Maternal dietary creatine supplementation does not alter the capacity for creatine synthesis in the newborn spiny mouse

We have previously reported that maternal creatine supplementation protects the neonate from hypoxic injury. Here, we investigated whether maternal creatine supplementation altered expression of the creatine synthesis enzymes (arginine:glycine amidinotransferase [AGAT], guanidinoaceteate methyltransferase [GAMT]) and the creatine transporter (solute carrier family 6 [neurotransmitter transporter, creatine] member 8: SLC6A8) in the term offspring. Pregnant spiny mice were fed a 5% creatine monohydrate diet from midgestation (day 20) to term (39 days). Placentas and neonatal kidney, liver, heart, and brain collected at 24 hours of age underwent quantitative polymerase chain reaction and Western blot analysis. Maternal creatine had no effect on the expression of AGAT and GAMT in neonatal kidney and liver, but mRNA expression of AGAT in brain tissues was significantly decreased in both male and female neonates born to mothers who were fed the creatine diet. SLC6A8 expression was not affected by maternal dietary creatine loading in any tissues. Maternal dietary creatine supplementation from midgestation in the spiny mouse did not alter the capacity for creatine synthesis or transport.

Addition of magnesium chloride to enhance mono-dispersity of a coiled-coil recombinant mouse macrophage protein

X-ray crystallography for the determination of three-dimensional structures of protein macromolecules represents an important tool in function assignment of uncharacterized proteins. However, crystallisation is often difficult to achieve. A protein sample fully characterized in terms of dispersity may increase the likelihood of successful crystallisation by improving the predictability of the crystallisation process. To maximize the probability of crystallisation of a novel mouse macrophage protein (rMMP), target molecule was characterized and refined to improve monodispersity. Addition of MgCl2 at low concentrations resolves the rMMP into a monodisperse solution, and finally successful crystallization of rMMP was achieved. The effect of MgCl2 was studied using gel filtration chromatography and dynamic light scattering.

Effect of Direct and Indirect Cues of Predation Risk on the Foraging Behavior of the White-Footed Mouse (Peromyscus leucopus)

Understanding predator-prey dynamics requires an understanding of how prey assess predation risk. This study tested the effect of microhabitat, moon stages, and mammalian predator urines (Vulpes vulpes [Red Fox], Mustela vison [Mink], and Procyon lotor [Raccoon]) on the degree of predation risk perceived by Peromyscus leucopus (White-footed Mouse). Giving-up densities from artificial food patches were used to quantify perceived predation risk. White-footed Mice exhibited a strong preference for cover microhabitat and for the new moon stage. However, the mice did not significantly alter their foraging behavior in response to the predator urines compared to a water control. Additionally, mice foraged less on colder nights. The results suggest that mammalian predator urines may not provide reliable information on actual predation risk for the White-footed Mice and that the mice extensively use indirect cues to assess predation risk.

Tissue specific roles for the Ribosome Biogenesis Factor Wdr43 in zebrafish development

During vertebrate craniofacial development, neural crest cells (NCCs) contribute to most of the craniofacial pharyngeal skeleton. Defects in NCC specification, migration and differentiation resulting in malformations in the craniofacial complex are associated with human craniofacial disorders including Treacher-Collins Syndrome, caused by mutations in TCOF1. It has been hypothesized that perturbed ribosome biogenesis and resulting p53 mediated neuroepithelial apoptosis results in NCC hypoplasia in mouse Tcof1 mutants. However, the underlying mechanisms linking ribosome biogenesis and NCC development remain poorly understood. Here we report a new zebrafish mutant, fantome (fan), which harbors a point mutation and predicted premature stop codon in zebrafish wdr43, the ortholog to yeast UTP5. Although wdr43 mRNA is widely expressed during early zebrafish development, and its deficiency triggers early neural, eye, heart and pharyngeal arch defects, later defects appear fairly restricted to NCC derived craniofacial cartilages. Here we show that the C-terminus of Wdr43, which is absent in fan mutant protein, is both necessary and sufficient to mediate its nucleolar localization and protein interactions in metazoans. We demonstrate that Wdr43 functions in ribosome biogenesis, and that defects observed in fan mutants are mediated by a p53 dependent pathway. Finally, we show that proper localization of a variety of nucleolar proteins, including TCOF1, is dependent on that of WDR43. Together, our findings provide new insight into roles for Wdr43 in development, ribosome biogenesis, and also ribosomopathy-induced craniofacial phenotypes including Treacher-Collins Syndrome.

