Plasmodium falciparum Cyclic GMP-Dependent Protein Kinase Interacts with a Subunit of the Parasite Proteasome.

Malaria is caused by the protozoan parasite Plasmodium, which undergoes a complex life cycle in a human host and a mosquito vector. The parasite's cyclic GMP (cGMP)-dependent protein kinase (PKG) is essential at multiple steps of the life cycle. Phosphoproteomic studies in Plasmodium falciparum erythrocytic stages and Plasmodium berghei ookinetes have identified proteolysis as a major biological pathway dependent on PKG activity. To further understand PKG's mechanism of action, we screened a yeast two-hybrid library for P. falciparum proteins that interact with P. falciparum PKG (PfPKG) and tested peptide libraries to identify its phosphorylation site preferences. Our data suggest that PfPKG has a distinct phosphorylation site and that PfPKG directly phosphorylates parasite RPT1, one of six AAA+ ATPases present in the 19S regulatory particle of the proteasome. PfPKG and RPT1 interact in vitro, and the interacting fragment of RPT1 carries a PfPKG consensus phosphorylation site; a peptide carrying this consensus site competes with the RPT1 fragment for binding to PfPKG and is efficiently phosphorylated by PfPKG. These data suggest that PfPKG's phosphorylation of RPT1 could contribute to its regulation of parasite proteolysis. We demonstrate that proteolysis plays an important role in a biological process known to require Plasmodium PKG: invasion by sporozoites of hepatocytes. A small-molecule inhibitor of proteasomal activity blocks sporozoite invasion in an additive manner when combined with a Plasmodium PKG-specific inhibitor. Mining the previously described parasite PKG-dependent phosphoproteomes using the consensus phosphorylation motif identified additional proteins that are likely to be direct substrates of the enzyme.

The effect of flurbiprofen oral spray and ibuprofen vs ibuprofen alone on postoperative tonsillectomy pain: An open, randomised, controlled trial.

OBJECTIVE: This prospective clinical study was carried out to evaluate the analgesic efficacy and safety of oral spray form of flurbiprofen in the treatment of postoperative pain in tonsillectomy patients. STUDY DESIGN: Open, randomised, controlled clinical study. SETTING: Tertiary care training and research hospital. PARTICIPANTS: One hundred (53 males, 47 females) with an age range of 18-53 years old (mean 27.4 ± 9.3 SD) undergoing tonsillectomy were enrolled in this prospective controlled study. MAIN OUTCOME MEASURES: Patients receiving oral ibuprofen and flurbiprofen as spray form were enrolled as study group (53), whereas patients receiving only oral ibuprofen were enrolled as control group (47) in postoperative period. Postoperative pain was evaluated through visual analogue scale on 12th hour, first, third and seventh days after surgery. RESULTS: The mean maximal pain score of patients who have received flurbiprofen spray and ibuprofen was 3.36 ± 1.93 SD that was statistically lower than the mean maximal pain score of patients who were medicated with only ibuprofen which was 4.06 ± 1.29 SD on postoperative seventh day (P = .013). CONCLUSION: This study revealed that addition of flurbiprofen spray to oral ibuprofen is effective in the management of postoperative pain in tonsillectomy patients with no notable complications.

Genotoxic evaluation of poly(anhydride) nanoparticles in the gastrointestinal tract of mice.

Gantrez® AN 119-based NPs have been developed as oral drug carriers due to their strong bioadhesive interaction with components of the gastrointestinal mucosa and to their adaptable surface. The use of mannosamine to coat Gantrez® AN 119-based NPs results in a high mucus-permeable carrier, able to reach the gastrointestinal epithelium. Although their efficacy to transport a therapeutic agent has been demonstrated, their safety has not yet been thoroughly studied. They have proved to be non-cytotoxic, non-genotoxic and non-mutagenic in vitro; however, the in vivo toxicity profile has not yet been determined. In this study, the in vivo genotoxic potential of Gantrez® AN 119 NPs coated with mannosamine (GN-MA-NP) has been assessed using the in vivo comet assay in combination with the enzyme formamidopyrimidine DNA glycosylase in mice, following the OECD test guideline 489. To determine the relevant organs to analyse and the sampling times, an in vivo biodistribution study was also carried out. Results showed a statistically significant induction of DNA strand breaks and oxidized bases in the duodenum of animals exposed to 2000 mg/kg bw. However, this effect was not observed at lower doses (i.e. 500 and 1000 mg/kg which are closer to the potential therapeutic doses) or in other organs. In conclusion, GN-MA-NP are promising nanocarriers as oral drug delivery systems.

