Design considerations to ensure accuracy when using the resazurin reduction assay to noninvasively quantify cell expansion within perfused extracellular matrix scaffolds

Analysis of perfusion-based bioreactors for organ engineering and a detailed evaluation of dynamic changes within maturing cell-laden scaffolds are critical components of ex vivo tissue development that remain understudied topics in the tissue and organ engineering literature. Precise measurement of cell numbers within bioartificial tissues and extracellular matrix scaffolds is necessary to provide measurement assurance and rigorous characterization of cell behavior within three-dimensional (3D) scaffolds. Accurate benchmarking of tissue function and biosynthetic activity to cell number facilitates comparison of data across experiments and between laboratories to increase rigor and reproducibility in tissue engineering and biofabrication. Soluble, fluorescent indicators of metabolic activity are valuable, noninvasive tools for estimating viable cell number. We investigated experimental conditions in which resazurin is a reliable indicator of cell content within 3D extracellular matrix kidney and liver scaffolds, and we present recommendations on experimental methodology for its optimal use. Resazurin is reduced to resorufin in proportion to metabolic activity of viable cells. Using three renal cell lines and one hepatic cell line, we show that correlation of viable cell number with the rate of resorufin generation may deviate from linearity at higher cell density, low resazurin working volumes, and/or longer incubation times – all of which contribute to depleting the working pool of resazurin. Importantly, we also show that the resazurin reduction rate in cell-conditioned medium is about double that in fresh culture medium. This finding has the potential to increase assay sensitivity, while saving expensive media. In conclusion, while the resazurin reduction assay provides a powerful, noninvasive readout for cell growth within extracellular matrix scaffolds, assay conditions may strongly influence its applicability for accurate quantification of cell number. The approach and recommendations developed in this study to maintain the pool of reducible resazurin may be used as a guide for application-specific optimization of the resazurin reduction assay to obtain accurate measurements of cell content in bioengineered tissues

Hepatic tristetraprolin promotes insulin resistance through RNA destabilization of FGF21

The role of posttranscriptional metabolic gene regulatory programs in diabetes is not well understood. Here, we show that the RNA-binding protein tristetraprolin (TTP) is reduced in the livers of diabetic mice and humans and is transcriptionally induced in response to insulin treatment in murine livers in vitro and in vivo. Liver-specific Ttp-KO (lsTtp-KO) mice challenged with high-fat diet (HFD) have improved glucose tolerance and peripheral insulin sensitivity compared with littermate controls. Analysis of secreted hepatic factors demonstrated that fibroblast growth factor 21 (FGF21) is posttranscriptionally repressed by TTP. Consistent with increased FGF21, lsTtp-KO mice fed HFD have increased brown fat activation, peripheral tissue glucose uptake, and adiponectin production compared with littermate controls. Downregulation of hepatic Fgf21 via an adeno-associated virus-driven shRNA in mice fed HFD reverses the insulin-sensitizing effects of hepatic Ttp deletion. Thus, hepatic TTP posttranscriptionally regulates systemic insulin sensitivity in diabetes through liver-derived FGF21

Evolutionary Analyses of Staphylococcus aureus Identify Genetic Relationships between Nasal Carriage and Clinical Isolates

Nasal carriage of Staphylococcus aureus has long been hypothesized to be a major vector for the transmission of virulent strains throughout the community. To address this hypothesis, we have analyzed the relatedness between a cohort of nasal carriage strains and clinical isolates to understand better the genetic conformity therein. To assess the relatedness between nasal carriage and clinical isolates of S. aureus, a genetic association study was conducted using multilocus sequence typing (MLST) and typing of the hypervariable regions of clumping factor and fibronectin binding protein genes. At all loci analyzed, genetic associations between both nasal carriage and clinical isolates were observed. Computational analyses of MLST data indicate that nasal carriage and clinical isolates belong to the same genetic clusters (clades), despite differences in sequence type assignments. Genetic analyses of the hypervariable regions from the clumping factor and fibronectin binding protein genes revealed that not only do clinically relevant strains belong to identical genetic lineages as the nasal carriage isolates within our cohort, but they also exhibit 100% sequence similarity within these regions. The findings of this report indicate that strains of S. aureus being carried asymptomatically throughout the community via nasal colonization are genetically related to those responsible for high levels of morbidity and mortality

