Boca Resorts Inc.-A Valuation Report

This paper presents the valuation of Boca Resorts Inc. a luxury recreational lodging business based in the United States. The McKinsey model was implemented in order to conduct this valuation. The final equity value obtained in this valuation is lower than the market value of the firm and this discrepancy reflects several alternative explanations. The value of the firm was found to be quite sensitive to several factors and thus different scenarios which reflect changes in these assumptions have been tested. The paper concludes with possible explanations for the firm’s market value.

Differential Roles for STIM1 and STIM2 in Store-Operated Calcium Entry in Rat Neurons

The interaction between Ca2+ sensors STIM1 and STIM2 and Ca2+ channel-forming protein ORAI1 is a crucial element of store-operated calcium entry (SOCE) in non-excitable cells. However, the molecular mechanism of SOCE in neurons remains unclear. We addressed this issue by establishing the presence and function of STIM proteins. Real-time polymerase chain reaction from cortical neurons showed that these cells contain significant amounts of Stim1 and Stim2 mRNA. Thapsigargin (TG) treatment increased the amount of both endogenous STIM proteins in neuronal membrane fractions. The number of YFP-STIM1/ORAI1 and YFP-STIM2/ORAI1 complexes was also enhanced by such treatment. The differences observed in the number of STIM1 and STIM2 complexes under SOCE conditions and the differential sensitivity to SOCE inhibitors suggest their distinct roles. Endoplasmic reticulum (ER) store depletion by TG enhanced intracellular Ca2+ levels in loaded with Fura-2 neurons transfected with YFP-STIM1 and ORAI1, but not with YFP-STIM2 and ORAI1, which correlated well with the number of complexes formed. Moreover, the SOCE inhibitors ML-9 and 2-APB reduced Ca2+ influx in neurons expressing YFP-STIM1/ORAI1 but produced no effect in cells transfected with YFP-STIM2/ORAI1. Moreover, in neurons transfected with YFP-STIM2/ORAI1, the increase in constitutive calcium entry was greater than with YFP-STIM1/ORAI1. Our data indicate that both STIM proteins are involved in calcium homeostasis in neurons. STIM1 mainly activates SOCE, whereas STIM2 regulates resting Ca2+ levels in the ER and Ca2+ leakage with the additional involvement of STIM1

TCF7L2 mediates the cellular and behavioral response to chronic lithium treatment in animal models

AbstractThe mechanism of lithium's therapeutic action remains obscure, hindering the discovery of safer treatments for bipolar disorder. Lithium can act as an inhibitor of the kinase GSK3α/β, which in turn negatively regulates β-catenin, a co-activator of LEF1/TCF transcription factors. However, unclear is whether therapeutic levels of lithium activate β-catenin in the brain, and whether this activation could have a therapeutic significance. To address this issue we chronically treated mice with lithium. Although the level of non-phospho-β-catenin increased in all of the brain areas examined, β-catenin translocated into cellular nuclei only in the thalamus. Similar results were obtained when thalamic and cortical neurons were treated with a therapeutically relevant concentration of lithium in vitro. We tested if TCF7L2, a member of LEF1/TCF family that is highly expressed in the thalamus, facilitated the activation of β-catenin. Silencing of Tcf7l2 in thalamic neurons prevented β-catenin from entering the nucleus, even when the cells were treated with lithium. Conversely, when Tcf7l2 was ectopically expressed in cortical neurons, β-catenin shifted to the nucleus, and lithium augmented this process. Lastly, we silenced tcf7l2 in zebrafish and exposed them to lithium for 3 days, to evaluate whether TCF7L2 is involved in the behavioral response. Lithium decreased the dark-induced activity of control zebrafish, whereas the activity of zebrafish with tcf7l2 knockdown was unaltered. We conclude that therapeutic levels of lithium activate β-catenin selectively in thalamic neurons. This effect is determined by the presence of TCF7L2, and potentially contributes to the therapeutic response