Attachment and growth of dental pulp stem cells on dentin in presence of extra calcium.

OBJECTIVE: We aimed to differentiate dental pulp stem cells (DPSC) to odontoblast-like cells (ODPSC) and to investigate their attachment and growth on dentin in the presence of extra calcium by colorimetric assay and scanning electron microscopy (SEM). METHODS: After isolation of DPSC, they were differentiated to ODPSC. Standard dentin discs from human molar teeth were prepared. While the dentin discs in Group 1 did not receive any extra treatment, the discs in Group 2 were treated with acidic calcium phosphate precipitation (CPP) solution. In Group 3, the discs were suspended in phosphate buffered saline containing calcium. DPSC or ODPSC (3×10(4) cells/mL) were seeded on all discs and incubated for 7, 14 or 21 days. Attachment and growth of 7-day cell cultures on extra dentin samples were examined by SEM. MTT assay showed that number of cells on dentin surfaces was increased by time periods regardless of type of treatment and cells (p<0.05). RESULTS: While DPSC and ODPSC showed similar proliferation rates at 7 and 14days (p>0.05), the number of ODPSC was higher than DPSC in 21-day samples (p=0.039). MTT assay showed that number of cells on dentin surfaces was increased by time periods regardless of type of treatment and cells (p<0.05). Calcium-treated dentin surfaces always had lower number of cells; being significant for only CPP-treated surfaces (p<0.01). Both types of cells demonstrated good attachment and proliferation on dentin surfaces regardless of type of dentin treatment. CONCLUSIONS: Because the nature of dentin surface itself showed good adhesive characteristics with ODPSC and DPSC, additional calcium treatment of dentin surfaces may not be necessary.

Investigation of telomerase activity and apoptosis on invasive ductal carcinoma of the breast using immunohistochemical and Western blot methods.

OBJECTIVE: Invasive ductal carcinoma (IDC) comprises the largest group of breast cancers. This study aimed to investigate telomerase activity and apoptosis using immunohistochemical and Western blot methods. PATIENTS AND METHODS: In total, 75 cases that had been diagnosed as IDC and 20 cases that had undergone a freezing procedure were included. The histological sections were stained with Bax, Bcl-2, hTERT and BNIP3. The ages of the patients, as well as their hormonal status and tumour sizes and grades were evaluated, as well as the staining characteristics of the antibodies in question. RESULTS: A decrease in Bcl-2 positivity and an increase in Bax positivity were found immunohistochemically with increasing tumour grades. The data obtained by western blot method showed that Bcl-2 was highest in grade 1 tumours although these results were not statistically significant. The relationship between estrogen and progesterone receptor positivity and Bcl-2 was statistically significant, suggesting there is hormonal control through apoptosis. BNIP3 was found to be decreased with increasing tumour grades. Similarly, BNIP3 was found to be having the lowest value in grade 3 tumours by western blot method. Furthermore, hTERT was found to be increased with increasing tumour grades. In the western blot method, hTERT increased nearly four-fold compared to the control. In addition, hTERT, which was seen in very high levels in tumours, may be a helpful cancer marker. Both hTERT and BNIP3 are important markers that can provide information about prognosis. CONCLUSIONS: Big improvements can be achieved in tumour progression control with new treatment modalities that stop telomerase activity and hypoxic cell death.

Gene electrotransfer of plasmid AMEP, an integrin-targeted therapy, has antitumor and antiangiogenic action in murine B16 melanoma.

