Small regulatory RNAs are mediators of the Streptococcus mutans SloR regulon.

Dental caries is among the most prevalent chronic diseases worldwide. Streptococcus mutans, the chief causative agent of caries, uses a 25-kDa manganese-dependent SloR protein to coordinate the uptake of essential manganese with the transcription of its virulence attributes. Small non-coding RNAs (sRNAs) can either enhance or repress gene expression, and reports in the literature ascribe an emerging role for sRNAs in the environmental stress response. Herein, we focused our attention on 18-50 nt sRNAs as mediators of the S. mutans SloR and manganese regulons. Specifically, the results of RNA sequencing revealed 19 sRNAs in S. mutans, which were differentially transcribed in the SloR-proficient UA159 and SloR-deficient GMS584 strains, and 10 sRNAs that were differentially expressed in UA159 cells grown in the presence of low vs high manganese. We describe SmsR1532 and SmsR1785 as SloR- and manganese-responsive sRNAs that are processed from large transcripts and that bind SloR directly in their promoter regions. The predicted targets of these sRNAs include regulators of metal ion transport, growth management via a toxin-antitoxin operon, and oxidative stress tolerance. These findings support a role for sRNAs in coordinating intracellular metal ion homeostasis with virulence gene control in an important oral cariogen. IMPORTANCE Small regulatory RNAs (sRNAs) are critical mediators of environmental signaling, particularly in bacterial cells under stress, but their role in Streptococcus mutans is poorly understood. S. mutans, the principal causative agent of dental caries, uses a 25-kDa manganese-dependent protein, called SloR, to coordinate the regulated uptake of essential metal ions with the transcription of its virulence genes. In the present study, we identified and characterized sRNAs that are both SloR and manganese responsive. Taken together, this research can elucidate the details of regulatory networks that engage sRNAs in an important oral pathogen and that can enable the development of an effective anti-caries therapeutic.

Spatial variation in high temperature-regulated gene expression predicts evolution of plasticity with climate change in the scarlet monkeyflower.

A major way that organisms can adapt to changing environmental conditions is by evolving increased or decreased phenotypic plasticity. In the face of current global warming, more attention is being paid to the role of plasticity in maintaining fitness as abiotic conditions change over time. However, given that temporal data can be challenging to acquire, a major question is whether evolution in plasticity across space can predict adaptive plasticity across time. In growth chambers simulating two thermal regimes, we generated transcriptome data for western North American scarlet monkeyflowers (Mimulus cardinalis) collected from different latitudes and years (2010 and 2017) to test hypotheses about how plasticity in gene expression is responding to increases in temperature, and if this pattern is consistent across time and space. Supporting the genetic compensation hypothesis, individuals whose progenitors were collected from the warmer-origin northern 2017 descendant cohort showed lower thermal plasticity in gene expression than their cooler-origin northern 2010 ancestors. This was largely due to a change in response at the warmer (40°C) rather than cooler (20°C) treatment. A similar pattern of reduced plasticity, largely due to a change in response at 40°C, was also found for the cooler-origin northern versus the warmer-origin southern population from 2017. Our results demonstrate that reduced phenotypic plasticity can evolve with warming and that spatial and temporal changes in plasticity predict one another.

Salt Stress Enhances Early Symbiotic Gene Expression in Medicago truncatula and Induces a Stress-Specific Set of Rhizobium-Responsive Genes.

Salt stress is a major agricultural concern inhibiting not only plant growth but also the symbiotic association between legume roots and the soil bacteria rhizobia. This symbiotic association is initiated by a molecular dialogue between the two partners, leading to the activation of a signaling cascade in the legume host and, ultimately, the formation of nitrogen-fixing root nodules. Here, we show that a moderate salt stress increases the responsiveness of early symbiotic genes in Medicago truncatula to its symbiotic partner, Sinorhizobium meliloti while, conversely, inoculation with S. meliloti counteracts salt-regulated gene expression, restoring one-third to control levels. Our analysis of early nodulin 11 (ENOD11) shows that salt-induced expression is dynamic, Nod-factor dependent, and requires the ionic but not the osmotic component of salt. We demonstrate that salt stimulation of rhizobium-induced gene expression requires NSP2, which functions as a node to integrate the abiotic and biotic signals. In addition, our work reveals that inoculation with S. meliloti succinoglycan mutants also hyperinduces ENOD11 expression in the presence or absence of salt, suggesting a possible link between rhizobial exopolysaccharide and the plant response to salt stress. Finally, we identify an accessory set of genes that are induced by rhizobium only under conditions of salt stress and have not been previously identified as being nodulation-related genes. Our data suggest that interplay of core nodulation genes with different accessory sets, specific for different abiotic conditions, functions to establish the symbiosis. Together, our findings reveal a complex and dynamic interaction between plant, microbe, and environment.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Implications of region-specific gene expression for development of the partially fused petunia corolla.

