Whole-body galactose oxidation as a robust functional assay to assess the efficacy of gene-based therapies in a mouse model of Galactosemia.

Despite the implementation of lifesaving newborn screening programs and a galactose-restricted diet, many patients with classic galactosemia develop long-term debilitating neurological deficits and primary ovarian insufficiency. Previously, we showed that the administration of human GALT mRNA predominantly expressed in the GalT gene-trapped mouse liver augmented the expression of hepatic GALT activity, which decreased not only galactose-1 phosphate (gal-1P) in the liver but also peripheral tissues. Since each peripheral tissue requires distinct methods to examine the biomarker and/or GALT effect, this highlights the necessity for alternative strategies to evaluate the overall impact of therapies. In this study, we established that whole-body galactose oxidation (WBGO) as a robust, noninvasive, and specific method to assess the in vivo pharmacokinetic and pharmacodynamic parameters of two experimental gene-based therapies that aimed to restore GALT activity in a mouse model of galactosemia. Although our results illustrated the long-lasting efficacy of AAVrh10-mediated GALT gene transfer, we found that GALT mRNA therapy that targets the liver predominantly is sufficient to sustain WBGO. The latter could have important implications in the design of novel targeted therapy to ensure optimal efficacy and safety.

Integrated stress response control of granulosa cell translation and proliferation during normal ovarian follicle development.

Mechanisms that directly control mammalian ovarian primordial follicle (PF) growth activation and the selection of individual follicles for survival are largely unknown. Follicle cells produce factors that can act as potent inducers of cellular stress during normal function. Consistent with this, we show here that normal, untreated ovarian cells, including pre-granulosa cells of dormant PFs, express phenotype and protein markers of the activated integrated stress response (ISR), including stress-specific protein translation (phospho-Serine 51 eukaryotic initiation factor 2α; P-EIF2α), active DNA damage checkpoints, and cell-cycle arrest. We further demonstrate that mRNAs upregulated in primary (growing) follicles versus arrested PFs mostly include stress-responsive upstream open reading frames (uORFs). Treatment of a granulosa cell (GC) line with the PF growth trigger tumor necrosis factor alpha results in the upregulation of a 'stress-dependent' translation profile. This includes further elevated P-eIF2α and a shift of uORF-containing mRNAs to polysomes. Because the active ISR corresponds to slow follicle growth and PF arrest, we propose that repair and abrogation of ISR checkpoints (e.g. checkpoint recovery) drives the GC cell cycle and PF growth activation (PFGA). If cellular stress is elevated beyond a threshold(s) or, if damage occurs that cannot be repaired, cell and follicle death ensue, consistent with physiological atresia. These data suggest an intrinsic quality control mechanism for immature and growing follicles, where PFGA and subsequent follicle growth and survival depend causally upon ISR resolution, including DNA repair and thus the proof of genomic integrity.

Discovery of Novel Inhibitors Targeting Multi-UDP-hexose Pyrophosphorylases as Anticancer Agents.

To minimize treatment toxicities, recent anti-cancer research efforts have switched from broad-based chemotherapy to targeted therapy, and emerging data show that altered cellular metabolism in cancerous cells can be exploited as new venues for targeted intervention. In this study, we focused on, among the altered metabolic processes in cancerous cells, altered glycosylation due to its documented roles in cancer tumorigenesis, metastasis and drug resistance. We hypothesize that the enzymes required for the biosynthesis of UDP-hexoses, glycosyl donors for glycan synthesis, could serve as therapeutic targets for cancers. Through structure-based virtual screening and kinetic assay, we identified a drug-like chemical fragment, GAL-012, that inhibit a small family of UDP-hexose pyrophosphorylases-galactose pyro-phosphorylase (GALT), UDP-glucose pyrophosphorylase (UGP2) and UDP-N-acetylglucosamine pyrophosphorylase (AGX1/UAP1) with an IC50 of 30 µM. The computational docking studies supported the interaction of GAL-012 to the binding sites of GALT at Trp190 and Ser192, UGP2 at Gly116 and Lys127, and AGX1/UAP1 at Asn327 and Lys407, respectively. One of GAL-012 derivatives GAL-012-2 also demonstrated the inhibitory activity against GALT and UGP2. Moreover, we showed that GAL-012 suppressed the growth of PC3 cells in a dose-dependent manner with an EC50 of 75 µM with no effects on normal skin fibroblasts at 200 µM. Western blot analysis revealed reduced expression of pAKT (Ser473), pAKT (Thr308) by 77% and 72%, respectively in the treated cells. siRNA experiments against the respective genes encoding the pyrophosphorylases were also performed and the results further validated the proposed roles in cancer growth inhibition. Finally, synergistic relationships between GAL-012 and tunicamycin, as well as bortezomib (BTZ) in killing cultured cancer cells were observed, respectively. With its unique scaffold and relatively small size, GAL-012 serves as a promising early chemotype for optimization to become a safe, effective, multi-target anti-cancer drug candidate which could be used alone or in combination with known therapeutics.

