Machine learning-informed and synthetic biology-enabled semi-continuous algal cultivation to unleash renewable fuel productivity.

Algal biofuel is regarded as one of the ultimate solutions for renewable energy, but its commercialization is hindered by growth limitations caused by mutual shading and high harvest costs. We overcome these challenges by advancing machine learning to inform the design of a semi-continuous algal cultivation (SAC) to sustain optimal cell growth and minimize mutual shading. An aggregation-based sedimentation (ABS) strategy is then designed to achieve low-cost biomass harvesting and economical SAC. The ABS is achieved by engineering a fast-growing strain, Synechococcus elongatus UTEX 2973, to produce limonene, which increases cyanobacterial cell surface hydrophobicity and enables efficient cell aggregation and sedimentation. SAC unleashes cyanobacterial growth potential with 0.1 g/L/hour biomass productivity and 0.2 mg/L/hour limonene productivity over a sustained period in photobioreactors. Scaling-up the SAC with an outdoor pond system achieves a biomass yield of 43.3 g/m2/day, bringing the minimum biomass selling price down to approximately $281 per ton.

An Albumin-Binding T1- T2 Dual-Modal MRI Contrast Agents for Improved Sensitivity and Accuracy in Tumor Imaging.

Magnetic resonance imaging (MRI) diagnosis is better assisted by contrast agents that can augment the signal contrast in the imaging appearance. However, this technique is still limited by the inherently low sensitivity on the recorded signal changes in conventional T1 or T2 MRI in a qualitative manner. Here, we provide a new paradigm of MRI diagnosis using T1- T2 dual-modal MRI contrast agents for contrast-enhanced postimaging computations on T1 and T2 relaxation changes. An albumin-binding molecule (i.e., truncated Evans blue) chelated with paramagnetic manganese ion was developed as a novel T1- T2 dual-modal MRI contrast agent at high magnetic field (7 T). Furthermore, the postimaging computations on T1- T2 dual-modal MRI led to greatly enhanced signal-to-noise ratios (SNR) and contrast-to-noise ratios (CNR) in both subcutaneous and orthotopic brain tumor models compared with traditional MRI methods. The T1- T2 dual-modal MRI computations have great potential to eliminate suspicious artifacts and false-positive signals in mouse brain imaging. This study may open new avenues for contrast-enhanced MRI diagnosis and holds great promise for precision medicine.

Repurposing a novel parathyroid hormone analogue to treat hypoparathyroidism.

BACKGROUND AND PURPOSE: Human parathyroid hormone (PTH) is critical for maintaining physiological calcium homeostasis and plays an important role in the formation and maintenance of the bone. Full-length PTH and a truncated peptide form are approved for treatment of hypoparathyroidism and osteoporosis respectively. Our initial goal was to develop an improved PTH therapy for osteoporosis, but clinical development was halted. The novel compound was then repurposed as an improved therapy for hypoparathyroidism. EXPERIMENTAL APPROACH: A longer-acting form of PTH was synthesised by altering the peptide to increase cell surface residence time of the bound ligand to its receptor. In vitro screening identified a compound, which was tested in an animal model of osteoporosis before entering human trials. This compound was subsequently tested in two independent animal models of hypoparathyroidism. KEY RESULTS: The peptide identified, LY627-2K, exhibited delayed internalization kinetics. In an ovariectomy-induced bone loss rat model, LY627-2K demonstrated improved vertebral bone mineral density and biomechanical properties at skeletal sites and a modest increase in serum calcium. In a Phase I clinical study, dose-dependent increases in serum calcium were reproduced. These observations prompted us to explore a second indication, hypoparathyroidism. In animal models of this disease, LY627-2K restored serum calcium, comparing favourably to treatment with wild-type PTH. CONCLUSIONS AND IMPLICATIONS: We summarize the repositioning of a therapeutic candidate with substantial preclinical and clinical data. Our results support its repurposing and continued development, from a common indication (osteoporosis) to a rare disease (hypoparathyroidism) by exploiting a shared molecular target. LINKED ARTICLES: This article is part of a themed section on Inventing New Therapies Without Reinventing the Wheel: The Power of Drug Repurposing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.2/issuetoc.