Gene therapy with Plasmid AMEP (antiangiogenic metargidin peptide) has recently been studied as a potential targeted therapy for melanoma. This plasmid is designed to downregulate α5ß1 and αvß3 integrins. In our study, electroporation was used as a nonviral delivery system. We investigated the antiangiogenic and direct antitumor effectiveness of this gene therapy on low and highly metastatic B16 melanoma variants. In vitro, the antiangiogenic effectiveness as determined by tube formation assay on endothelial cells was predominantly dependent on AMEP expression levels. In vivo, antitumor effectiveness was mediated by the inhibition of proliferation, migration and invasion of melanoma cells and correlated with the expression of integrins on tumor cells after intratumor delivery. In addition, reduced metastatic potential was shown. Intramuscular gene electrotransfer of Plasmid AMEP, for AMEP systemic distribution, had no antitumor effect with this specific preventive treatment protocol, confirming that direct tumor delivery was more effective. This study confirms our previous in vitro data that the expression levels of integrins on melanoma cells could be used as a biomarker for antitumor effectiveness in integrin-targeted therapies, whereas the expression levels of AMEP peptide could be a predictive factor for antiangiogenic effectiveness of Plasmid AMEP in the treatment of melanoma.

Stefin B deficiency reduces tumor growth via sensitization of tumor cells to oxidative stress in a breast cancer model.

Lysosomal cysteine cathepsins contribute to proteolytic events promoting tumor growth and metastasis. Their enzymatic activity, however, is tightly regulated by endogenous inhibitors. To investigate the role of cathepsin inhibitor stefin B (Stfb) in mammary cancer, Stfb null mice were crossed with transgenic polyoma virus middle T oncogene (PyMT) breast cancer mice. We show that ablation of Stfb resulted in reduced size of mammary tumors but did not affect their rate of metastasis. Importantly, decrease in tumor growth was correlated with an increased incidence of dead cell islands detected in tumors of Stfb-deficient mice. Ex vivo analysis of primary PyMT tumor cells revealed no significant effects of ablation of Stfb expression on proliferation, angiogenesis, migration and spontaneous cell death as compared with control cells. However, upon treatment with the lysosomotropic agent Leu-Leu-OMe, cancer cells lacking Stfb exhibited a significantly higher sensitivity to apoptosis. Moreover, Stfb-ablated tumor cells were significantly more prone to cell death under increased oxidative stress. These results indicate an in vivo role for Stfb in protecting cancer cells by promoting their resistance to oxidative stress and to apoptosis induced through the lysosomal pathway.

Dermoscopic nevus patterns in skin of colour: a prospective, cross-sectional, morphological study in individuals with skin type V and VI.

BACKGROUND: Most of the knowledge on the prevailing dermoscopic patterns of acquired melanocytic nevi (AMV) is based on studies in Caucasians, while little research focuses on the dermoscopic variability in nevi in skin of colour. OBJECTIVE: To analyse the prevalent dermoscopic nevus patterns in subjects with a skin type (ST) V and VI. METHODS: Prospective, cross-sectional, morphological study was conducted in six clinics with enrolment of consecutive individuals with a ST V or VI. Digital dermoscopic images of selected representative AMN were assessed for dermoscopic colours, morphological patterns and pigment distribution. RESULTS: Analysis of 300 nevi from subjects with ST V and VI revealed significant differences in the nevus pattern between these two groups. The majority of nevi in ST V revealed a reticular pattern, whereas persons with ST VI more frequently exhibited a structureless pattern. Black, blue and grey were more frequent in ST VI, whereas the vast majority of nevi in ST V individuals showed dark brown colour. CONCLUSIONS: Our study provides new insights into the nevus pattern in individuals with a dark pigmentary trait, which may aid the diagnosis and management of nevi in this patients group.

Siramesine triggers cell death through destabilisation of mitochondria, but not lysosomes.