Angiosperm petal fusion (sympetaly) has evolved multiple times independently and is associated with increased specificity between plants and their pollinators. To uncover developmental genetic changes that might have led to the evolution of sympetaly in the asterid core eudicot genus Petunia (Solanaceae), we carried out global and fine-scale gene expression analyses in different regions of the corolla. We found that, despite several similarities with the choripetalous model species Arabidopsis thaliana in the proximal-distal transcriptome, the Petunia axillaris fused and proximal corolla tube expresses several genes that in A. thaliana are associated with the distal petal region. This difference aligns with variation in petal shape and fusion across ontogeny of the two species. Moreover, differential gene expression between the unfused lobes and fused tube of P. axillaris petals revealed three strong candidate genes for sympetaly based on functional annotation in organ boundary specification. Partial silencing of one of these, the HANABA TARANU (HAN)-like gene PhGATA19, resulted in reduced fusion of Petunia hybrida petals, with silencing of both PhGATA19 and its close paralog causing premature plant senescence. Finally, detailed expression analyses for the previously characterized organ boundary gene candidate NO APICAL MERISTEM (NAM) supports the hypothesis that it establishes boundaries between most P. axillaris floral organs, with the exception of boundaries between petals.

An in silico proteomics screen to predict and prioritize protein-protein interactions dependent on post-translationally modified motifs.

Motivation: The development of proteomic methods for the characterization of domain/motif interactions has greatly expanded our understanding of signal transduction. However, proteomics-based binding screens have limitations including that the queried tissue or cell type may not harbor all potential interacting partners or post-translational modifications (PTMs) required for the interaction. Therefore, we sought a generalizable, complementary in silico approach to identify potentially novel motif and PTM-dependent binding partners of high priority. Results: We used as an initial example the interaction between the Src homology 2 (SH2) domains of the adaptor proteins CT10 regulator of kinase (CRK) and CRK-like (CRKL) and phosphorylated-YXXP motifs. Employing well-curated, publicly-available resources, we scored and prioritized potential CRK/CRKL-SH2 interactors possessing signature characteristics of known interacting partners. Our approach gave high priority scores to 102 of the >9000 YXXP motif-containing proteins. Within this 102 were 21 of the 25 curated CRK/CRKL-SH2-binding partners showing a more than 80-fold enrichment. Several predicted interactors were validated biochemically. To demonstrate generalized applicability, we used our workflow to predict protein-protein interactions dependent upon motif-specific arginine methylation. Our data demonstrate the applicability of our approach to, conceivably, any modular binding domain that recognizes a specific post-translationally modified motif. Supplementary information: Supplementary data are available at Bioinformatics online.

Kinetic and kinematic analysis of stamping impacts during simulated rucking in rugby union.

Laceration injuries account for up to 23% of injuries in rugby union. They are frequently caused by studded footwear as a result of a player stamping onto another player during the ruck. Little is known about the kinetics and kinematics of rugby stamping impacts; current test methods assessing laceration injury risk of stud designs therefore lack informed test parameters. In this study, twelve participants stamped on an anthropomorphic test device in a one-on-one simulated ruck setting. Velocity and inclination angle of the foot prior to impact was determined from high-speed video footage. Total stamping force and individual stud force were measured using pressure sensors. Mean foot inbound velocity was 4.3 m ∙ s-1 (range 2.1-6.3 m ∙ s-1). Mean peak total force was 1246 N and mean peak stud force was 214 N. The total mean effective mass during stamping was 6.6 kg (range: 1.6-13.5 kg) and stud effective mass was 1.2 kg (range: 0.5-2.9 kg). These results provide representative test parameters for mechanical test devices designed to assess laceration injury risk of studded footwear for rugby union.

