The long-term effects of hard alloplastic implants when put on bone.

The resorption of bone beneath 4 alloplastic materials has been studied for periods up to 18 months in rabbit skulls. When the effects of pressure were negated, there was no significant difference in bone resorption attributable to placement above or below the periosteum. Dacron backing or polyurethane backing were associated with more inflammation and greater bone reaction than were plain silicone or stainless steel. The changes noted at 18 months were essentially the same as those noted at 6 months.     

An approach to confirmatory testing of subpopulations in clinical trials

In oncology studies with immunotherapies, populations of “super‐responders” (patients in whom the treatment works particularly well) are often suspected to be related to biomarkers. In this paper, we explore various ways of confirmatory statistical hypothesis testing for joint inference on the subpopulation of putative “super‐responders” and the full study population. A model‐based testing framework is proposed, which allows to define, up‐front, the strength of evidence required from both full and subpopulations in terms of clinical efficacy. This framework is based on a two‐way analysis of variance (ANOVA) model with an interaction in combination with multiple comparison procedures. The ease of implementation of this model‐based approach is emphasized and details are provided for the practitioner who would like to adopt this approach. The discussion is exemplified by a hypothetical trial that uses an immune‐marker in oncology to define the subpopulation and tumor growth as the primary endpoint.     

Hydroxyapatite as a concentrating probe for phosphoproteomic analyses

A novel method for the selective enrichment of casein phosphoproteins/phosphopeptides (CPP) from complex mixtures is reported herein. This method employs ceramic hydroxyapatite (HA) as a solid-phase adsorbent to efficiently capture phosphoproteins and CPP from complex media. Casein was chosen as the model phosphoprotein to test the protocol. CPP immobilized on HA microgranules formed a complex that was included in the matrix-assisted laser desorption/ionization mass spectrometry (MALDI) matrix before desorbing directly from the well plate. Casein fractions with different levels of phosphorylation were desorbed based upon the specific concentration of trifluoroacetic acid (TFA) included in the MALDI matrix. The HA-bound casein enzymolysis was performed in situ with trypsin to remove non-phosphorylated peptides and isolate the immobilized CPP. The latter were recovered by centrifugation, dried, and co-crystallized with a 1% phosphoric acid (PA) solution in the matrix that was appropriate for detecting CPP in MALDI-MS spectra. This approach for the selection of casein/CPP resulted in the identification of 32 CPP by MALDI-time of flight (TOF). The analytical process involved two steps requiring ∼2 h, excluding the time required for the enzymatic reaction. The alkaline phosphatase (AP)-assisted de-phosphorylation of tryptic CPP allowed the phosphorylation level of peptides to be calculated concurrently with MALDI-TOF MS and liquid chromatography-electrospray ionization–mass spectrometry (LC–ESI–MS/MS). The effectiveness of the extraction procedure assayed on eggshell phosphoproteins resulted in the identification of 5 phosphoproteins and 14 derived phosphopeptides with a phosphoprotein global recovery of ∼70% at least.     

Anticandidal effect of berry juices and extracts from Ribes species

The biological activities of fruit juices and pomace (skin, seeds) extracts from blackcurrant (Ribes nigrum), gooseberry (Ribes uva-crispa) and their hybrid plant (jostaberry, Ribes × nidigrolaria) were evaluated against the most frequently isolated twelve human pathogenic Candida species by broth dilution tests. The phenolic content of juice, water and methanol extracts were measured and the relationship with antifungal activity was assessed. Growth of the most Candida species was inhibited, with the exception of C. albicans, C. krusei, C. lusitaniae and C. pulcherrima. R. nigrum, with the highest phenol content, was observed to have the highest anticandidal activity, indicating a positive correlation between phenol content and antifungal activity.     

Analysis of epithelial–mesenchymal transition markers in psoriatic epidermal keratinocytes

Psoriasis is similar to endpoints of epithelial–mesenchymal transition (EMT), a process of epithelial cells transformed into fibroblast-like cells. The molecular epithelial and mesenchymal markers were analysed in psoriatic keratinocytes. No obvious alteration of epithelial markers E-cadherin (E-cad), keratin 10 (K10), K14 and K16 was detected in psoriatic keratinocytes. However, significantly increased expression of Vim, FN, plasminogen activator inhibitor 1 (PAI-1) and Slug was seen. IL-17A and IL-13 at 50 ng ml⁻¹ strongly decreased expression of K10, Vim and FN. TGF-β1 at 50 ng ml⁻¹ promoted the production of N-cad, Vim, FN and PAI-1. Slug was decreased by dexamethasone (Dex), but E-cad was upregulated by Dex. Silencing of ERK partially increased E-cad and K16, but remarkably inhibited K14, FN, Vim, β-catenin, Slug and α5 integrin. Moreover, inhibition of Rho and GSK3 by their inhibitors Y27632 and SB216763, respectively, strongly raised E-cad, β-catenin and Slug. Dex decreased Y27632-mediated increase of β-catenin. Dex at 2.0 µM inhibited SB216763-regulated E-cad, β-catenin and slug. In conclusion, EMT in psoriatic keratinocytes may be defined as an intermediate phenotype of type 2 EMT. ERK, Rho and GSK3 play active roles in the process of EMT in psoriatic keratinocytes.     