A sigma-2 receptor agonist siramesine has been shown to trigger cell death of cancer cells and to exhibit a potent anticancer activity in vivo. However, its mechanism of action is still poorly understood. We show that siramesine can induce rapid cell death in a number of cell lines at concentrations above 20 µM. In HaCaT cells, cell death was accompanied by caspase activation, rapid loss of mitochondrial membrane potential (MMP), cytochrome c release, cardiolipin peroxidation and typical apoptotic morphology, whereas in U-87MG cells most apoptotic hallmarks were not notable, although MMP was rapidly lost. In contrast to the rapid loss of MMP above 20 µM siramesine, a rapid increase in lysosomal pH was observed at all concentrations tested (5-40 µM); however, it was not accompanied by lysosomal membrane permeabilisation (LMP) and the release of lysosomal enzymes into the cytosol. Increased lysosomal pH reduced the lysosomal degradation potential as indicated by the accumulation of immature forms of cysteine cathepsins. The lipophilic antioxidant α-tocopherol, but not the hydrophilic antioxidant N-acetyl-cysteine, considerably reduced cell death and destabilisation of mitochondrial membranes, but did not prevent the increase in lysosomal pH. At concentrations below 15 µM, siramesine triggered cell death after 2 days or later, which seems to be associated with a general metabolic and energy imbalance due to defects in the endocytic pathway, intracellular trafficking and energy production, and not by a specific molecular event. Overall, we show that cell death in siramesine-treated cells is induced by destabilisation of mitochondria and is independent of LMP and the release of cathepsins into the cytosol. Moreover, it is unlikely that siramesine acts exclusively through sigma-2 receptors, but rather through multiple molecular targets inside the cell. Our findings are therefore of significant importance in designing the next generation of siramesine analogues with high anticancer potential.

Melanoma arising in chronic ulceration associated with lymphoedema.

Chronic ulceration can be complicated by development of a malignancy. The most frequent associated malignances are squamous cell carcinoma and basal cell carcinoma, although melanoma, leiomyosarcoma and adenocarcinoma are less commonly seen. Chronic lymphoedema may also predispose to development of some malignancies, including lymphangiosarcoma, squamous cell carcinoma and Kaposi's sarcoma. Here, we report the case of a 77-year-old man with primary lymphoedema, who developed melanoma in a chronic foot ulcer of 60 years' duration. The patient underwent wide excision for the melanoma, and remains free from metastases at 1-year follow up.

Rectus abdominal muscle endometriosis in a patient with cesarian scar: case report.

Endometriosis is the existence of endometrial tissue out of the intrauterine cavity. Abdominal wall endometrioma is a well-defined mass composed of endometrial glands and stroma that may develop after gynecologic and obstetrical surgeries. A cyclic painful mass at the site of a cesarean section scar is most likely due to an endometrioma, and wide local excision is the advisable treatment. The authors present a case of endometrioma in the abdominal wall, which was treated with local excision.

Random/aligned electrospun PCL/PCL-collagen nanofibrous membranes: comparison of neural differentiation of rat AdMSCs and BMSCs.

In this study, the aligned (A) and randomly oriented (R) polycaprolactone (PCL-A and PCL-R) and PCL/collagen (PCL/Col-A and PCL/Col-R) nanofibers were electrospun onto smooth PCL membranes (PCLMs) prepared by solvent casting. In order to investigate the effects of chemical composition and nanotopography of fibrous surfaces on proliferation and on neural differentiation of mesenchymal stem cells (MSCs), adipose and bone marrow-derived rat MSCs (AdMSCs and BMSCs) were cultivated in suitable media i.e. inducing medium containing basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF), and cell maintenance medium (CMM). BMSCs adhered and proliferated on all nanofibrous membranes more efficiently than AdMSCs. PCL/Col-A was found as the most convenient surface supporting proliferation in both cell types. Immunofluorescence staining indicated that BMSCs and AdMSCs are prone for differentiation to oligodendrocytes more than they differentiate to other neuronal cell types. PCL-A nanofibrous membranes supported differentiation of MSCs to O4(+) (an oligodendrocytes surface antigen) cells in both culture media. The intensity of immunoreactivity of O4(+) cells differentiated from BMSCs on PCL-A was highest when compared with the other groups (p < 0.001). Some BIII-T signed neural cells were investigated on PCL-A nanofibrous membranes, but the intensity of immunoreactivity was lower than that of O4(+) cells. In conclusion, this study can be evaluated to establish the cell therapy strategies in neurodegenerative disorders, which are relevant to oligodendrocyte abstinence using BMSCs or AdMSCs on aligned nanofibrous membranes.