Identification of target genes downstream of semaphorin6A/PlexinA2 signaling in zebrafish.

BACKGROUND: Semaphorin (Sema)/Plexin (Plxn) signaling is important for many aspects of neuronal development, however, the transcriptional regulation imposed by this signaling pathway is unknown. Previously, we identified an essential role for Sema6A/PlxnA2 signaling in regulating proliferation and cohesion of retinal precursor cells (RPCs) during early eye development. This study used RNA isolated from control, Sema6A-deficient and PlxnA2-deficient zebrafish embryos in a microarray analysis to identify genes that were differentially expressed when this signaling pathway was disrupted. RESULTS: We uncovered a set of 58 transcripts, and all but 1 were up-regulated in both sema6A and plxnA2 morphants. We validated gene expression changes in subset of candidates that are suggested to be involved in proliferation, migration or neuronal positioning. We further functionally evaluated one gene, rasl11b, as contributing to disrupted proliferation in sema6A and plxna2 morphants. Our results suggest rasl11b negatively regulates proliferation of RPCs in the developing zebrafish eye. CONCLUSIONS: Microarray analysis has generated a resource of target genes downstream of Sema6A/PlxnA2 signaling, which can be further investigated to elucidate the downstream effects of this well-studied neuronal and vascular guidance signaling pathway. Developmental Dynamics 246:539-549, 2017. © 2017 Wiley Periodicals, Inc.

The phylogeny of Heliconia (Heliconiaceae) and the evolution of floral presentation.

Heliconia (Heliconiaceae, order Zingiberales) is among the showiest plants of the Neotropical rainforest and represent a spectacular co-evolutionary radiation with hummingbirds. Despite the attractiveness and ecological importance of many Heliconia, the genus has been the subject of limited molecular phylogenetic studies. We sample seven markers from the plastid and nuclear genomes for 202 samples of Heliconia. This represents ca. 75% of accepted species and includes coverage of all taxonomic subgenera and sections. We date this phylogeny using fossils associated with other families in the Zingiberales; in particular we review and evaluate the Eocene fossil Ensete oregonense. We use this dated phylogenetic framework to evaluate the evolution of two components of flower orientation that are hypothesized to be important for modulating pollinator discrimination and pollen placement: resupination and erect versus pendant inflorescence habit. Our phylogenetic results suggest that the monophyletic Melanesian subgenus Heliconiopsis and a small clade of Ecuadorian species are together the sister group to the rest of Heliconia. Extant diversity of Heliconia originated in the Late Eocene (39Ma) with rapid diversification through the Early Miocene, making it the oldest known clade of hummingbird-pollinated plants. Most described subgenera and sections are not monophyletic, though closely related groups of species, often defined by shared geography, mirror earlier morphological cladistic analyses. Evaluation of changes in resupination and inflorescence habit suggests that these characters are more homoplasious than expected, and this largely explains the non-monophyly of previously circumscribed subgenera, which were based on these characters. We also find strong evidence for the correlated evolution of resupination and inflorescence habit. The correlated model suggests that the most recent common ancestor of all extant Heliconia had resupinate flowers and erect inflorescences. Finally, we note our nearly complete species sampling and dated phylogeny allow for an assessment of taxonomic history in terms of phylogenetic diversity. We find approximately half of the currently recognized species, corresponding to half of the phylogenetic diversity, have been described since 1975, highlighting the continued importance of basic taxonomic research and conservation initiatives to preserve both described and undiscovered species of Heliconia.

Acetate supplementation as a means of inducing glioblastoma stem-like cell growth arrest.