Association of RNA with the uracil-DNA-degrading factor has major conformational effects and is potentially involved in protein folding

Recently, a novel uracil-DNA-degrading factor protein (UDE) was identified in Drosophila melanogaster, with homologues only in pupating insects. Its unique uracil-DNA-degrading activity and a potential domain organization pattern have been described. UDE seems to be the first representative of a new protein family with unique enzyme activity that has a putative role in insect development. In addition, UDE may also serve as potential tool in molecular biological applications. Owing to lack of homology with other proteins with known structure and/or function, de novo data are required for a detailed characterization of UDE structure and function. Here, experimental evidence is provided that recombinant protein is present in two distinct conformers. One of these contains a significant amount of RNA strongly bound to the protein, influencing its conformation. Detailed biophysical characterization of the two distinct conformational states (termed UDE and RNA-UDE) revealed essential differences. UDE cannot be converted into RNA-UDE by addition of the same RNA, implying putatively joint processes of RNA binding and protein folding in this conformational species. By real-time PCR and sequencing after random cloning, the bound RNA pool was shown to consist of UDE mRNA and the two ribosomal RNAs, also suggesting cotranslational RNA-assisted folding. This finding, on the one hand, might open a way to obtain a conformationally homogeneous UDE preparation, promoting successful crystallization; on the other hand, it might imply a further molecular function of the protein. In fact, RNA-dependent complexation of UDE was also demonstrated in a fruit fly pupal extract, suggesting physiological relevance of RNA binding of this DNA-processing enzyme.     

Corn hybrids display lower metabolite variability and complex metabolite inheritance patterns

We conducted a comparative analysis of the root metabolome of six parental maize inbred lines and their 14 corresponding hybrids showing fresh weight heterosis. We demonstrated that the metabolic profiles not only exhibit distinct features for each hybrid line compared with its parental lines, but also separate reciprocal hybrids. Reconstructed metabolic networks, based on robust correlations between metabolic profiles, display a higher network density in most hybrids as compared with the corresponding inbred lines. With respect to metabolite level inheritance, additive, dominant and overdominant patterns are observed with no specific overrepresentation. Despite the observed complexity of the inheritance pattern, for the majority of metabolites the variance observed in all 14 hybrids is lower compared with inbred lines. Deviations of metabolite levels from the average levels of the hybrids correlate negatively with biomass, which could be applied for developing predictors of hybrid performance based on characteristics of metabolite patterns.     

The role of Sprouty1 in the proliferation,differentiation and apoptosis of epidermal keratinocytes

Objectives

Sprouty (SPRY) 1 is one of the SPRY proteins that inhibits signalling from various growth factors pathways and has also been known as a tumour suppressor in various malignancies. However, no study elucidates the role of SPRY1 in the skin. Our study was conducted to determine the function of SPRY1 in human keratinocytes and the epidermis.

Materials and methods

In vitro primary cultured epidermal keratinocytes were used to investigate the proliferation, differentiation and apoptosis of these cells. We also established overexpression of SPRY1 in vitro and K14‐SPRY1 transgenic mice.

Results

SPRY1 was mainly located in the cytoplasm of the epidermal keratinocytes from the granular epidermal layer of the skin and cultured cells. Overexpressed SPRY1 in keratinocytes resulted in up‐regulation of P21, P27 and down‐regulation of cyclin B1; decrease in MMP3 and integrin α6. SPRY1‐overexpressed primary keratinocytes exhibited a lower proliferation and migration capability and higher rates of apoptosis. Epidermis of SPRY1‐TG mice represented delayed wound healing. Proteomics analysis and GO enrichment showed DEPs of SPRY1 TG mice epidermis is significantly enriched in immune‐ and inflammatory‐associated biological process.

Conclusions

In summary, SPRY1 expression was inversely correlated with cell proliferation, migration and promote cell apoptosis of keratinocytes. SPRY1 maybe a negative feedback regulator in normal human epidermal keratinocytes and cutaneous inflammatory responses. Our study raised the possibility that enhancing expression of SPRY1 may have the potential to promote anti‐inflammatory effects.

     

Protein Expression Profile of HT-29 Human Colon Cancer Cells after Treatment with a Cytotoxic Daunorubicin-GnRH-III Derivative Bioconjugate

Targeted delivery of chemotherapeutic agents is a new approach for the treatment of cancer, which provides increased selectivity and decreased systemic toxicity. We have recently developed a promising drug delivery system, in which the anticancer drug daunorubicin (Dau) was attached via oxime bond to a gonadotropin-releasing hormone-III (GnRH-III) derivative used as a targeting moiety (Glp-His-Trp-Lys(Ac)-His-Asp-Trp-Lys(Dau = Aoa)-Pro-Gly-NH₂; Glp = pyroglutamic acid, Ac = acetyl; Aoa = aminooxyacetyl). This bioconjugate exerted in vitro cytostatic/cytotoxic effect on human breast, prostate and colon cancer cells, as well as significant in vivo tumor growth inhibitory effect on colon carcinoma bearing mice. In our previous studies, H-Lys(Dau = Aoa)-OH was identified as the smallest metabolite produced in the presence of rat liver lysosomal homogenate, which was able to bind to DNA in vitro. To get a deeper insight into the mechanism of action of the bioconjugate, changes in the protein expression profile of HT-29 human colon cancer cells after treatment with the bioconjugate or free daunorubicin were investigated by mass spectrometry-based proteomics. Our results indicate that several metabolism-related proteins, molecular chaperons and proteins involved in signaling are differently expressed after targeted chemotherapeutic treatment, leading to the conclusion that the bioconjugate exerts its cytotoxic action by interfering with multiple intracellular processes.