Time course of disassociation of bone formation signals with bone mass and bone strength in sclerostin antibody treated ovariectomized rats.

Sclerostin antibodies increase bone mass by stimulating bone formation. However, human and animal studies show that bone formation increases transiently and returns to pre-treatment level despite ongoing antibody treatment. To understand its mechanism of action, we studied the time course of bone formation, correlating the rate and extent of accrual of bone mass and strength after sclerostin antibody treatment. Ovariectomized (OVX) rats were treated with a sclerostin-antibody (Scle-ab) at 20mg/kg sc once weekly and sacrificed at baseline and 2, 3, 4, 6, and 8weeks post-treatment. In Scle-ab treated rats, serum PINP and OCN rapidly increased at week 1, peaked around week 3, and returned to OVX control levels by week 6. Transcript analyses from the distal femur revealed an early increase in bone formation followed by a sustained decrease in bone resorption genes. Lumbar vertebral (LV) osteoblast surface increased 88% by week 2, and bone formation rate (BFR/BS) increased 138% by week 4. Both parameters were below OVX control by week 8. Bone formation was primarily a result of modeling based formation. Endocortical and periosteal BFR/BS peaked around week 4 at 313% and 585% of OVX control, respectively. BFR/BS then declined but remained higher than OVX control on both surfaces through week 8. Histomorphometric analyses showed LV-BV/TV did not further increase after week 4, while BMD continued to increase at LV, mid femur (MF), and femoral neck (FN) through week 8. Biomechanical tests showed a similar improvement in bone strength through 8weeks in MF and FN, but bone strength plateaued between weeks 6 and 8 for LV. Our data suggest that bone formation with Scle-ab treatment is rapid and modeling formation dominated in OVX rats. Although transient, the bone formation response persists longer in cortical than trabecular bone.

Vitamin D and/or calcium deficient diets may differentially affect muscle fiber neuromuscular junction innervation.

INTRODUCTION: There is evidence that supports a role for Vitamin D (Vit. D) in muscle. The exact mechanism by which Vit. D deficiency impairs muscle strength and function is not clear. METHODS: Three-week-old mice were fed diets with varied combinations of Vit. D and Ca2+ deficiency. Behavioral testing, genomic and protein analysis, and muscle histology were performed with a focus on neuromuscular junction (NMJ) -related genes. RESULTS: Vit. D and Ca2+ deficient mice performed more poorly on given behavioral tasks than animals with Vit. D deficiency alone. Genomic and protein analysis of the soleus and tibialis anterior muscles revealed changes in several Vit. D metabolic, NMJ-related, and protein chaperoning and refolding genes. CONCLUSIONS: These data suggest that detrimental effects of a Vit. D deficient or a Vit. D and Ca2+ deficient diet may be a result of differential alterations in the structure and function of the NMJ and a lack of a sustained stress response in muscles. Muscle Nerve 54: 1120-1132, 2016.

Molecular targets of androgen signaling that characterize skeletal muscle recovery and regeneration.