Glioblastoma (GBM), the most common primary adult malignant brain tumor, is associated with a poor prognosis due, in part, to tumor recurrence mediated by chemotherapy and radiation resistant glioma stem-like cells (GSCs). The metabolic and epigenetic state of GSCs differs from their non-GSC counterparts, with GSCs exhibiting greater glycolytic metabolism and global hypoacetylation. However, little attention has been focused on the potential use of acetate supplementation as a therapeutic approach. N-acetyl-l-aspartate (NAA), the primary storage form of brain acetate, and aspartoacylase (ASPA), the enzyme responsible for NAA catalysis, are significantly reduced in GBM tumors. We recently demonstrated that NAA supplementation is not an appropriate therapeutic approach since it increases GSC proliferation and pursued an alternative acetate source. The FDA approved food additive Triacetin (glyceryl triacetate, GTA) has been safely used for acetate supplementation therapy in Canavan disease, a leukodystrophy due to ASPA mutation. This study characterized the effects of GTA on the proliferation and differentiation of six primary GBM-derived GSCs relative to established U87 and U251 GBM cell lines, normal human cerebral cortical astrocytes, and murine neural stem cells. GTA reduced proliferation of GSCs greater than established GBM lines. Moreover, GTA reduced growth of the more aggressive mesenchymal GSCs greater than proneural GSCs. Although sodium acetate induced a dose-dependent reduction of GSC growth, it also reduced cell viability. GTA-mediated growth inhibition was not associated with differentiation, but increased protein acetylation. These data suggest that GTA-mediated acetate supplementation is a novel therapeutic strategy to inhibit GSC growth.

Pumilio-2 regulates translation of Nav1.6 to mediate homeostasis of membrane excitability.

The ability to regulate intrinsic membrane excitability, to maintain consistency of action potential firing, is critical for stable neural circuit activity. Without such mechanisms, Hebbian-based synaptic plasticity could push circuits toward activity saturation or, alternatively, quiescence. Although now well documented, the underlying molecular components of these homeostatic mechanisms remain poorly understood. Recent work in the fruit fly, Drosophila melanogaster, has identified Pumilio (Pum), a translational repressor, as an essential component of one such mechanism. In response to changing synaptic excitation, Pum regulates the translation of the voltage-gated sodium conductance, leading to a concomitant adjustment in action potential firing. Although similar homeostatic mechanisms are operational in mammalian neurons, it is unknown whether Pum is similarly involved. In this study, we report that Pum2 is indeed central to the homeostatic mechanism regulating membrane excitability in rat visual cortical pyramidal neurons. Using RNA interference, we observed that loss of Pum2 leads to increased sodium current (I(Na)) and action potential firing, mimicking the response by these neurons to being deprived of synaptic depolarization. In contrast, increased synaptic depolarization results in increased Pum2 expression and subsequent reduction in INa and membrane excitability. We further show that Pum2 is able to directly bind the predominant voltage-gated sodium channel transcript (NaV1.6) expressed in these neurons and, through doing so, regulates translation of this key determinant of membrane excitability. Together, our results show that Pum2 forms part of a homeostatic mechanism that matches membrane excitability to synaptic depolarization in mammalian neurons.

Triacetin-based acetate supplementation as a chemotherapeutic adjuvant therapy in glioma.

Cancer is associated with epigenetic (i.e., histone hypoacetylation) and metabolic (i.e., aerobic glycolysis) alterations. Levels of N-acetyl-L-aspartate (NAA), the primary storage form of acetate in the brain, and aspartoacylase (ASPA), the enzyme responsible for NAA catalysis to generate acetate, are reduced in glioma; yet, few studies have investigated acetate as a potential therapeutic agent. This preclinical study sought to test the efficacy of the food additive Triacetin (glyceryl triacetate, GTA) as a novel therapy to increase acetate bioavailability in glioma cells. The growth-inhibitory effects of GTA, compared to the histone deacetylase inhibitor Vorinostat (SAHA), were assessed in established human glioma cell lines (HOG and Hs683 oligodendroglioma, U87 and U251 glioblastoma) and primary tumor-derived glioma stem-like cells (GSCs), relative to an oligodendrocyte progenitor line (Oli-Neu), normal astrocytes, and neural stem cells (NSCs) in vitro. GTA was also tested as a chemotherapeutic adjuvant with temozolomide (TMZ) in orthotopically grafted GSCs. GTA-induced cytostatic growth arrest in vitro comparable to Vorinostat, but, unlike Vorinostat, GTA did not alter astrocyte growth and promoted NSC expansion. GTA alone increased survival of mice engrafted with glioblastoma GSCs and potentiated TMZ to extend survival longer than TMZ alone. GTA was most effective on GSCs with a mesenchymal cell phenotype. Given that GTA has been chronically administered safely to infants with Canavan disease, a leukodystrophy due to ASPA mutation, GTA-mediated acetate supplementation may provide a novel, safe chemotherapeutic adjuvant to reduce the growth of glioma tumors, most notably the more rapidly proliferating, glycolytic and hypoacetylated mesenchymal glioma tumors.

