potentials ranged between 0.24 and 5.9 mg N kg-1 day-1 and N fixation capacity was found in both the above and below ground plant parts. Results confirmed previous reports of plant species-based variation and that Alpha-Proteobacteria were the dominant group of nifH-harboring taxa both in the belowground and aboveground parts of the three grass species. Results also showed a well-structured nifH-harboring community in all plant parts, an example for a functional endophytic community. Overall, the variation in the number and identity of module hubs and connectors among the different plant parts suggests that co-occurrence patterns within the nifH-harboring community specific to individual compartments and local environments of the niches within each plant part may dictate the overall composition of diazotrophs within a plant.

Developing defined substrates for stem cell culture and differentiation.

Over the past few decades, a variety of different reagents for stem cell maintenance and differentiation have been commercialized. These reagents share a common goal in facilitating the manufacture of products suitable for cell therapy while reducing the amount of non-defined components. Lessons from developmental biology have identified signalling molecules that can guide the differentiation process in vitro, but less attention has been paid to the extracellular matrix used. With the introduction of more biologically relevant and defined matrices, that better mimic specific cell niches, researchers now have powerful resources to fine-tune their in vitro differentiation systems, which may allow the manufacture of therapeutically relevant cell types. In this review article, we revisit the basics of the extracellular matrix, and explore the important role of the cell-matrix interaction. We focus on laminin proteins because they help to maintain pluripotency and drive cell fate specification.This article is part of the theme issue 'Designer human tissue: coming to a lab near you'.

Laminin 521 maintains differentiation potential of mouse and human satellite cell-derived myoblasts during long-term culture expansion.

BACKGROUND: Large-scale expansion of myogenic progenitors is necessary to support the development of high-throughput cellular assays in vitro and to advance genetic engineering approaches necessary to develop cellular therapies for rare muscle diseases. However, optimization has not been performed in order to maintain the differentiation capacity of myogenic cells undergoing long-term cell culture. Multiple extracellular matrices have been utilized for myogenic cell studies, but it remains unclear how different matrices influence long-term myogenic activity in culture. To address this challenge, we have evaluated multiple extracellular matrices in myogenic studies over long-term expansion. METHODS: We evaluated the consequence of propagating mouse and human myogenic stem cell progenitors on various extracellular matrices to determine if they could enhance long-term myogenic potential. For the first time reported, we comprehensively examine the effect of physiologically relevant laminins, laminin 211 and laminin 521, compared to traditionally utilized ECMs (e.g., laminin 111, gelatin, and Matrigel) to assess their capacity to preserve myogenic differentiation potential. RESULTS: Laminin 521 supported increased proliferation in early phases of expansion and was the only substrate facilitating high-level fusion following eight passages in mouse myoblast cell cultures. In human myoblast cell cultures, laminin 521 supported increased proliferation during expansion and superior differentiation with myotube hypertrophy. Counterintuitively however, laminin 211, the native laminin isoform in resting skeletal muscle, resulted in low proliferation and poor differentiation in mouse and human cultures. Matrigel performed excellent in short-term mouse studies but showed high amounts of variability following long-term expansion. CONCLUSIONS: These results demonstrate laminin 521 is a superior substrate for both short-term and long-term myogenic cell culture applications compared to other commonly utilized substrates. Since Matrigel cannot be used for clinical applications, we propose that laminin 521 could possibly be employed in the future to provide myoblasts for cellular therapy directed clinical studies.

Denitrifying and diazotrophic community responses to artificial warming in permafrost and tallgrass prairie soils.

Increasing temperatures have been shown to impact soil biogeochemical processes, although the corresponding changes to the underlying microbial functional communities are not well understood. Alterations in the nitrogen (N) cycling functional component are particularly important as N availability can affect microbial decomposition rates of soil organic matter and influence plant productivity. To assess changes in the microbial component responsible for these changes, the composition of the N-fixing (nifH), and denitrifying (nirS, nirK, nosZ) soil microbial communities was assessed by targeted pyrosequencing of functional genes involved in N cycling in two major biomes where the experimental effect of climate warming is under investigation, a tallgrass prairie in Oklahoma (OK) and the active layer above permafrost in Alaska (AK). Raw reads were processed for quality, translated with frameshift correction, and a total of 313,842 amino acid sequences were clustered and linked to a nearest neighbor using reference datasets. The number of OTUs recovered ranged from 231 (NifH) to 862 (NirK). The N functional microbial communities of the prairie, which had experienced a decade of experimental warming were the most affected with changes in the richness and/or overall structure of NifH, NirS, NirK and NosZ. In contrast, the AK permafrost communities, which had experienced only 1 year of warming, showed decreased richness and a structural change only with the nirK-harboring bacterial community. A highly divergent nirK-harboring bacterial community was identified in the permafrost soils, suggesting much novelty, while other N functional communities exhibited similar relatedness to the reference databases, regardless of site. Prairie and permafrost soils also harbored highly divergent communities due mostly to differing major populations.