The high regenerative capacity of adult skeletal muscle relies on a self-renewing depot of adult stem cells, termed muscle satellite cells (MSCs). Androgens, known mediators of overall body composition and specifically skeletal muscle mass, have been shown to regulate MSCs. The possible overlapping function of androgen regulation of muscle growth and MSC activation has not been carefully investigated with regards to muscle regeneration.Therefore, the aim of this study was to examine coinciding androgen-mediated genetic changes in an in vitro MSC model and clinically relevant in vivo models. A gene signature was established via microarray analysis for androgen-mediated MSC engagement and highlighted several markers including follistatin (FST), IGF-1, C-X-C chemokine receptor 4 (CXCR4), hepatocyte growth factor (HGF) and glucocorticoid receptor (GR). In an in vivo muscle atrophy model, androgen re-supplementation significantly increased muscle size and expression of IGF-1, FST, and HGF, while significantly decreasing expression of GR. Biphasic gene expression profiles over the 7-day re-supplementation period identified temporal androgen regulation of molecular targets involved in satellite cell engagement into myogenesis. In a muscle injury model, removal of androgens resulted in delayed muscle recovery and regeneration. Modifications in the androgen signaling gene signature, along with reduced Pax7 and MyoD expression, suggested that limited MSC activation and increased inflammation contributed to the delayed regeneration. However, enhanced MSC activation in the androgen-deplete mouse injury model was driven by an androgen receptor (AR) agonist. These results provide novel in vitro and in vivo evidence describing molecular targets of androgen signaling, while also increasing support for translational use of AR agonists in skeletal muscle recovery and regeneration.

Synthesis and characterization of gadolinium-Peptidomimetic complex as an αvß3 integrin targeted MR contrast agent.

There is growing interest in small and rigid peptidomimetic αvß3 integrin antagonists that are readily synthesized and characterized and amenable to physiological conditions. Peptidomimetic 4-[2-(3,4,5,6-tetrahydropyrimidine-2-ylamino)ethyloxy]benzoyl-2-[N-(3-amino-neopenta-1-carbamyl)]-aminoethylsulfonyl-amino-ß-alanine (IAC) was successfully conjugated to DOTA, complexed with Gd(III) and radiolabeled with (153)Gd. Radioassay results demonstrated specificity of the labeled conjugate by blocking ∼95% binding with the addition of a 50-fold molar excess of cold IAC to the reaction solution. Relaxometry was used to support the hypothesis that the specificity of the Gd-peptidomimetic targeting αvß3 integrin would increase the contrast and therefore enhance the sensitivity of an MRI scan of αvß3 integrin positive tissues. Magnetic resonance imaging of cell pellets (M21 human melanoma) was also performed, and the images clearly show that cells reacted with Gd(III)-DOTA-IAC display a brighter image than cells without the Gd(III)-DOTA-IAC contrast agent. In addition, Gd(III)-DOTA-IAC and IAC, with IC50 of 300nM and 230nM, respectively, are 2.1 and 2.7 times more potent than c(RGDfK) whose IC50 is 625nM. This promising preliminary data fuels further investigation of DOTA-IAC conjugates for targeting tumor associated angiogenesis and αvß3 integrin positive tumors using magnetic resonance imaging.

Application of aziridinium ring opening for preparation of optically active diamine and triamine analogues: Highly efficient synthesis and evaluation of DTPA-based MRI contrast enhancement agents.

Ring opening of aziridinium ions with nitrogen nucleophiles was applied to the highly efficient synthesis of optically active vicinal diamines and diethylene triamine pentaacetic acid (DTPA) analogues as potential magnetic resonance imaging (MRI) contrast enhancement agents. The synthetic method features a column-free isolation of the regiospecific and stereospecific nucleophilic substitution products of enantiomerically enriched aziridinium ions in excellent yield.

Manganese G8 dendrimers targeted to oxidation-specific epitopes: in vivo MR imaging of atherosclerosis.