Self-Assemblage and Quorum in the Earthworm Eisenia fetida (Oligochaete, Lumbricidae)

Despite their ubiquity and ecological significance in temperate ecosystems, the behavioural ecology of earthworms is not well described. This study examines the mechanisms that govern aggregation behaviour specially the tendency of individuals to leave or join groups in the compost earthworm Eisenia fetida, a species with considerable economic importance, especially in waste management applications. Through behavioural assays combined with mathematical modelling, we provide the first evidence of self-assembled social structures in earthworms and describe key mechanisms involved in cluster formation. We found that the probability of an individual joining a group increased with group size, while the probability of leaving decreased. Moreover, attraction to groups located at a distance was observed, suggesting a role for volatile cues in cluster formation. The size of earthworm clusters appears to be a key factor determining the stability of the group. These findings enhance our understanding of intra-specific interactions in earthworms and have potential implications for extraction and collection of earthworms in vermicomposting processes

The Functions of Grainy Head-Like Proteins in Animals and Fungi and the Evolution of Apical Extracellular Barriers

The Grainy head (GRH) family of transcription factors are crucial for the development and repair of epidermal barriers in all animals in which they have been studied. This is a high-level functional conservation, as the known structural and enzymatic genes regulated by GRH proteins differ between species depending on the type of epidermal barrier being formed. Interestingly, members of the CP2 superfamily of transcription factors, which encompasses the GRH and LSF families in animals, are also found in fungi – organisms that lack epidermal tissues. To shed light on CP2 protein function in fungi, we characterized a Neurospora crassa mutant lacking the CP2 member we refer to as grainy head-like (grhl). We show that Neurospora GRHL has a DNA-binding specificity similar to that of animal GRH proteins and dissimilar to that of animal LSF proteins. Neurospora grhl mutants are defective in conidial-spore dispersal due to an inability to remodel the cell wall, and we show that grhl mutants and the long-known conidial separation-2 (csp-2) mutants are allelic. We then characterized the transcriptomes of both Neurospora grhl mutants and Drosophila grh mutant embryos to look for similarities in the affected genes. Neurospora grhl appears to play a role in the development and remodeling of the cell wall, as well as in the activation of genes involved in defense and virulence. Drosophila GRH is required to activate the expression of many genes involved in cuticular/epidermal-barrier formation. We also present evidence that GRH plays a role in adult antimicrobial defense. These results, along with previous studies of animal GRH proteins, suggest the fascinating possibility that the apical extracellular barriers of some animals and fungi might share an evolutionary connection, and that the formation of physical barriers in the last common ancestor was under the control of a transcriptional code that included GRH-like proteins

Music and the brain: disorders of musical listening

The study of the brain bases for normal musical listening has advanced greatly in the last 30 years. The evidence from basic and clinical neuroscience suggests that listening to music involves many cognitive components with distinct brain substrates. Using patient cases reported in the literature, we develop an approach for understanding disordered musical listening that is based on the systematic assessment of the perceptual and cognitive analysis of music and its emotional effect. This approach can be applied both to acquired and congenital deficits of musical listening, and to aberrant listening in patients with musical hallucinations. Both the bases for normal musical listening and the clinical assessment of disorders now have a solid grounding in systems neuroscience

From the Philadelphia chromosome to the cytoskeleton: The role of BCR -ABL in modulating cell adhesion and initiating leukemia