Prevalence of epithelial abnormalities and high-risk human papilloma virus in cervicovaginal Pap smears of population subgroups as a guide toward evidence-based best practice.

BACKGROUND: The 2012 American Society for Colposcopy and Cervical Pathology Consensus Guidelines provide information for managing abnormal cervical cancer screening tests and cancer precursors. According to these guidelines for Pap smear diagnosis of Atypical squamous cells of undetermined significance, reflex high risk (HR) human papilloma virus (HPV) genotyping is required among women 21 years of age or older. Whereas, in women of 30 to 65 years of age, HR-HPV can be ordered by the clinicians as part of co-testing with any diagnosis and every 5 years with a negative Cervico-Vaginal Pap test (CVPT). METHODS: A retrospective review of the CoPath database of the Pathology Department at the University of Florida, College of Medicine Jacksonville, FL, was performed to identify North Florida (NF) women who underwent CVPT and HR-HPV testing between 2006 and 2014. The women were stratified by race and age, respectively. RESULTS: The study included 19,933 CVPTs. Significant differences in the outcomes' distributions were found among age and race groups, respectively. Highest prevalence of HPV positivity was found in African American women, and in 14- to 20-year-old women, respectively. Twenty- to 30-year-old women had the highest percentage (59%) of epithelial abnormality. The most common HR-HPV genotypic distribution was other HR-HPV. CONCLUSIONS: This study underscores the importance of using both HR-HPV and CVPT for screening for cervical cancer, and confirms the need for special focus on managing high-risk populations subgroups, such as African American women, and women of ages 14 to 20 years especially in high-risk populations.

The Leloir Pathway of Galactose Metabolism - A Novel Therapeutic Target for Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the most lethal types of cancer worldwide, with poor prognosis and limited treatments. In order to identify novel therapeutic targets that will lead to development of effective therapies with manageable side effects, we tested the hypothesis that knocking-down galactokinase (GALK1) or galactose-1 phosphate uridylyltransferase (GALT) gene expression would control the growth of cultured hepatoma cells. Our results showed small interfering RNA (siRNA) against GALK1 or GALT inhibited the growth of HepG2 cells in culture. Western blot analysis revealed simultaneous down-regulation of multiple players of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) growth signaling pathway, as well as heat-shock protein 90 (HSP90) and poly ADP ribose polymerase (PARP). Reverse transcription-polymerase chain reaction (RT-PCR) data, however, showed no significant mRNA reduction of the encoded genes. Our study thus not only supports GALK1 and GALT as being possible novel targets for treating HCC, but also uncovers new post-transcriptional regulatory mechanisms that link the galactose metabolic pathway to protein expression of the PI3K/AKT pathway in hepatoma.

Galactose-1 phosphate uridylyltransferase (GalT) gene: A novel positive regulator of the PI3K/Akt signaling pathway in mouse fibroblasts.

The vital importance of the Leloir pathway of galactose metabolism has been repeatedly demonstrated by various uni-/multicellular model organisms, as well human patients who have inherited deficiencies of the key GAL enzymes. Yet, other than the obvious links to the glycolytic pathway and glycan biosynthetic pathways, little is known about how this metabolic pathway interacts with the rest of the metabolic and signaling networks. In this study, we compared the growth and the expression levels of the key components of the PI3K/Akt growth signaling pathway in primary fibroblasts derived from normal and galactose-1 phosphate uridylyltransferase (GalT)-deficient mice, the latter exhibited a subfertility phenotype in adult females and growth restriction in both sexes. The growth potential and the protein levels of the pAkt(Thr308), pAkt(Ser473), pan-Akt, pPdk1, and Hsp90 proteins were significantly reduced by 62.5%, 60.3%, 66%, 66%, and 50%, respectively in the GalT-deficient cells. Reduced expression of phosphorylated Akt proteins in the mutant cells led to diminished phosphorylation of Gsk-3ß (-74%). Protein expression of BiP and pPten were 276% and 176% higher respectively in cells with GalT-deficiency. Of the 24 genes interrogated using QIAGEN RT(2) Profiler PCR Custom Arrays, the mRNA abundance of Akt1, Pdpk1, Hsp90aa1 and Pi3kca genes were significantly reduced at least 2.03-, 1.37-, 2.45-, and 1.78-fold respectively in mutant fibroblasts. Both serum-fasted normal and GalT-deficient cells responded to Igf-1-induced activation of Akt phosphorylation at +15 min, but the mutant cells have lower phosphorylation levels. The steady-state protein abundance of Igf-1 receptor was also significantly reduced in mutant cells. Our results thus demonstrated that GalT deficiency can effect down-regulation of the PI3K/Akt growth signaling pathway in mouse fibroblasts through distinct mechanisms targeting both gene and protein expression levels.