An evaluation of oligonucleotide-based therapeutic strategies for polyQ diseases

<p>Abstract</p> <p>Background</p> <p>RNA interference (RNAi) and antisense strategies provide experimental therapeutic agents for numerous diseases, including polyglutamine (polyQ) disorders caused by CAG repeat expansion. We compared the potential of different oligonucleotide-based strategies for silencing the genes responsible for several polyQ diseases, including Huntington's disease and two spinocerebellar ataxias, type 1 and type 3. The strategies included nonallele-selective gene silencing, gene replacement, allele-selective SNP targeting and CAG repeat targeting.</p> <p>Results</p> <p>Using the patient-derived cell culture models of polyQ diseases, we tested various siRNAs, and antisense reagents and assessed their silencing efficiency and allele selectivity. We showed considerable allele discrimination by several SNP targeting siRNAs based on a weak G-G or G-U pairing with normal allele and strong G-C pairing with mutant allele at the site of RISC-induced cleavage. Among the CAG repeat targeting reagents the strongest allele discrimination is achieved by miRNA-like functioning reagents that bind to their targets and inhibit their translation without substantial target cleavage. Also, morpholino analog performs well in mutant and normal allele discrimination but its efficient delivery to cells at low effective concentration still remains a challenge.</p> <p>Conclusions</p> <p>Using three cellular models of polyQ diseases and the same experimental setup we directly compared the performance of different oligonucleotide-based treatment strategies that are currently under development. Based on the results obtained by us and others we discussed the advantages and drawbacks of these strategies considering them from several different perspectives. The strategy aimed at nonallele-selective inhibiting of causative gene expression by targeting specific sequence of the implicated gene is the easiest to implement but relevant benefits are still uncertain. The gene replacement strategy that combines the nonallele-selective gene silencing with the expression of the exogenous normal allele is a logical extension of the former and it deserves to be explored further. Both allele-selective RNAi approaches challenge cellular RNA interference machinery to show its ability to discriminate between similar sequences differing in either single base substitutions or repeated sequence length. Although both approaches perform well in allele discrimination most of our efforts are focused on repeat targeting due to its potentially higher universality.</p

Gender differences in post-operative human skin

Although the impact of age, gender, and obesity on the skin wound healing process has been extensively studied, the data related to gender differences in aspects of skin scarring are limited. The present study performed on abdominal human intact and scar skin focused on determining gender differences in extracellular matrix (ECM) composition, dermal white adipose tissue (dWAT) accumulation, and Foxn1 expression as a part of the skin response to injury. Scar skin of men showed highly increased levels of COLLAGEN 1A1, COLLAGEN 6A3, and ELASTIN mRNA expression, the accumulation of thick collagen I-positive fibers, and the accumulation of $\alpha$-SMA-positive cells in comparison to the scar skin of women. However, post-injured skin of women displayed an increase (in comparison to post-injured men’s skin) in collagen III accumulation in the scar area. On the contrary, women’s skin samples showed a tendency towards higher levels of adipogenic-related genes ($PPAR\gamma$, FABP4, LEPTIN) than men, regardless of intact or scar skin. Intact skin of women showed six times higher levels of LEPTIN mRNA expression in comparison to men intact (p < 0.05), men post-injured (p < 0.05), or women post-injured scar (p < 0.05) skin. Higher levels of FOXN1 mRNA and protein were also detected in women than in men’s skin. In conclusion, the present data confirm and extend (dWAT layer) the data related to the presence of differences between men and women in the skin, particularly in scar tissues, which may contribute to the more effective and gender-tailored improvement of skin care interventions

Hypoxia reveals a new function of Foxn1 in the keratinocyte antioxidant defense system