PURPOSE: To determine if manganese (Mn) G8 dendrimers targeted to oxidation-specific epitopes (OSE) allow for in vivo detection of atherosclerotic lesions. MATERIALS AND METHODS: OSE have been identified as key factors in atherosclerotic plaque progression and destabilization. Mn offers a potentially clinically translatable alternative to gadolinium-based agents when bioretention and potential toxicity of gadolinium is anticipated. However, to be effective, high payloads of Mn must accumulate intracellularly in macrophages. It was hypothesized that G8 dendrimers targeted to OSE may allow delivery of high Mn payloads, thereby enabling in vivo detection of macrophage-rich plaques. G8 dendrimers were modified to allow conjugation with MnDTPA (758 Mn ion) and the antibody MDA2 that is targeted to malondialdehyde (MDA)-lysine epitopes. Both the untargeted and targeted G8 dendrimers were characterized and their in vivo efficacy evaluated in apoE(-/-) mice over a 96-hour time period after bolus administration of a 0.05 mmol Mn/kg dose using a clinical MR system (3T). RESULTS: Significant enhancement (normalized enhancement >60%, P = 0.0013) of atherosclerotic lesions was observed within a 72-hour time period following administration of the targeted dendrimers. The presence of Mn within atherosclerotic lesions was confirmed using spectroscopic methods (>8 µg Mn/g). Limited signal attenuation (<18%) and Mn deposition (<1 µg Mn/g) was observed in the arterial wall following injection of the untargeted material. CONCLUSION: This study demonstrates that manganese-labeled dendrimers, allowing a high Mn payload, targeted to OSE may allow in vivo image of atherosclerotic lesions.

Follistatin: a novel therapeutic for the improvement of muscle regeneration.

Follistatin (FST) is a member of the tissue growth factor ß family and is a secreted glycoprotein that antagonizes many members of the family, including activin A, growth differentiation factor 11, and myostatin. The objective of this study was to explore the use of an engineered follistatin therapeutic created by fusing FST315 lacking heparin binding activity to the N terminus of a murine IgG1 Fc (FST315-ΔHBS-Fc) as a systemic therapeutic agent in models of muscle injury. Systemic administration of this molecule was found to increase body weight and lean muscle mass after weekly administration in normal mice. Subsequently, we tested this agent in several models of muscle injury, which were chosen based on their severity of damage and their ability to reflect clinical settings. FST315-ΔHBS-Fc treatment proved to be a potent inducer of muscle remodeling and regeneration. FST315-ΔHBS-Fc induced improvements in muscle repair after injury/atrophy by modulating the early inflammatory phase allowing for increased macrophage density, and Pax7-positive cells leading to an accelerated restoration of myofibers and muscle function. Collectively, these data demonstrate the benefits of a therapeutically viable form of FST that can be leveraged as an alternate means of ameliorating muscle regeneration.

The magnetic, relaxometric, and optical properties of gadolinium-catalyzed single walled carbon nanotubes.

We report the magnetic behavior, relaxometry, phantom magnetic resonance imaging (MRI), and near-infrared (NIR) photoluminescence spectroscopy of gadolinium (Gd) catalyzed single-walled carbon nanotubes (Gd-SWCNTs). Gd-SWCNTs are paramagnetic with an effective magnetic moment of 7.29 µB . Gd-SWCNT solutions show high r1 and r2 relaxivities at very low (0.01 MHz) to clinically relevant (61 MHz) magnetic fields (r1 ≥ 130 mM-1 s-1, r2 ≥ 160 mM-1 s-1). Analysis of nuclear magnetic resonance dispersion profiles using Solomon, Bloembergen, and Morgan equations suggests that multiple structural and dynamic parameters such as rotational correlation time [Formula: see text], rate of water exchange [Formula: see text], and the number of fast-exchanging water molecules within the inner sphere q may be responsible for the increase in r1 and r2 relaxivity. The T1 weighted MRI signal intensity (gradient echo sequence; repetition time (TR) = 66 ms, echo time (TE) = 3 ms, flop angle = 108°) of Gd-SWCNT phantom solution is 14 times greater than the Gd-based clinical MRI contrast agent Magnevist. Additionally, these nanotubes exhibit near infrared fluorescence with distinct E11 transitions of several semiconducting SWCNTs. Taken together, these results demonstrate that Gd-SWCNTs have potential as a novel, highly efficacious, multimodal MRI-NIR optical imaging contrast agent.

Hydrophobic Interactions Improve Selectivity to ERα for Ben-zothiophene SERMs.