A finite element model for predicting impact-induced damage to a skin simulant.

A finite element model was developed for assessing the efficacy of rugby body padding in reducing the risk of sustaining cuts and abrasions. The model was developed to predict the onset of damage to a soft tissue simulant from concentrated impact loading (i.e., stud impact) and compared against a corresponding experiment. The damage modelling techniques involved defining an element deletion criterion, whereby those on the surface of the surrogate were deleted if their maximum principal stress reached a predefined value. Candidate maximum principal stress values for element deletion criteria were identified independently from puncture test simulations on the soft tissue simulant. Experimental impacts with a stud were carried out at three energies (2, 4 and 6 J), at three angular orientations (0°, 15° and 30°) and compared to corresponding simulations. Suitable maximum principal stress values for element deletion criteria settings were first identified for the 4 J impact, selecting the candidates that best matched the experimental results. The same element deletion settings were then applied in simulations at 2 and 6 J and the validity of the model was further assessed (difference < 15% for the force at tear and < 30% for time to tear). The damage modelling techniques presented here could be applied to other skin simulants to assess the onset of skin injuries and the ability of padding to prevent them.

Small regulatory RNAs are mediators of the Streptococcus mutans SloR regulon.

Dental caries is among the most prevalent chronic infectious diseases worldwide. Streptococcus mutans , the chief causative agent of caries, uses a 25 kDa manganese dependent SloR protein to coordinate the uptake of essential manganese with the transcription of its virulence attributes. Small non-coding RNAs (sRNAs) can either enhance or repress gene expression and reports in the literature ascribe an emerging role for sRNAs in the environmental stress response. Herein, we identify 18-50 nt sRNAs as mediators of the S. mutans SloR and manganese regulons. Specifically, the results of sRNA-seq revealed 56 sRNAs in S. mutans that were differentially transcribed in the SloR-proficient UA159 and SloR-deficient GMS584 strains, and 109 sRNAs that were differentially expressed in UA159 cells grown in the presence of low versus high manganese. We describe SmsR1532 and SmsR1785 as SloR- and/or manganese-responsive sRNAs that are processed from large transcripts, and that bind SloR directly in their promoter regions. The predicted targets of these sRNAs include regulators of metal ion transport, growth management via a toxin-antitoxin operon, and oxidative stress tolerance. These findings support a role for sRNAs in coordinating intracellular metal ion homeostasis with virulence gene control in an important oral cariogen. IMPORTANCE: Small regulatory RNAs (sRNAs) are critical mediators of environmental signaling, particularly in bacterial cells under stress, but their role in Streptococcus mutans is poorly understood. S. mutans, the principal causative agent of dental caries, uses a 25 kDa manganese-dependent protein, called SloR, to coordinate the regulated uptake of essential metal ions with the transcription of its virulence genes. In the present study, we identified and characterize sRNAs that are both SloR- and manganese-responsive. Taken together, this research can elucidate the details of regulatory networks that engage sRNAs in an important oral pathogen, and that can enable the development of an effective anti-caries therapeutic.

Myeloma-Modified Adipocytes Exhibit Metabolic Dysfunction and a Senescence-Associated Secretory Phenotype.

