Stabilization of Notch1 and β-catenin in response to ER- breast cancer-specific up-regulation of PSAT1 mediates distant metastasis

Breast cancer has the highest incidence in women worldwide, with a mortality rate second only to lung cancer. Distant metastasis is the major cause of breast cancer-induced death. While upregulation of phosphoserine aminotransferase 1 (PSAT1) has been reported in several cancer types, its specific roles in breast cancer and potential involvement in distant metastasis remain unclear. In our study, PSAT1 was upregulated in metastatic breast cancer and promoted distant metastasis both in vitro and in vivo. Data obtained from transwell and wound healing, colony, sphere assays and detection of various malignant phenotypic markers showed that PSAT1 mediates distant metastasis by promoting invasion, migration, proliferation, anti-apoptosis, stemness and angiogenesis in breast cancer cells. Mechanistically, PSAT1 activated Notch and β-catenin signaling pathways, leading to enhanced distant metastasis. The clinical relevance of PSAT1 in breast cancer was additionally investigated, which revealed associations of poorer patient prognosis with high PSAT1 mRNA and protein expression. In summary, PSAT1 is a critical molecular regulator of distant metastasis that may effectively serve as a marker of poor prognosis in breast cancer.     

Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice: A Model for Immunotherapy Development

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy and/or cytotoxic chemotherapy. To induce bladder cancer, C57BL/6 mice (MUC1.Tg and wild type) were treated orally with the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN) at 3.0 mg/day, 5 days/week for 12 weeks. To assess the effects of OH-BBN on serum cytokine profile during tumor development, whole blood was collected via submandibular bleeds prior to treatment and every four weeks. In addition, a MUC1-targeted peptide vaccine and placebo were administered to groups of mice weekly for eight weeks. Multiplex fluorometric microbead immunoanalyses of serum cytokines during tumor development and following vaccination were performed. At termination, interferon gamma (IFN-γ)/interleukin-4 (IL-4) ELISpot analysis for MUC1 specific T-cell immune response and histopathological evaluations of tumor type and grade were performed. The results showed that: (1) the incidence of bladder cancer in both MUC1.Tg and wild type mice was 67%; (2) transitional cell carcinomas (TCC) developed at a 2:1 ratio compared to squamous cell carcinomas (SCC); (3) inflammatory cytokines increased with time during tumor development; and (4) administration of the peptide vaccine induces a Th1-polarized serum cytokine profile and a MUC1 specific T-cell response. All tumors in MUC1.Tg mice were positive for MUC1 expression, and half of all tumors in MUC1.Tg and wild type mice were invasive. In conclusion, using a team approach through the coordination of the efforts of pharmacologists, immunologists, pathologists and molecular biologists, we have developed an immune intact transgenic mouse model of bladder cancer that expresses hMUC1.     

Stretch in Brain Microvascular Endothelial Cells (cEND) as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying mechanisms involved in it are useful for treatment. There are both in vivo and in vitro methods available for this purpose. In vivo models can mimic actual head injury as it occurs during TBI. However, in vivo techniques may not be exploited for studies at the cell physiology level. Hence, in vitro methods are more advantageous for this purpose since they provide easier access to the cells and the extracellular environment for manipulation.Our protocol presents an in vitro model of TBI using stretch injury in brain microvascular endothelial cells. It utilizes pressure applied to the cells cultured in flexible-bottomed wells. The pressure applied may easily be controlled and can produce injury that ranges from low to severe. The murine brain microvascular endothelial cells (cEND) generated in our laboratory is a well-suited model for the blood brain barrier (BBB) thus providing an advantage to other systems that employ a similar technique. In addition, due to the simplicity of the method, experimental set-ups are easily duplicated. Thus, this model can be used in studying the cellular and molecular mechanisms involved in TBI at the BBB.     

