AccuStart II Mouse Genotyping Kit

Fzd4 Haploinsufficiency Delays Retinal Revascularization in the Mouse Model of Oxygen Induced Retinopathy

11, PLOS ONE – 2016

ABSTRACT
Mutations in genes that code for components of the Norrin-FZD4 ligand-receptor complex cause the inherited childhood blinding disorder familial exudative vitreoretinopathy (FEVR). Statistical evidence from studies of patients at risk for the acquired disease retinopathy of prematurity (ROP) suggest that rare polymorphisms in these same genes increase the risk of developing severe ROP, implying that decreased Norrin-FZD4 activity predisposes patients to more severe ROP. To test this hypothesis, we measured the development and recovery of retinopathy in wild type and Fzd4 heterozygous mice in the absence or presence of ocular ischemic retinopathy (OIR) treatment. Avascular and total retinal vascular areas and patterning were determined, and vessel number and caliber were quantified. In room air, there was a small delay in retinal vascularization in Fzd4 heterozygous mice that resolved as mice reached maturity suggestive of a slight defect in retinal vascular development. Subsequent to OIR treatment there was no difference between wild type and Fzd4 heterozygous mice in the vaso-obliterated area following exposure to high oxygen. Importantly, after return of Fzd4 heterozygous mice to room air subsequent to OIR treatment, there was a substantial delay in retinal revascularization of the avascular area surrounding the optic nerve, as well as delayed vascularization toward the periphery of the retina. Our study demonstrates that a small decrease in Norrin-Fzd4 dependent retinal vascular development lengthens the period during which complications from OIR could occur.

A novel mouse model for the identification of thioredoxin-1 protein interactions

Michelle L. Booze, Free Radical Biology and Medicine – 2016

ABSTRACT
Thiol switches are important regulators of cellular signaling and are coordinated by several redox enzyme systems including thioredoxins, peroxiredoxins, and glutathione. Thioredoxin-1 (Trx1), in particular, is an important signaling molecule not only in response to redox perturbations, but also in cellular growth, regulation of gene expression, and apoptosis. The active site of this enzyme is a highly conserved C-G-P-C motif and the redox mechanism of Trx1 is rapid which presents a challenge in determining specific substrates. Numerous in vitro approaches have identified Trx1-dependent thiol switches; however, these findings may not be physiologically relevant and little is known about Trx1 interactions in vivo. In order to identify Trx1 targets in vivo, we generated a transgenic mouse with inducible expression of a mutant Trx1 transgene to stabilize intermolecular disulfides with protein substrates. Expression of the Trx1 “substrate trap” transgene did not interfere with endogenous thioredoxin or glutathione systems in brain, heart, lung, liver, and kidney. Following immunoprecipitation and proteomic analysis, we identified 41 homeostatic Trx1 interactions in perinatal lung, including previously described Trx1 substrates such as members of the peroxiredoxin family and collapsin response mediator protein 2. Using perinatal hyperoxia as a model of oxidative injury, we found 17 oxygen-induced interactions which included several cytoskeletal proteins which may be important to alveolar development. The data herein validates this novel mouse model for identification of tissue- and cell-specific Trx1-dependent pathways that regulate physiological signals in response to redox perturbations.

Intensified vmPFC surveillance over PTSS under perturbed microRNA-608/AChE interaction

T. Lin, Translational Psychiatry – 2016

ABSTRACT
Trauma causes variable risk of posttraumatic stress symptoms (PTSS) owing to yet-unknown genome–neuronal interactions. Here, we report co-intensified amygdala and ventromedial prefrontal cortex (vmPFC) emotional responses that may overcome PTSS in individuals with the single-nucleotide polymorphism (SNP) rs17228616 in the acetylcholinesterase (AChE) gene. We have recently shown that in individuals with the minor rs17228616 allele, this SNP interrupts AChE suppression by microRNA (miRNA)-608, leading to cortical elevation of brain AChE and reduced cortisol and the miRNA-608 target GABAergic modulator CDC42, all stress-associated. To examine whether this SNP has effects on PTSS and threat-related brain circuits, we exposed 76 healthy Israel Defense Forces soldiers who experienced chronic military stress to a functional magnetic resonance imaging task of emotional and neutral visual stimuli. Minor allele individuals predictably reacted to emotional stimuli by hyperactivated amygdala, a hallmark of PTSS and a predisposing factor of posttraumatic stress disorder (PTSD). Despite this, minor allele individuals showed no difference in PTSS levels. Mediation analyses indicated that the potentiated amygdala reactivity in minor allele soldiers promoted enhanced vmPFC recruitment that was associated with their limited PTSS. Furthermore, we found interrelated expression levels of several miRNA-608 targets including CD44, CDC42 and interleukin 6 in human amygdala samples (N=7). Our findings suggest that miRNA-608/AChE interaction is involved in the threat circuitry and PTSS and support a model where greater vmPFC regulatory activity compensates for amygdala hyperactivation in minor allele individuals to neutralize their PTSS susceptibility.