The discovery, pharmacology, and biophysical characterization of an ERα selective benzothiophene (BTPα) is described. BTPα (4) is a high affinity ligand with 140-fold greater selectivity for ERα (K(i)=0.25 nM) over ERbeta (K(i)=35 nM). In rodent models of estrogen action, BTPα blocks the effects of estrogen in the uterus but mimics the effects estrogen on bone. The basis of ERα selectivity for BTPα was evaluated by using protein crystallography and hydrogen/deuterium exchange (HDX) mass spectrometry. HDX data supports that the n-butyl chain of BTPα stabilizes helix 7 in ERα relative to that of ERß which we propose leads to an enhancement of affinity to the alpha receptor sub-type.

Teriparatide [rhPTH (1-34)], but not strontium ranelate, demonstrated bone anabolic efficacy in mature, osteopenic, ovariectomized rats.

We compared teriparatide (TPTD) and strontium ranelate (SR) efficacy on bone formation activity in a mature rat model of estrogen-deficiency bone loss. Rats were ovariectomized (OVX) at age 6 months and permitted to lose bone for 2 months to establish osteopenia before initiation of treatment with TPTD (5 or 15 µg/kg · d sc) or SR (150 or 450 mg/kg · d oral gavage). After 3 wk, RT-PCR analyses of bone formation genes in the distal femur metaphysis showed significant elevation of collagen 1α2, osteocalcin, bone sialoprotein, alkaline phosphatase, and Runx2 gene expression at both TPTD doses, relative to OVX controls. SR had no significant effect on expression of these genes. TPTD treatment for 12 wk dose dependently increased lumbar vertebral (LV) and femoral midshaft bone mineral content (BMC) and bone mineral density over pretreatment and age-matched OVX controls. SR 150 increased BMC, and SR 450 increased BMC and bone mineral density of femoral midshaft and LV over OVX controls. There were significant dose-dependent TPTD increases of LV and femoral neck strength, and TPTD 15 also increased midshaft strength compared with pretreatment and age-matched OVX controls. SR did not enhance bone strength relative to pretreatment or age-matched OVX controls. Histomorphometry of the proximal tibial metaphysis showed dose-dependent effects of TPTD on trabecular area, number, width, and osteoblast surface, bone mineralizing surface, and bone formation rate relative to pretreatment and age-matched OVX controls, whereas SR had no effect on these parameters. These findings confirmed the bone anabolic efficacy of teriparatide, but not SR in mature, osteopenic, OVX rats.

Formulation and characterisation of magnetic resonance imageable thermally sensitive liposomes for use with magnetic resonance-guided high intensity focused ultrasound.

PURPOSE: Objectives of this study were to: 1) develop iLTSL, a low temperature sensitive liposome co-loaded with an MRI contrast agent (ProHance® Gd-HP-DO3A) and doxorubicin, 2) characterise doxorubicin and Gd-HP-DO3A release from iLTSL and 3) investigate the ability of magnetic resonance-guided high intensity focused ultrasound (MR-HIFU) to induce and monitor iLTSL content release in phantoms and in vivo. METHODS: iLTSL was passively loaded with Gd-HP-DO3A and actively loaded with doxorubicin. Doxorubicin and Gd-HP-DO3A release was quantified by fluorescence and spectroscopic techniques, respectively. Release with MR-HIFU was examined in tissue-mimicking phantoms containing iLTSL and in a VX2 rabbit tumour model. RESULTS: iLTSL demonstrated consistent size and doxorubicin release kinetics after storage at 4°C for 7 days. Release of doxorubicin and Gd-HP-DO3A from iLTSL was minimal at 37°C but fast when heated to 41.3°C. The magnitude of release was not significantly different between doxorubicin and Gd-HP-DO3A over 10 min in HEPES buffer and plasma at 37°, 40° and 41.3°C (p > 0.05). Relaxivity of iLTSL increased significantly (p < 0.0001) from 1.95 ± 0.05 to 4.01 ± 0.1 mMs⁻¹ when heated above the transition temperature. Signal increase corresponded spatially and temporally to MR-HIFU-heated locations in phantoms. Signal increase was also observed in vivo after iLTSL injection and after each 10-min heating (41°C), with greatest increase in the heated tumour region. CONCLUSION: An MR imageable liposome formulation co-loaded with doxorubicin and an MR contrast agent was developed. Stability, imageability, and MR-HIFU monitoring and control of content release suggest that MR-HIFU combined with iLTSL may enable real-time monitoring and spatial control of content release.