Bone marrow adipocytes (BMAd) have recently been implicated in accelerating bone metastatic cancers, such as acute myelogenous leukemia and breast cancer. Importantly, bone marrow adipose tissue (BMAT) expands with aging and obesity, two key risk factors in multiple myeloma disease prevalence, suggesting that BMAds may influence and be influenced by myeloma cells in the marrow. Here, we provide evidence that reciprocal interactions and cross-regulation of myeloma cells and BMAds play a role in multiple myeloma pathogenesis and treatment response. Bone marrow biopsies from patients with multiple myeloma revealed significant loss of BMAT with myeloma cell infiltration of the marrow, whereas BMAT was restored after treatment for multiple myeloma. Myeloma cells reduced BMAT in different preclinical murine models of multiple myeloma and in vitro using myeloma cell-adipocyte cocultures. In addition, multiple myeloma cells altered adipocyte gene expression and cytokine secretory profiles, which were also associated with bioenergetic changes and induction of a senescent-like phenotype. In vivo, senescence markers were also increased in the bone marrow of tumor-burdened mice. BMAds, in turn, provided resistance to dexamethasone-induced cell-cycle arrest and apoptosis, illuminating a new possible driver of myeloma cell evolution in a drug-resistant clone. Our findings reveal that bidirectional interactions between BMAds and myeloma cells have significant implications for the pathogenesis and treatment of multiple myeloma. Targeting senescence in the BMAd or other bone marrow cells may represent a novel therapeutic approach for treatment of multiple myeloma. SIGNIFICANCE: This study changes the foundational understanding of how cancer cells hijack the bone marrow microenvironment and demonstrates that tumor cells induce senescence and metabolic changes in adipocytes, potentially driving new therapeutic directions.

Seasonal Ely Copper Mine Superfund site shotgun metagenomic and metatranscriptomic data analysis.

High throughput sequencing data collected from acid rock drainage (ARD) communities can reveal the active taxonomic and functional diversity of these extreme environments, which can be exploited for bioremediation, pharmaceutical, and industrial applications. Here, we report a seasonal comparison of a microbiome and transcriptome in Ely Brook (EB-90M), a confluence of clean water and upstream tributaries that drains the Ely Copper Mine Superfund site in Vershire, VT, USA. Nucleic acids were extracted from EB-90M water and sediment followed by shotgun sequencing using the Illumina NextSeq platform. Approximately 575,933 contigs with a total length of 1.54 Gbp were generated. Contigs of at least a size of 3264 (N50) or greater represented 50% of the sequences and the longest contig was 488,568 bp in length. Using Centrifuge against the NCBI "nt" database 141 phyla, including candidate phyla, were detected. Roughly 380,000 contigs were assembled and ∼1,000,000 DNA and ∼550,000 cDNA sequences were identified and functionally annotated using the Prokka pipeline. Most expressed KEGG-annotated microbial genes were involved in amino acid metabolism and several KEGG pathways were differentially expressed between seasons. Biosynthetic gene clusters involved in secondary metabolism as well as metal- and antibiotic-resistance genes were annotated, some of which were differentially expressed, colocalized, and coexpressed. These data can be used to show how ecological stimuli, such as seasonal variations and metal concentrations, affect the ARD microbiome and select taxa to produce novel natural products. The data reported herein is supporting information for the research article "Characterization of an acid rock drainage microbiome and transcriptome at the Ely Copper Mine Superfund site" by Giddings et al. [1].

Characterization of an acid rock drainage microbiome and transcriptome at the Ely Copper Mine Superfund site.

The microbial oxidation of metal sulfides plays a major role in the formation of acid rock drainage (ARD). We aimed to broadly characterize the ARD at Ely Brook, which drains the Ely Copper Mine Superfund site in Vermont, USA, using metagenomics and metatranscriptomics to assess the metabolic potential and seasonal ecological roles of microorganisms in water and sediment. Using Centrifuge against the NCBI "nt" database, ~25% of reads in sediment and water samples were classified as acid-tolerant Proteobacteria (61 ± 4%) belonging to the genera Pseudomonas (2.6-3.3%), Bradyrhizobium (1.7-4.1%), and Streptomyces (2.9-5.0%). Numerous genes (12%) were differentially expressed between seasons and played significant roles in iron, sulfur, carbon, and nitrogen cycling. The most abundant RNA transcript encoded the multidrug resistance protein Stp, and most expressed KEGG-annotated transcripts were involved in amino acid metabolism. Biosynthetic gene clusters involved in secondary metabolism (BGCs, 449) as well as metal- (133) and antibiotic-resistance (8501) genes were identified across the entire dataset. Several antibiotic and metal resistance genes were colocalized and coexpressed with putative BGCs, providing insight into the protective roles of the molecules BGCs produce. Our study shows that ecological stimuli, such as metal concentrations and seasonal variations, can drive ARD taxa to produce novel bioactive metabolites.