Subfertility and growth restriction in a new galactose-1 phosphate uridylyltransferase (GALT) - deficient mouse model.

The first GalT gene knockout (KO) mouse model for Classic Galactosemia (OMIM 230400) accumulated some galactose and its metabolites upon galactose challenge, but was seemingly fertile and symptom free. Here we constructed a new GalT gene-trapped mouse model by injecting GalT gene-trapped mouse embryonic stem cells into blastocysts, which were later implanted into pseudo-pregnant females. High percentage GalT gene-trapped chimera obtained were used to generate heterozygous and subsequently, homozygous GalT gene-trapped mice. Biochemical assays confirmed total absence of galactose-1 phosphate uridylyltransferase (GALT) activity in the homozygotes. Although the homozygous GalT gene-trapped females could conceive and give birth when fed with normal chow, they had smaller litter size (P=0.02) and longer time-to-pregnancy (P=0.013) than their wild-type littermates. Follicle-stimulating hormone levels of the mutant female mice were not significantly different from the age-matched, wild-type females, but histological examination of the ovaries revealed fewer follicles in the homozygous mutants (P=0.007). Administration of a high-galactose (40% w/w) diet to lactating homozygous GalT gene-trapped females led to lethality in over 70% of the homozygous GalT gene-trapped pups before weaning. Cerebral edema, abnormal changes in the Purkinje and the outer granular cell layers of the cerebellum, as well as lower blood GSH/GSSG ratio were identified in the galactose-intoxicated pups. Finally, reduced growth was observed in GalT gene-trapped pups fed with normal chow and all pups fed with high-galactose (20% w/w) diet. This new mouse model presents several of the complications of Classic Galactosemia and will be useful to investigate pathogenesis and new therapies.

Use of hyperspectral imaging to distinguish normal, precancerous, and cancerous cells.

BACKGROUND: The objective of the current study was to test the hypothesis that the cytologic diagnosis of cancer cells can be enhanced by the technique of hyperspectral imaging (HSI). METHODS: As a proof of principle, HSI was employed to obtain hyperspectrum from a normal human fibroblast, as well as its telomerase-immortalized and SV40-transformed derivatives. Novel algorithms were developed to differentiate among these cell models based on spectral and spatial differences. Using the same technique with modified algorithms, the authors were able to differentiate among normal and precancerous (low-grade [LG] and high-grade [HG]) cervical cells and squamous cell carcinoma (SCC) on liquid-based Papanicolaou (Pap) test slides. RESULTS: The specificity for identifying normal fibroblast cell type based on spatial and spectral algorithms was 74.2%. The sensitivity for identifying telomerase-immortalized and SV40-transformed cells was 100% and 90.3%, respectively. The system identified normal cervical cells with a specificity of 95.8%. With regard to LG precancerous cells and HG precancerous cells, the sensitivity was 66.7% and 93.5%, respectively. The sensitivity detected for SCC was 98.6%. CONCLUSIONS: HSI can be utilized in prescreening liquid-based Pap test slides to improve efficiency in Pap test diagnoses with the goal of ultimately reducing the mortality from cervical cancer while reducing health care costs.

Alternative pathways of galactose assimilation: could inverse metabolic engineering provide an alternative to galactosemic patients?

The galactose assimilation pathway has been extensively studied as an example of a genetic regulatory switch. Besides the importance of this pathway as a tool in basic biological research, unraveling its structure and regulation is also of major medical importance. Impairment of galactose assimilation is the cause of the genetic metabolic disease known as "galactosemia", while the in vivo activity of the pathway affects the production of glycans. The latter have been connected to tumor metastasis, anti-cancer drug resistance and various cardiovascular diseases. Despite the vast amount of studies, however, galactose assimilation and its interaction with other parts of the metabolic network have not been fully elucidated yet. In yeast and higher eukaryotes, it is still being studied as comprising only the linear Leloir pathway. Recent observations, however, indicate that alternative pathways of galactose assimilation identified in prokaryotes and fungi might also be present in yeast. Such a result is valuable per se, because it could lead to the discovery of these pathways in humans. Even more importantly, these pathways provide alternative phenotypes with known genetic fingerprints that can be used in the context of classical and inverse metabolic engineering to examine and treat the mechanisms of defects of galactose assimilation.