Skin exposed to environmental threats, including injuries and oxidative stress, develops an efficient but not fully recognized system of repair and antioxidant protection. Here, using mass spectrometry analysis (LC–MS/MS), followed by in vitro and in vivo experiments, we provided evidence that Foxn1 in keratinocytes regulates elements of the electron transport chain and participates in the thioredoxin system (Txn2, Txnrd3, and Srxn1) induction, particularly in a hypoxic environment. We first showed that Foxn1 in keratinocytes upregulates glutathione thioredoxin reductase 3 (Txnrd3) protein expression, and high levels of Txnrd3 mRNA were detected in injured skin of Foxn1+/+ mice. We also showed that Foxn1 strongly downregulated the Ccn2 protein expression, participating in epidermal reconstruction after injury. An in vitro assay revealed that Foxn1 controls keratinocyte migration, stimulating it under normoxia and suppressing it under hypoxia. Keratinocytes overexpressing Foxn1 and exposed to hypoxia displayed a reduced ability to promote angiogenesis by downregulating Vegfa expression. In conclusion, this study showed a new mechanism in which Foxn1, along with hypoxia, participates in the activation of antioxidant defense and controls the functional properties of keratinocytes. </p

The RNA-binding landscape of HAX1 protein indicates its involvement in translation and ribosome assembly

HAX1 is a human protein with no known homologues or structural domains. Mutations in the HAX1 gene cause severe congenital neutropenia through mechanisms that are poorly understood. Previous studies reported the RNA-binding capacity of HAX1, but the role of this binding in physiology and pathology remains unexplained. Here, we report the transcriptome-wide characterization of HAX1 RNA targets using RIP-seq and CRAC, indicating that HAX1 binds transcripts involved in translation, ribosome biogenesis, and rRNA processing. Using CRISPR knockouts, we find that HAX1 RNA targets partially overlap with transcripts downregulated in HAX1 KO, implying a role in mRNA stabilization. Gene ontology analysis demonstrated that genes differentially expressed in HAX1 KO (including genes involved in ribosome biogenesis and translation) are also enriched in a subset of genes whose expression correlates with HAX1 expression in four analyzed neoplasms. The functional connection to ribosome biogenesis was also demonstrated by gradient sedimentation ribosome profiles, which revealed differences in the small subunit:monosome ratio in HAX1 WT/KO. We speculate that changes in HAX1 expression may be important for the etiology of HAX1-linked diseases through dysregulation of translation

Evaluation of manure management systems in Europe

The gross value of the agricultural goods in 2014 amounted up to 370 billion Euros. Almost 50% of all agricultural production is provided by livestock farming.Postprint (published version

New amniotic membrane based biocomposite for future application in reconstructive urology

OBJECTIVE Due to the capacity of the amniotic membrane (Am) to support re-epithelisation and inhibit scar formation, Am has a potential to become a considerable asset for reconstructive urology i.e., reconstruction of ureters and urethrae. The application of Am in reconstructive urology is limited due to a poor mechanical characteristic. Am reinforcement with electrospun nanofibers offers a new strategy to improve Am mechanical resistance, without affecting its unique bioactivity profile. This study evaluated biocomposite material composed of Am and nanofibers as a graft for urinary bladder augmentation in a rat model. MATERIAL AND METHODS Sandwich-structured biocomposite material was constructed from frozen Am and covered on both sides with two-layered membranes prepared from electrospun poly-(L-lactide-co-Ecaprolactone) (PLCL). Wistar rats underwent hemicystectomy and bladder augmentation with the biocomposite material. RESULTS Immunohistohemical analysis (hematoxylin and eosin [H&E], anti-smoothelin and Masson’s trichrome staining [TRI]) revealed effective regeneration of the urothelial and smooth muscle layers. Anti-smoothelin staining confirmed the presence of contractile smooth muscle within a new bladder wall. Sandwich-structured biocomposite graft material was designed to regenerate the urinary bladder wall, fulfilling the requirements for normal bladder tension, contraction, elasticity and compliance. Mechanical evaluation of regenerated bladder wall conducted based on Young’s elastic modulus reflected changes in the histological remodeling of the augmented part of the bladder. The structure of the biocomposite material made it possible to deliver an intact Am to the area for regeneration. An unmodified Am surface supported regeneration of the urinary bladder wall and the PLCL membranes did not disturb the regeneration process. CONCLUSIONS Am reinforcement with electrospun nanofibers offers a new strategy to improve Am mechanical resistance without affecting its unique bioactivity profile.http://www.plosone.orgam2016Internal Medicin