Methionine Adenosyltransferase 1a (MAT1A) Enhances Cell Survival During Chemotherapy Treatment and is Associated with Drug Resistance in Bladder Cancer PDX Mice.

Bladder cancer is among the top ten most common cancers, with about ~380,000 new cases and ~150,000 deaths per year worldwide. Tumor relapse following chemotherapy treatment has long been a significant challenge towards completely curing cancer. We have utilized a patient-derived bladder cancer xenograft (PDX) platform to characterize molecular mechanisms that contribute to relapse following drug treatment in advanced bladder cancer. Transcriptomic profiling of bladder cancer xenograft tumors by RNA-sequencing analysis, before and after relapse, following a 21-day cisplatin/gemcitabine drug treatment regimen identified methionine adenosyltransferase 1a (MAT1A) as one of the significantly upregulated genes following drug treatment. Survey of patient tumor sections confirmed elevated levels of MAT1A in individuals who received chemotherapy. Overexpression of MAT1A in 5637 bladder cancer cells increased tolerance to gemcitabine and stalled cell proliferation rates, suggesting MAT1A upregulation as a potential mechanism by which bladder cancer cells persist in a quiescent state to evade chemotherapy

Global Gene Expression Analysis Identifies Age-Related Differences in Knee Joint Transcriptome during the Development of Post-Traumatic Osteoarthritis in Mice.

Aging and injury are two major risk factors for osteoarthritis (OA). Yet, very little is known about how aging and injury interact and contribute to OA pathogenesis. In the present study, we examined age- and injury-related molecular changes in mouse knee joints that could contribute to OA. Using RNA-seq, first we profiled the knee joint transcriptome of 10-week-old, 62-week-old, and 95-week-old mice and found that the expression of several inflammatory-response related genes increased as a result of aging, whereas the expression of several genes involved in cartilage metabolism decreased with age. To determine how aging impacts post-traumatic arthritis (PTOA) development, the right knee joints of 10-week-old and 62-week-old mice were injured using a non-invasive tibial compression injury model and injury-induced structural and molecular changes were assessed. At six-week post-injury, 62-week-old mice displayed significantly more cartilage degeneration and osteophyte formation compared with young mice. Although both age groups elicited similar transcriptional responses to injury, 62-week-old mice had higher activation of inflammatory cytokines than 10-week-old mice, whereas cartilage/bone metabolism genes had higher expression in 10-week-old mice, suggesting that the differential expression of these genes might contribute to the differences in PTOA severity observed between these age groups

SOST Inhibits Prostate Cancer Invasion.

Inhibitors of Wnt signaling have been shown to be involved in prostate cancer (PC) metastasis; however the role of Sclerostin (Sost) has not yet been explored. Here we show that elevated Wnt signaling derived from Sost deficient osteoblasts promotes PC invasion, while rhSOST has an inhibitory effect. In contrast, rhDKK1 promotes PC elongation and filopodia formation, morphological changes characteristic of an invasive phenotype. Furthermore, rhDKK1 was found to activate canonical Wnt signaling in PC3 cells, suggesting that SOST and DKK1 have opposing roles on Wnt signaling in this context. Gene expression analysis of PC3 cells co-cultured with OBs exhibiting varying amounts of Wnt signaling identified CRIM1 as one of the transcripts upregulated under highly invasive conditions. We found CRIM1 overexpression to also promote cell-invasion. These findings suggest that bone-derived Wnt signaling may enhance PC tropism by promoting CRIM1 expression and facilitating cancer cell invasion and adhesion to bone. We concluded that SOST and DKK1 have opposing effects on PC3 cell invasion and that bone-derived Wnt signaling positively contributes to the invasive phenotypes of PC3 cells by activating CRIM1 expression and facilitating PC-OB physical interaction. As such, we investigated the effects of high concentrations of SOST in vivo. We found that PC3-cells overexpressing SOST injected via the tail vein in NSG mice did not readily metastasize, and those injected intrafemorally had significantly reduced osteolysis, suggesting that targeting the molecular bone environment may influence bone metastatic prognosis in clinical settings

Conditional Deletion of Sost in MSC‐derived lineages Identifies Specific Cell Type Contributions to Bone Mass and B Cell Development