A nonsecosteroidal vitamin D receptor ligand with improved therapeutic window of bone efficacy over hypercalcemia.

Vitamin D(3) analogues were shown to be beneficial for osteoporosis and other indications, but their narrow therapeutic window between efficacy and hypercalcemia has limited their clinical utility. A nonsecosteroidal, tissue-selective, orally bioavailable, vitamin D receptor (VDR) ligand was ascertained to be efficacious in bone while having modest calcemic effects in vivo. This compound (VDRM2) potently induced Retinoid X Receptor alpha (RXR)-VDR heterodimerization (EC(50) = 7.1 +/- 1.6 nM) and induced osteocalcin promoter activity (EC(50) = 1.9 +/- 1.6 nM). VDRM2 was less potent in inducing Ca(2+) channel transient receptor potential cation channel, subfamily V, member 6 (TRPV6) expression (EC(50) = 37 +/- 12 nM). VDRM2 then was evaluated in osteopenic ovariectomized (OVX) rats and shown to dose-dependently restore vertebral bone mineral density (BMD) from OVX to sham levels at 0.08 microg/kg per day. Hypercalcemia was observed at a dose of 4.6 microg/kg per day of VDRM2, suggesting a safety margin of 57 [90% confidence interval (CI) 35-91]. 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D], ED71, and alfacalcidol restored BMD at 0.030, 0.0055, and 0.046 microg/kg per day, respectively, whereas hypercalcemia was observed at 0.22, 0.027, and 0.23 microg/kg per day, indicating a safety margin of 7.3, 4.9, and 5.0, respectively (90% CIs 4.1-13, 3.2-7.7, and 3.5-6.7, respectively). Histomorphometry showed that VDRM2 increased cortical bone area and stimulated the periosteal bone-formation rate relative to OVX at doses below the hypercalcemic dose. By contrast, ED71 increased the periosteal bone-formation rate only above the hypercalcemic dose. VDRM2 suppressed eroded surface on trabecular bone surfaces at normal serum calcium dosage levels, suggesting dual anabolic and antiresorptive activity. In summary, vitamin D analogues were more potent than VDRM2, but VDRM2 had a greater safety margin, suggesting possible therapeutic potential.

Changes in osteoblast, chondrocyte, and adipocyte lineages mediate the bone anabolic actions of PTH and small molecule GSK-3 inhibitor.

Parathyroid hormone (PTH) and glycogen synthase kinase-3 (GSK-3) inhibitor 603281-31-8, administered once daily increased bone formation in vivo. We investigated the molecular mechanisms of the anabolic responses of PTH and 603281-31-8 in rat osteopenia model. Female 6-month-old rats were ovariectomized (Ovx) and permitted to lose bone for 1 month, followed by treatment with PTH (1-38) at 10 microg/kg/day s.c. or 603281-31-8 at 3 mg/kg/day p.o. for 60 days. Twenty-four hours after the last treatment, RNA from distal femur metaphysis was subjected to gene expression analysis. Differentially expressed genes (P<0.05) were subjected to pathway analysis to delineate relevant bio-processes involved in skeletal biology. Genes involved in morphogenesis, cell growth/differentiation, and apoptosis were significantly altered by Ovx and the treatments. Analysis of morphogenesis genes showed an overrepresentation of genes involved in osteogenesis, chondrogenesis, and adipogenesis. A striking finding was that Ovx decreased several markers of osteogenesis/chondrogenesis and increased markers of adipogenesis/lipid metabolism. Treatment with either PTH or the GSK-3 inhibitor reversed these effects, albeit at different levels. Histological analysis confirmed that osteopenia in Ovx animals was associated with three-fold increase in marrow adiposity. PTH and GSK-3 inhibitor restored bone volume, and reversed or normalized marrow adiposity. Ex vivo studies showed that PTH and GSK-3 inhibitor increased the ratio of colony forming marrow stromal progenitors (CFU-fs) that were alkaline phosphatase positive (putative osteoblasts). Our results suggest that the bone anabolic actions of PTH and GSK-3 inhibitor in vivo involve concerted effects on mesenchymal lineages; osteoblasts, chondrocytes, and adipocytes.