Endothelial progenitor cells control the angiogenic switch in mouse lung metastasis

Angiogenesis-mediated progression of micrometastasis to lethal macrometastasis is the major cause of death in cancer patients. Here, using mouse models of pulmonary metastasis, we identify bone marrow (BM)–derived endothelial progenitor cells (EPCs) as critical regulators of this angiogenic switch. We show that tumors induce expression of the transcription factor Id1 in the EPCs and that suppression of Id1 after metastatic colonization blocked EPC mobilization, caused angiogenesis inhibition, impaired pulmonary macrometastases, and increased survival of tumor-bearing animals. These findings establish the role of EPCs in metastatic progression in preclinical models and suggest that selective targeting of EPCs may merit investigation as a therapy for cancer patients with lung metastases.

The release and induced immune responses of a plant-made and delivered antigen in the mouse gut

This study investigated the site of release of a model vaccine antigen from plant cells and the corresponding induced immune response. Three plant tissues (leaf, fruit and hairy root) and two formulations (aqueous and lipid) were compared in two mouse trials. A developed technique that enabled detection of antigen release by plant cells determined that antigen release occurred at early sites of the gastrointestinal tract when delivered in leaf material and at later sites when delivered in hairy roots. Lipid formulations delayed antigen release from all plant materials tested. While encapsulation in the plant cell provided some protection of the antigen in the gastrointestinal tract and influenced antigen release, formulation medium was also an important consideration with regard to vaccine delivery and immunogenicity. Systemic immune responses induced from the orally delivered vaccine benefited from late release of antigen in the mouse gastrointestinal tract. The influences to the mucosal immune response induced by these vaccines were too complex to be determined by studies performed here with no clear trend regarding plant tissue site of release or formulation medium. Expression and delivery of the model antigen in plant material prepared in an aqueous formulation provided the optimal systemic and mucosal, antigen-specific immune responses.

SurR9-C84A mutant nanocarrier and milk protein therapeutics for neurological disorders

 The current project revealed that the novel nanoformulation of SurR9-C84A was able to rescue the neurons following the Alzheimer’s related ß-amyloid toxicity and inflammation. In addition, bovine lactoferrin was found to have potential differentiating effect in the tumor cells and hence is a valuable therapeutic for strengthening the degenerating neurons.

Response of BRAF-mutant melanoma to BRAF inhibition is mediated by a network of transcriptional regulators of glycolysis.

Deregulated glucose metabolism fulfills the energetic and biosynthetic requirements for tumor growth driven by oncogenes. Because inhibition of oncogenic BRAF causes profound reductions in glucose uptake and a strong clinical benefit in BRAF-mutant melanoma, we examined the role of energy metabolism in responses to BRAF inhibition. We observed pronounced and consistent decreases in glycolytic activity in BRAF-mutant melanoma cells. Moreover, we identified a network of BRAF-regulated transcription factors that control glycolysis in melanoma cells. Remarkably, this network of transcription factors, including hypoxia-inducible factor-1α, MYC, and MONDOA (MLXIP), drives glycolysis downstream of BRAF(V600), is critical for responses to BRAF inhibition, and is modulated by BRAF inhibition in clinical melanoma specimens. Furthermore, we show that concurrent inhibition of BRAF and glycolysis induces cell death in BRAF inhibitor (BRAFi)-resistant melanoma cells. Thus, we provide a proof-of-principle for treatment of melanoma with combinations of BRAFis and glycolysis inhibitors.

EPO-receptor is present in mouse C2C12 and human primary skeletal muscle cells but EPO does not influence myogenesis.

Abstract The role and regulation of the pleiotropic cytokine erythropoietin (EPO) in skeletal muscle are controversial. EPO exerts its effects by binding its specific receptor (EPO-R), which activates intracellular signaling and gene transcription in response to internal and external stress signals. EPO is suggested to play a direct role in myogenesis via the EPO-R, but several studies have questioned the effect of EPO treatment in muscle in vitro and in vivo. The lack of certainty surrounding the use of nonspecific EPO-R antibodies contributes to the ambiguity of the field. Our study demonstrates that the EPO-R gene and protein are expressed at each stage of mouse C2C12 and human skeletal muscle cell proliferation and differentiation and validates a specific antibody for the detection of the EPO-R protein. However, in our experimental conditions, EPO treatment had no effect on mouse C2C12 and human muscle cell proliferation, differentiation, protein synthesis or EPO-R expression. While an increase in Akt and MAPK phosphorylation was observed, we demonstrate that this effect resulted from the stress caused by changing medium and not from EPO treatment. We therefore suggest that skeletal muscle EPO-R might be present in a nonfunctional form, or too lowly expressed to play a role in muscle cell function.