Sclerostin (Sost) is a negative regulator of bone formation and blocking its function via antibodies has shown great therapeutic promise by increasing both bone mass in humans and animal models. Sclerostin deletion in Sost knockout mice (Sost‐/‐) causes high bone mass (HBM) similar to Sclerosteosis patients. Sost‐/‐ mice have been shown to display an up to 300% increase in bone volume/total volume (BV/TV), relative to aged matched controls, and it has been postulated that the main source of skeletal Sclerostin is the osteocyte. To understand the cell‐type specific contributions to the HBM phenotype described in Sost‐/‐ mice, as well as to address the endocrine and paracrine mode of action of sclerostin, we examined the skeletal phenotypes of conditional Sost loss‐of‐function (SostiCOIN/iCOIN) mice with specific deletions in (1) the limb mesenchyme (Prx1‐Cre; targets osteoprogenitors and their progeny); (2) mid‐stage osteoblasts and their progenitors (Col1‐Cre); (3) mature osteocytes (Dmp1‐Cre) and (4) hypertrophic chondrocytes and their progenitors (ColX‐Cre). All conditional alleles resulted in significant increases in bone mass in trabecular bone in both the femur and lumbar vertebrae, but only Prx1‐Cre deletion fully recapitulated the amplitude of the HBM phenotype in the appendicular skeleton and the B cell defect described in the global knockout. Despite wildtype expression of Sost in the axial skeleton of Prx1‐Cre deleted mice, these mice also had a significant increase in bone mass in the vertebrae, but the Sclerostin released in circulation by the axial skeleton did not affect bone parameters in the appendicular skeleton. Also, both Col1 and Dmp1 deletion resulted in a similar 80% significant increase in trabecular bone mass, but only Col1 and Prx1 deletion resulted in a significant increase in cortical thickness. We conclude that several cell types within the Prx1‐osteoprogenitor derived lineages contribute significant amounts of Sclerostin protein to the paracrine pool of Sost, in bone

Localization of a gene for nonsyndromic renal hypodysplasia to chromosome 1p32-33.

Nonsyndromic defects in the urinary tract are the most common cause of end-stage renal failure in children and account for a significant proportion of adult nephropathy. The genetic basis of these disorders is not fully understood. We studied seven multiplex kindreds ascertained via an index case with a nonsyndromic solitary kidney or renal hypodysplasia. Systematic ultrasonographic screening revealed that many family members harbor malformations, such as solitary kidneys, hypodysplasia, or ureteric abnormalities (in a total of 29 affected individuals). A genomewide scan identified significant linkage to a 6.9-Mb segment on chromosome 1p32-33 under an autosomal dominant model with reduced penetrance (peak LOD score 3.5 at D1S2652 in the largest kindred). Altogether, three of the seven families showed positive LOD scores at this interval, demonstrating heterogeneity of the trait (peak HLOD 3.9, with 45% of families linked). The chromosome 1p32-33 interval contains 52 transcription units, and at least 23 of these are expressed at stage E12.5 in the murine ureteric bud and/or metanephric mesenchyme. These data show that autosomal dominant nonsyndromic renal hypodysplasia and associated urinary tract malformations are genetically heterogeneous and identify a locus for this common cause of human kidney failure

Optimization of a genomic editing system using CRISPR/Cas9-induced site-specific gene integration

The CRISPR-Cas system is an adaptive immune system found in bacteria which helps protect against the invasion of other microorganisms. This system induces double stranded breaks at precise genomic loci (1) in which repairs are initiated and insertions of a target are completed in the process. This mechanism can be used in eukaryotic cells in combination with sgRNAs (1) as a tool for genome editing. By using this CRISPR-Cas system, in addition to the “safe harbor locus,” ROSAβ26, the incorporation of a target gene into a site that is not susceptible to gene silencing effects can be achieved through few simple steps. PCR amplification of the target genes , ROSA26 and mKate2, with a sgRNA scaffold and T7 promoter followed by in vitro transcription aim to produce an RNA product. This sgRNA product can be run through a digestion with Cas9 to validate cleavage of the genomic ROSA DNA template or mKate plasmid. Osteoblast mouse cells are transfected and labeled through partial uptake by the CRISPR mechanism, by cutting in the ROSA loci and repairing with pieces of the fluorescent mKate2 plasmid. These cells were measured via flow cytometry to give a percentage of red cells. This data shows the scaffolding construct created is targeted by the Cas9 endonuclease and through homologous repair the cells will incorporate the mKate2 target gene in vitro in MC3T3 mouse cells

Computational Modeling of Silicate Glasses: A Quantitative Structure-Property Relationship Perspective

This article reviews the present state of Quantitative Structure-Property Relationships (QSPR) in glass design and gives an outlook into future developments. First an overview is given of the statistical methodology, with particular emphasis to the integration of QSPR with molecular dynamics simulations to derive informative structural descriptors. Then, the potentiality of this approach as a tool for interpretative and predictive purposes is highlighted by a number of recent inspiring applications