Structure-activity relationships of SERMs optimized for uterine antagonism and ovarian safety.

Structure-activity relationship studies are described, which led to the discovery of novel selective estrogen receptor modulators (SERMs) for the potential treatment of uterine fibroids. The SAR studies focused on limiting brain exposure and were guided by computational properties. Compounds with limited impact on the HPO axis were selected using serum estrogen levels as a biomarker for ovarian stimulation.

DNA microarray data integration by ortholog gene analysis reveals potential molecular mechanisms of estrogen-dependent growth of human uterine fibroids.

BACKGROUND: Uterine fibroids or leiomyoma are a common benign smooth muscle tumor. The tumor growth is well known to be estrogen-dependent. However, the molecular mechanisms of its estrogen-dependency is not well understood. METHODS: Differentially expressed genes in human uterine fibroids were either retrieved from published papers or from our own statistical analysis of downloaded array data. Probes for the same genes on different Affymetrix chips were mapped based on probe comparison information provided by Affymetrix. Genes identified by two or three array studies were submitted for ortholog analysis. Human and rat ortholog genes were identified by using ortholog gene databases, HomoloGene and TOGA and were confirmed by synteny analysis with MultiContigView tool in the Ensembl genome browser. RESULTS: By integrated analysis of three recently published DNA microarray studies with human tissue, thirty-eight genes were found to be differentially expressed in the same direction in fibroid compared to adjacent uterine myometrium by at least two research groups. Among these genes, twelve with rat orthologs were identified as estrogen-regulated from our array study investigating uterine expression in ovariectomized rats treated with estrogen. Functional and pathway analyses of the twelve genes suggested multiple molecular mechanisms for estrogen-dependent cell survival and tumor growth. Firstly, estrogen increased expression of the anti-apoptotic PCP4 gene and suppressed the expression of growth inhibitory receptors PTGER3 and TGFBR2. Secondly, estrogen may antagonize PPARgamma signaling, thought to inhibit fibroid growth and survival, at two points in the PPAR pathway: 1) through increased ANXA1 gene expression which can inhibit phospholipase A2 activity and in turn decrease arachidonic acid synthesis, and 2) by decreasing L-PGDS expression which would reduce synthesis of PGJ2, an endogenous ligand for PPARgamma. Lastly, estrogen affects retinoic acid (RA) synthesis and mobilization by regulating expression of CRABP2 and ALDH1A1. RA has been shown to play a significant role in the development of uterine fibroids in an animal model. CONCLUSION: Integrated analysis of multiple array datasets revealed twelve human and rat ortholog genes that were differentially expressed in human uterine fibroids and transcriptionally responsive to estrogen in the rat uterus. Functional and pathway analysis of these genes suggest multiple potential molecular mechanisms for the poorly understood estrogen-dependent growth of uterine fibroids. Fully understanding the exact molecular interactions among these gene products requires further study to validate their roles in uterine fibroids. This work provides new avenues of study which could influence the future direction of therapeutic intervention for the disease.

Orally bioavailable GSK-3alpha/beta dual inhibitor increases markers of cellular differentiation in vitro and bone mass in vivo.