Nanoformulated cell-penetrating survivin mutant and its dual actions

In this study, we investigated the differential actions of a dominant-negative survivin mutant (SurR9-C84A) against cancerous SK-N-SH neuroblastoma cell lines and differentiated SK-N-SH neurons. In both the cases, the mutant protein displayed dual actions, where its effects were cytotoxic toward cancerous cells and proliferative toward the differentiated neurons. This can be explained by the fact that tumorous (undifferentiated SK-N-SH) cells have a high endogenous survivin pool and upon treatment with mutant SuR9-C84A causes forceful survivin expression. These events significantly lowered the microtubule dynamics and stability, eventually leading to apoptosis. In the case of differentiated SK-N-SH neurons that express negligible levels of wild-type survivin, the mutant indistinguishably behaved in a wild-type fashion. It also favored cell-cycle progression, forming the chromosome-passenger complex, and stabilized the microtubule-organizing center. Therefore, mutant SurR9-C84A represents a novel therapeutic with its dual actions (cytotoxic toward tumor cells and protective and proliferative toward neuronal cells), and hence finds potential applications against a variety of neurological disorders. In this study, we also developed a novel poly(lactic-co-glycolic acid) nanoparticulate formulation to surmount the hurdles associated with the delivery of SurR9-C84A, thus enhancing its effective therapeutic outcome.

Comparative analysis of caveolins in mouse and tammar wallaby: role in regulating mammary gland function.

Recent studies using the mouse showed an inverse correlation between the Caveolin 1 gene expression and lactation, and this was regulated by prolactin. However, current study using mammary explants from pregnant mice showed that while insulin (I), cortisol (F) and prolactin (P) resulted in maximum induction of the β-casein gene, FP and IFP resulted in the downregulation of Caveolin 1. Additionally, IF, FP and IFP resulted in the downregulation of Caveolin 2. Immunohistochemistry confirmed localisation of Caveolin 1 specific to myoepithelial cells and adipocytes. Comparative studies with the tammar wallaby showed Caveolin 1 and 2 had 70-80% homology with the mouse proteins. However, in contrast to the mouse, Caveolin 1 and 2 genes showed a significantly increased level of expression in the mammary gland during lactation. The regulation of tammar Caveolin 1 and 2 gene expression was examined in mammary explants from pregnant tammars, and no significant difference was observed either in the absence or in the presence of IFP.

Short-chain fatty acids increase expression and secretion of stromal cell-derived factor-1 in mouse and human pre-adipocytes

Objective: Stromal cell-derived factor-1 (SDF-1) is expressed in pre-adipocytes but its role is unknown. We investigated butyrate (a histone deacetylase inhibitor - HDACi) and other short-chain fatty acids (SCFA) in the regulation of SDF-1. We further investigated whether effects of SCFA were signalled through G protein-coupled receptors FFA2 and FFA3. Design and Results: SDF-1 mRNA expression and protein secretion were studied in 3T3-L1 cells and human pre-adipocytes. SDF-1 was abundant, with mRNA and protein levels increased by butyrate. This was replicated with acetate and propionate, but not with trichostatin or valproate. Trichostatin inhibited SDF-1 secretion. Pertussis toxin blocked stimulation by butyrate. The order of potency of SCFA in stimulating SDF-1 (C3 > C4 > C2) is consistent with action through FFA3. Silencing the FFA3 gene abolished butyrate-stimulated SDF-1 expression and secretion. FFA3 was expressed in both pre-adipocytes and adipocytes, while FFA2 was expressed in adipocytes only. SDF-1 expression was low in murine macrophage J774.2 cells, while the SDF-1 receptor CXCR4 was absent from 3T3-L1 cells but abundant in J774.2 macrophages. In human pre-adipocytes, FFA3 was also expressed and SCFA increased SDF-1 secretion. Conclusions: SDF-1 and CXCR4 may mediate the interaction between adipose stromal cells and macrophages. Effects of SCFA are mediated through FFA3, but not histone deacetylase inhibition.