Multiple Myeloma Cells Alter Adipogenesis, Increase Senescence-Related and Inflammatory Gene Transcript Expression, and Alter Metabolism in Preadipocytes.

Within the bone marrow microenvironment, mesenchymal stromal cells (MSCs) are an essential precursor to bone marrow adipocytes and osteoblasts. The balance between this progenitor pool and mature cells (adipocytes and osteoblasts) is often skewed by disease and aging. In multiple myeloma (MM), a cancer of the plasma cell that predominantly grows within the bone marrow, as well as other cancers, MSCs, preadipocytes, and adipocytes have been shown to directly support tumor cell survival and proliferation. Increasing evidence supports the idea that MM-associated MSCs are distinct from healthy MSCs, and their gene expression profiles may be predictive of myeloma patient outcomes. Here we directly investigate how MM cells affect the differentiation capacity and gene expression profiles of preadipocytes and bone marrow MSCs. Our studies reveal that MM.1S cells cause a marked decrease in lipid accumulation in differentiating 3T3-L1 cells. Also, MM.1S cells or MM.1S-conditioned media altered gene expression profiles of both 3T3-L1 and mouse bone marrow MSCs. 3T3-L1 cells exposed to MM.1S cells before adipogenic differentiation displayed gene expression changes leading to significantly altered pathways involved in steroid biosynthesis, the cell cycle, and metabolism (oxidative phosphorylation and glycolysis) after adipogenesis. MM.1S cells induced a marked increase in 3T3-L1 expression of MM-supportive genes including Il-6 and Cxcl12 (SDF1), which was confirmed in mouse MSCs by qRT-PCR, suggesting a forward-feedback mechanism. In vitro experiments revealed that indirect MM exposure prior to differentiation drives a senescent-like phenotype in differentiating MSCs, and this trend was confirmed in MM-associated MSCs compared to MSCs from normal donors. In direct co-culture, human mesenchymal stem cells (hMSCs) exposed to MM.1S, RPMI-8226, and OPM-2 prior to and during differentiation, exhibited different levels of lipid accumulation as well as secreted cytokines. Combined, our results suggest that MM cells can inhibit adipogenic differentiation while stimulating expression of the senescence associated secretory phenotype (SASP) and other pro-myeloma molecules. This study provides insight into a novel way in which MM cells manipulate their microenvironment by altering the expression of supportive cytokines and skewing the cellular diversity of the marrow.

Advanced biofilm analysis in streams receiving organic deicer runoff.

Prolific heterotrophic biofilm growth is a common occurrence in airport receiving streams containing deicers and anti-icers, which are composed of low-molecular weight organic compounds. This study investigated biofilm spatiotemporal patterns and responses to concurrent and antecedent (i.e., preceding biofilm sampling) environmental conditions at stream sites upstream and downstream from Milwaukee Mitchell International Airport in Milwaukee, Wisconsin, during two deicing seasons (2009-2010; 2010-2011). Biofilm abundance and community composition were investigated along spatial and temporal gradients using field surveys and microarray analyses, respectively. Given the recognized role of Sphaerotilus in organically enriched environments, additional analyses were pursued to specifically characterize its abundance: a consensus sthA sequence was determined via comparison of whole metagenome sequences with a previously identified sthA sequence, the primers developed for this gene were used to characterize relative Sphaerotilus abundance using quantitative real-time PCR, and a Sphaerotilus strain was isolated to validate the determined sthA sequence. Results indicated that biofilm abundance was stimulated by elevated antecedent chemical oxygen demand concentrations, a surrogate for deicer concentrations, with minimal biofilm volumes observed when antecedent chemical oxygen demand concentrations remained below 48 mg/L. Biofilms were composed of diverse communities (including sheathed bacterium Thiothrix) whose composition appeared to shift in relation to antecedent temperature and chemical oxygen demand. The relative abundance of sthA correlated most strongly with heterotrophic biofilm volume (positive) and dissolved oxygen (negative), indicating that Sphaerotilus was likely a consistent biofilm member and thrived under low oxygen conditions. Additional investigations identified the isolate as a new strain of Sphaerotilus montanus (strain KMKE) able to use deicer components as carbon sources and found that stream dissolved oxygen concentrations related inversely to biofilm volume as well as to antecedent temperature and chemical oxygen demand. The airport setting provides insight into potential consequences of widescale adoption of organic deicers for roadway deicing.