Core Binding Factor-β Knockdown Alters Ovarian Gene Expression and Function in the Mouse – me.2015-1312

Kalin Wilson, Original Research – 2016

ABSTRACT
Core binding factor (CBF) is a heterodimeric transcription factor complex composed of a DNA binding subunit, one of three runt-related transcription factor (RUNX) factors, and a non-DNA binding subunit, CBF. CBF is critical for DNA binding and stability of the CBF transcription factor complex. In the ovary, the LH surge increases the expression of Runx1 and Runx2 in periovulatory follicles, implicating a role for CBFs in the periovulatory process. The present study investigated the functional significance of CBFs (RUNX1/CBF and RUNX2/CBF ) in the ovary by examining the ovarian phenotype of granulosa cell-specific CBF knockdown mice; CBF f/f * Cyp19 cre. The mutant female mice exhibited significant reductions in fertility, with smaller litter sizes, decreased progesterone during gestation, and fewer cumulus oocyte complexes collected after an induced superovulation. RNA sequencing and transcriptome assembly revealed altered expression of more than 200 mRNA transcripts in the granulosa cells of Cbfb knockdown mice after human chorionic gonadotropin stimulation in vitro. Among the affected transcripts are known regulators of ovulation and luteinization including Sfrp4,Sgk1,Lhcgr,Prlr,Wnt4, and Edn2 as well as many genes not yet characterized in the ovary. Cbf knockdown mice also exhibited decreased expression of key genes within the corpora lutea and morphological changes in the ovarian structure, including the presence of large antral follicles well into the luteal phase. Overall, these data suggest a rolefor CBFs as significant regulators of gene expression, ovulatory processes, and luteal development in the ovary.(Molecular Endocrinology 30: 733–747, 2016)

The Adhesion G-Protein-Coupled Receptor, GPR56/ADGRG1, Inhibits Cell–Extracellular Matrix Signaling to Prevent Metastatic Melanoma Growth

Michelle W. Millar, Frontiers in Oncology – 2018

ABSTRACT
Metastatic growth is considered a rate-limiting step in cancer progression, and upregulation of extracellular matrix (ECM) deposition and cell–ECM signaling are major drivers of this process. Mechanisms to reverse ECM upregulation in cancer could potentially facilitate its prevention and treatment but they are poorly understood. We previously reported that the adhesion G-protein-coupled receptor GPR56/ADGRG1 is downregulated in melanoma metastases. Its re-expression inhibited melanoma growth and metastasis and reduced the deposition of fibronectin, a major ECM component. We hypothesize that its effect on fibronectin deposition contributes to its inhibitory role on metastatic growth. To test this, we investigated the function of GPR56 on cell–fibronectin adhesion and its relationship with metastatic growth in melanoma. Our results reveal that GPR56 inhibits melanoma metastatic growth by impeding the expansion of micrometastases to macrometastases. Meanwhile, we present evidence that GPR56 inhibits fibronectin deposition and its downstream signaling, such as phosphorylation of focal adhesion kinase (FAK), during this process. Administration of the FAK inhibitor Y15 perturbed the proliferation of melanoma metastases, supporting a causative link between the cell adhesion defect induced by GPR56 and its inhibition of metastatic growth. Taken together, our results suggest that GPR56 in melanoma metastases inhibits ECM accumulation and adhesion, which contributes to its negative effects on metastatic growth.

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