UNLABELLED: GSK-3, a component of the canonical Wnt signaling pathway, is implicated in regulation of bone mass. The effect of a small molecule GSK-3 inhibitor was evaluated in pre-osteoblasts and in osteopenic rats. GSK-3 inhibitor induced osteoblast differentiation in vitro and increased markers of bone formation in vitro and in vivo with concomitant increased bone mass and strength in rats. INTRODUCTION: Inactivation of glycogen synthase kinase -3 (GSK-3) leads to stabilization, accumulation, and translocation of beta-catenin into the nucleus to activate downstream Wnt target genes. To examine whether GSK-3 directly regulates bone formation and mass we evaluated the effect of 603281-31-8, a small molecule GSK-3 alpha/beta dual inhibitor in preosteoblastic cells and in osteopenic rats. MATERIALS AND METHODS: Murine mesenchymal C3H10T1/2 cells were treated with GSK-3 inhibitor (603281-31-8) and assayed for beta-catenin levels, activity of Wnt-responsive promoter, expression of mRNA for bone formation, and adipogenic markers and alkaline phosphatase activity. In vivo, 6-month-old rats were ovariectomized (OVX), allowed to lose bone for 1 month, and treated with GSK-3 inhibitor at 3 mg/kg/day orally for 60 days. At the end of treatment, BMD was measured by DXA, bone formation rate by histomorphometry, vertebral strength (failure in compression), and the expression levels of osteoblast-related genes by real-time PCR. RESULTS: Treatment of C3H10T1/2 cells with the GSK-3 inhibitor increased the levels of beta-catenin accompanied by activation of Wnt-responsive TBE6-luciferase reporter gene. This was associated with an increased expression of mRNA for bone sialoprotein (1.4-fold), collagen alpha1 (I) (approximately 2-fold), osteocalcin (1.2-fold), collagen alpha1(V) (1.5-fold), alkaline phosphatase (approximately 160-fold), and runx2 (1.6-fold), markers of the osteoblast phenotype and bone formation activity. Alkaline phosphatase mRNA expression paralleled alkaline phosphatase activity. The mRNA levels of collagens alpha1 (I), alpha1 (V), biglycan, osteonectin, and runx-2 increased on treatment with the GSK-3 inhibitor in rat femur compared with the OVX control. DXA analyses revealed significant increases in BMC and BMD in cancellous and cortical bone of OVX rats treated with GSK-3 inhibitor. This was associated with increased strength (peak load, energy, and stiffness) assessed by lumbar vertebra load to failure in compression. Histomorphometric analyses showed that 603281-31-8 robustly increased bone formation but did not exclude a small effect on osteoclasts (resorption). CONCLUSIONS: An orally active, small molecule GSK-3 inhibitor induced osteoblast differentiation and increased markers of bone formation in vitro, and increased markers of bone formation, bone mass, and strength in vivo, consistent with a role for the canonical Wnt pathway in osteogenesis.

Regulation of bone mass by Wnt signaling.

Wnt proteins are a family of secreted proteins that regulate many aspects of cell growth, differentiation, function, and death. Considerable progress has been made in our understanding of the molecular links between Wnt signaling and bone development and remodeling since initial reports that mutations in the Wnt coreceptor low-density lipoprotein receptor-related protein 5 (LRP5) are causally linked to alterations in human bone mass. Of the pathways activated by Wnts, it is signaling through the canonical (i.e., Wnt/beta-catenin) pathway that increases bone mass through a number of mechanisms including renewal of stem cells, stimulation of preosteoblast replication, induction of osteoblastogenesis, and inhibition of osteoblast and osteocyte apoptosis. This pathway is an enticing target for developing drugs to battle skeletal diseases as Wnt/beta-catenin signaling is composed of a series of molecular interactions that offer potential places for pharmacological intervention. In considering opportunities for anabolic drug discovery in this area, one must consider multiple factors, including (a) the roles of Wnt signaling for development, remodeling, and pathology of bone; (b) how pharmacological interventions that target this pathway may specifically treat osteoporosis and other aspects of skeletal health; and (c) whether the targets within this pathway are amenable to drug intervention. In this Review we discuss the current understanding of this pathway in terms of bone biology and assess whether targeting this pathway might yield novel therapeutics to treat typical bone disorders.