Muscle side population cells from dystrophic or injured muscle adopt a fibro-adipogenic fate.

Muscle side population (SP) cells are rare multipotent stem cells that can participate in myogenesis and muscle regeneration upon transplantation. While they have been primarily studied for the development of cell-based therapies for Duchenne muscular dystrophy, little is known regarding their non-muscle lineage choices or whether the dystrophic muscle environment affects their ability to repair muscle. Unfortunately, the study of muscle SP cells has been challenged by their low abundance and the absence of specific SP cell markers. To address these issues, we developed culture conditions for the propagation and spontaneous multi-lineage differentiation of muscle SP cells. Using this approach, we show that SP cells from wild type muscle robustly differentiate into satellite cells and form myotubes without requiring co-culture with myogenic cells. Furthermore, this myogenic activity is associated with SP cells negative for immune (CD45) and vascular (CD31) markers but positive for Pax7, Sca1, and the mesenchymal progenitor marker PDGFRα. Additionally, our studies revealed that SP cells isolated from dystrophic or cardiotoxin-injured muscle fail to undergo myogenesis. Instead, these SP cells rapidly expand giving rise to fibroblast and adipocyte progenitors (FAPs) and to their differentiated progeny, fibroblasts and adipocytes. Our findings indicate that muscle damage affects the lineage choices of muscle SP cells, promoting their differentiation along fibro-adipogenic lineages while inhibiting myogenesis. These results have implications for a possible role of muscle SP cells in fibrosis and fat deposition in muscular dystrophy. In addition, our studies provide a useful in vitro system to analyze SP cell biology in both normal and pathological conditions.

A gene-targeted approach to investigate the intestinal butyrate-producing bacterial community.

BACKGROUND: Butyrate, which is produced by the human microbiome, is essential for a well-functioning colon. Bacteria that produce butyrate are phylogenetically diverse, which hinders their accurate detection based on conventional phylogenetic markers. As a result, reliable information on this important bacterial group is often lacking in microbiome research. RESULTS: In this study we describe a gene-targeted approach for 454 pyrotag sequencing and quantitative polymerase chain reaction for the final genes in the two primary bacterial butyrate synthesis pathways, butyryl-CoA:acetate CoA-transferase (but) and butyrate kinase (buk). We monitored the establishment and early succession of butyrate-producing communities in four patients with ulcerative colitis who underwent a colectomy with ileal pouch anal anastomosis and compared it with three control samples from healthy colons. All patients established an abundant butyrate-producing community (approximately 5% to 26% of the total community) in the pouch within the 2-month study, but patterns were distinctive among individuals. Only one patient harbored a community profile similar to the healthy controls, in which there was a predominance of but genes that are similar to reference genes from Acidaminococcus sp., Eubacterium sp., Faecalibacterium prausnitzii and Roseburia sp., and an almost complete absence of buk genes. Two patients were greatly enriched in buk genes similar to those of Clostridium butyricum and C. perfringens, whereas a fourth patient displayed abundant communities containing both genes. Most butyrate producers identified in previous studies were detected and the general patterns of taxa found were supported by 16S rRNA gene pyrotag analysis, but the gene-targeted approach provided more detail about the potential butyrate-producing members of the community. CONCLUSIONS: The presented approach provides quantitative and genotypic insights into butyrate-producing communities and facilitates a more specific functional characterization of the intestinal microbiome. Furthermore, our analysis refines but and buk reference annotations found in central databases.

BMP-9-induced muscle heterotopic ossification requires changes to the skeletal muscle microenvironment.

Heterotopic ossification (HO) is defined as the formation of bone inside soft tissue. Symptoms include joint stiffness, swelling, and pain. Apart from the inherited form, the common traumatic form generally occurs at sites of injury in damaged muscles and is often associated with brain injury. We investigated bone morphogenetic protein 9 (BMP-9), which possesses a strong osteoinductive capacity, for its involvement in muscle HO physiopathology. We found that BMP-9 had an osteoinductive influence on mouse muscle resident stromal cells by increasing their alkaline phosphatase activity and bone-specific marker expression. Interestingly, BMP-9 induced HO only in damaged muscle, whereas BMP-2 promoted HO in skeletal muscle regardless of its state. The addition of the soluble form of the ALK1 protein (the BMP-9 receptor) significantly inhibited the osteoinductive potential of BMP-9 in cells and HO in damaged muscles. BMP-9 thus should be considered a candidate for involvement in HO physiopathology, with its activity depending on the skeletal muscle microenvironment.