Chronic myelogenous leukemia (CML) is a biphasic neoplasm of the bone marrow that is precipitated by the Philadelphia Chromosome, a t(9;22) balanced translocation that encodes a constitutively activated nonreceptor tyrosine kinase termed P210BCR-ABL. This oncoprotein has several intracellular functions; however, the most important effect is phosphorylation of signaling molecules through a constitutively active tyrosine kinase domain. Despite extensive knowledge of the structure and functional domains of P210 BCR-ABL, its precise role in transformation is not known. This thesis utilizes two assays to better understand the functioning of P210BCR-ABL in leukemogenesis. In the first approach, we use a murine CML model in which all wild type P210BCR-ABL recipients develop a rapidly fatal myeloproliferative disease that shares many similarities with human CML. Using this model, we have shown that the N-terminal coiled-coil domain and the grb2 binding site at Tyr177 are required to induce murine CML. A second approach utilizes a quantitative cell detachment apparatus capable of measuring small changes in cell adhesion to investigate the mechanism by which P210BCR-ABL causes abnormal cell binding. We found that P210BCR-ABL leads to increased adhesion to fibronectin in a tyrosine kinase independent manner. To better understand the role vi abnormal adhesion may play in the development of leukemia, we have attempted to correlate the effects of functional P210BCR-ABL mutants in regulating adhesion and oncogenicity. We found that localization of P210BCR-ABL to F-actin, mediated through the actin binding domain or coiled-coil domain, as well as a region between as 163 to as 210 are necessary for P210 BCR-ABL-mediated increased adhesion. We compared our adhesion results with the ability of these mutants to cause the murine CML disease and found that enhanced adhesion to fibronectin did not correlate with the ability of these mutants to cause CML. Preventing the association of P210BCR-ABL with F-actin by deleting the C-terminal actin binding domain precipitates a CML-like disease that is indistinguishable from that caused by wild-type P210BCR-ABL. Together, our results suggest that F-actin localization may play a pivotal role in modulating adhesion, but that it is dispensable for CML development

From the Philadelphia chromosome to the cytoskeleton: The role of BCR -ABL in modulating cell adhesion and initiating leukemia

Chronic myelogenous leukemia (CML) is a biphasic neoplasm of the bone marrow that is precipitated by the Philadelphia Chromosome, a t(9;22) balanced translocation that encodes a constitutively activated nonreceptor tyrosine kinase termed P210BCR-ABL. This oncoprotein has several intracellular functions; however, the most important effect is phosphorylation of signaling molecules through a constitutively active tyrosine kinase domain. Despite extensive knowledge of the structure and functional domains of P210 BCR-ABL, its precise role in transformation is not known. This thesis utilizes two assays to better understand the functioning of P210BCR-ABL in leukemogenesis. In the first approach, we use a murine CML model in which all wild type P210BCR-ABL recipients develop a rapidly fatal myeloproliferative disease that shares many similarities with human CML. Using this model, we have shown that the N-terminal coiled-coil domain and the grb2 binding site at Tyr177 are required to induce murine CML. A second approach utilizes a quantitative cell detachment apparatus capable of measuring small changes in cell adhesion to investigate the mechanism by which P210BCR-ABL causes abnormal cell binding. We found that P210BCR-ABL leads to increased adhesion to fibronectin in a tyrosine kinase independent manner. To better understand the role vi abnormal adhesion may play in the development of leukemia, we have attempted to correlate the effects of functional P210BCR-ABL mutants in regulating adhesion and oncogenicity. We found that localization of P210BCR-ABL to F-actin, mediated through the actin binding domain or coiled-coil domain, as well as a region between as 163 to as 210 are necessary for P210 BCR-ABL-mediated increased adhesion. We compared our adhesion results with the ability of these mutants to cause the murine CML disease and found that enhanced adhesion to fibronectin did not correlate with the ability of these mutants to cause CML. Preventing the association of P210BCR-ABL with F-actin by deleting the C-terminal actin binding domain precipitates a CML-like disease that is indistinguishable from that caused by wild-type P210BCR-ABL. Together, our results suggest that F-actin localization may play a pivotal role in modulating adhesion, but that it is dispensable for CML development