Description and validation of the LocoWhisk system: Quantifying rodent exploratory, sensory and motor behaviours.

BACKGROUND: Previous studies have demonstrated that analysing whisker movements and locomotion allows us to quantify the behavioural consequences of sensory, motor and cognitive deficits in rodents. Independent whisker and feet trackers exist but there is no fully-automated, open-source software and hardware solution, that measures both whisker movements and gait. NEW METHOD: We present the LocoWhisk arena and new accompanying software (ARTv2) that allows the automatic detection and measurement of both whisker and gait information from high-speed video footage. RESULTS: We demonstrate the new whisker and foot detector algorithms on high-speed video footage of freely moving small mammals, and show that whisker movement and gait measurements collected in the LocoWhisk arena are similar to previously reported values in the literature. COMPARISON WITH EXISTING METHOD(S): We demonstrate that the whisker and foot detector algorithms, are comparable in accuracy, and in some cases significantly better, than readily available software and manual trackers. CONCLUSION: The LocoWhisk system enables the collection of quantitative data from whisker movements and locomotion in freely behaving rodents. The software automatically records both whisker and gait information and provides added statistical tools to analyse the data. We hope the LocoWhisk system and software will serve as a solid foundation from which to support future research in whisker and gait analysis.

Development of a 3D bone marrow adipose tissue model.

Over the past twenty years, evidence has accumulated that biochemically and spatially defined networks of extracellular matrix, cellular components, and interactions dictate cellular differentiation, proliferation, and function in a variety of tissue and diseases. Modeling in vivo systems in vitro has been undeniably necessary, but when simplified 2D conditions rather than 3D in vitro models are used, the reliability and usefulness of the data derived from these models decreases. Thus, there is a pressing need to develop and validate reliable in vitro models to reproduce specific tissue-like structures and mimic functions and responses of cells in a more realistic manner for both drug screening/disease modeling and tissue regeneration applications. In adipose biology and cancer research, these models serve as physiologically relevant 3D platforms to bridge the divide between 2D cultures and in vivo models, bringing about more reliable and translationally useful data to accelerate benchtop to bedside research. Currently, no model has been developed for bone marrow adipose tissue (BMAT), a novel adipose depot that has previously been overlooked as "filler tissue" but has more recently been recognized as endocrine-signaling and systemically relevant. Herein we describe the development of the first 3D, BMAT model derived from either human or mouse bone marrow (BM) mesenchymal stromal cells (MSCs). We found that BMAT models can be stably cultured for at least 3 months in vitro, and that myeloma cells (5TGM1, OPM2 and MM1S cells) can be cultured on these for at least 2 weeks. Upon tumor cell co-culture, delipidation occurred in BMAT adipocytes, suggesting a bidirectional relationship between these two important cell types in the malignant BM niche. Overall, our studies suggest that 3D BMAT represents a "healthier," more realistic tissue model that may be useful for elucidating the effects of MAT on tumor cells, and tumor cells on MAT, to identify novel therapeutic targets. In addition, proteomic characterization as well as microarray data (expression of >22,000 genes) coupled with KEGG pathway analysis and gene set expression analysis (GSEA) supported our development of less-inflammatory 3D BMAT compared to 2D culture. In sum, we developed the first 3D, tissue-engineered bone marrow adipose tissue model, which is a versatile, novel model that can be used to study numerous diseases and biological processes involved with the bone marrow.