Efficient production of retinol in Yarrowia lipolytica by increasing stability using antioxidant and detergent extraction

The demand for bio-based retinol (vitamin A) is currently increasing, however its instability represents a major bottleneck in microbial production. Here, we developed an efficient method to selectively produce retinol in Yarrowia lipolytica. The β-carotene 15,15′-dioxygenase (BCO) cleaves β-carotene into retinal, which is reduced to retinol by retinol dehydrogenase (RDH). Therefore, to produce retinol, we first generated β-carotene-producing strain based on a high-lipid-producer via overexpressing genes including heterologous β-carotene biosynthetic genes, GGS1^F43I mutant of endogenous geranylgeranyl pyrophosphate synthase isolated by directed evolution, and FAD1 encoding flavin adenine dinucleotide synthetase, while deleting several genes previously known to be beneficial for carotenoid production. To produce retinol, 11 copies of BCO gene from marine bacterium 66A03 (Mb.Blh) were integrated into the rDNA sites of the β-carotene overproducer. The resulting strain produced more retinol than retinal, suggesting strong endogenous promiscuous RDH activity in Y. lipolytica. The introduction of Mb.Blh led to a considerable reduction in β-carotene level, but less than 5% of the consumed β-carotene could be detected in the form of retinal or retinol, implying severe degradation of the produced retinoids. However, addition of the antioxidant butylated hydroxytoluene (BHT) led to a >20-fold increase in retinol production, suggesting oxidative damage is the main cause of intracellular retinol degradation. Overexpression of GSH2 encoding glutathione synthetase further improved retinol production. Raman imaging revealed co-localization of retinol with lipid droplets, and extraction of retinol using Tween 80 was effective in improving retinol production. By combining BHT treatment and extraction using Tween 80, the final strain CJ2104 produced 4.86 g/L retinol and 0.26 g/L retinal in fed-batch fermentation in a 5-L bioreactor, which is the highest retinol production titer ever reported. This study demonstrates that Y. lipolytica is a suitable host for the industrial production of bio-based retinol.     

DNA repair defects ascribed to pby1 are caused by disruption of Holliday junction resolvase Mus81-Mms4

PBY1 continues to be linked with DNA repair through functional genomics studies in yeast. Using the yeast knockout (YKO) strain collection, high-throughput genetic interaction screens have identified a large set of negative interactions between PBY1 and genes involved in genome stability. In drug sensitivity screens, the YKO collection pby1Δ strain exhibits a sensitivity profile typical for genes involved in DNA replication and repair. We show that these findings are not related to loss of Pby1. On the basis of genetic interaction profile similarity, we pinpoint disruption of Holliday junction resolvase Mus81-Mms4 as the mutation responsible for DNA repair phenotypes currently ascribed to pby1. The finding that Pby1 is not a DNA repair factor reconciles discrepancies in the data available for PBY1, and indirectly supports a role for Pby1 in mRNA metabolism. Data that has been collected using the YKO collection pby1Δ strain confirms and expands the chemical-genetic interactome of MUS81-MMS4.     

Optimizing Xylanase Production by a Newly Isolated Strain CAU44 of the Thermophile Thermomyces lanuginosus

Summary Thermomyces lanuginosus CAU44, a newly isolated thermophilic fungus strain, was used for the production of extracellular xylanase on various lignocellulosic materials under shake flask conditions. High-level production of xylanase by the strain was enhanced by optimizing the type of carbon sources, substrate concentration, particle size and surfactants in the culture medium. The titre of xylanase activity obtained of up to 4156 U ml⁻¹ was the highest ever reported.     

Direct epitranscriptomic regulation of mammalian translation initiation through N4-acetylcytidine

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PGC-1α senses the CBC of pre-mRNA to dictate the fate of promoter-proximally paused RNAPII

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Phytotoxicity of Citric Acid and Tween® 80 for Potential Use as Soil Amendments in Enhanced Phytoremediation

Enhanced phytoremediation adding biodegradable amendments like low molecular weight organic acids and surfactants is an interesting area of current research to overcome the limitation that represents low bioavailability of pollutants in soils. However, prior to their use in assisted phytoremediation, it is necessary to test if amendments per se exert any toxic effect to plants and to optimize their application mode. In this context, the present study assessed the effects of citric acid and Tween® 80 (polyethylene glycol sorbitan monooleate) on the development of alfalfa (Medicago sativa) plants, as influenced by their concentration and frequency of application, in order to evaluate the feasibility for their future use in enhanced phytoremediation of multi-contaminated soils. The results showed that citric acid negatively affected plant germination, while it did not have any significant effect on biomass or chlorophyll content. In turn, Tween® 80 did not affect plant germination and showed a trend to increase biomass, as well as it did not have any significant effect on chlorophyll levels. M. sativa appeared to tolerate citric acid and Tween® 80 at the tested concentrations, applied weekly. Consequently, citric acid and Tween® 80 could potentially be utilized to assist phytoremediation of contaminated soils vegetated with M. sativa.