MAGUKs, scaffolding proteins at cell junctions, are substrates of different proteases during apoptosis.

A major feature of apoptotic cell death is gross structural changes, one of which is the loss of cell-cell contacts. The caspases, executioners of apoptosis, were shown to cleave several proteins involved in the formation of cell junctions. The membrane-associated guanylate kinases (MAGUKs), which are typically associated with cell junctions, have a major role in the organization of protein-protein complexes at plasma membranes and are therefore potentially important caspase targets during apoptosis. We report here that MAGUKs are cleaved and/or degraded by executioner caspases, granzyme B and several cysteine cathepsins in vitro. When apoptosis was induced by UV-irradiation and staurosporine in different epithelial cell lines, caspases were found to efficiently cleave MAGUKs in these cell models, as the cleavages could be prevented by a pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(OMe)fluoromethylketone. Using a selective lysosomal disrupting agent L-leucyl-L-leucine methyl ester, which induces apoptosis through the lysosomal pathway, it was further shown that MAGUKs are also cleaved by the cathepsins in HaCaT and CaCo-2 cells. Immunohistological data showed rapid loss of MAGUKs at the sites of cell-cell contacts, preceding actual cell detachment, suggesting that cleavage of MAGUKs is an important step in fast and efficient cell detachment.

Eruptive disseminated Spitz naevi: dermatoscopic features.

Eruptive disseminated Spitz naevi is a rarely reported condition. Although the dermatoscopic features of nondisseminated, solitary forms of Spitz naevi are well known, there are no reports describing the dermatoscopic features of eruptive disseminated variant. We report an additional case and describe the dermatoscopic features. Two patterns were observed. In all pink lesions, the vascular pattern was seen, composed of dotted, linear or comma-like vessels located at the centre of the meshes of the reticular depigmentation. In all brown lesions, we observed only the reticular pattern, which is quite interesting as the reticular pattern is a rare feature of Spitz naevi. This observation may be a special feature particularly seen in the eruptive disseminated variant. A superficial black network also accompanied reticular pattern in some lesions. In dichromatic lesions, both patterns were observed in different areas of the body.

In vitro antimicrobial activity of calcium hydroxide mixed with different vehicles against Enterococcus faecalis and Candida albicans.

OBJECTIVE: The purpose of this in vitro study was to investigate antimicrobial activity of calcium hydroxide (CH) in combination with glycerin, chlorhexidine gluconate (CHX), cetrimide, or distilled water against Enterococcus faecalis and Candida albicans. STUDY DESIGN: Standard holes in the cultivated agar plates were filled with one of the CH preparations and control agents. The zones of microbial inhibition were measured after incubation period. RESULTS: The CH preparations with glycerin and CHX demonstrated more antifungal activity than CH preparations with cetrimide and distilled water. The CH-glycerin preparations had no effect against E. faecalis, and CH-CHX preparation was the most effective medication. CONCLUSION: Antimicrobial activity of CH may change with the type of the vehicle and against different microorganisms. Enterococcus faecalis was more resistant than C. albicans to CH preparations.

The effect of treatment of radicular dentin on colonization patterns of C. albicans.

OBJECTIVE: The aim of this study was to observe the colonization pattern of C. albicans on treated and untreated radicular dentin. STUDY DESIGN: Root sections of 10 human mandibular premolar teeth were longitudinally separated into halves. The 20 halves were separated into 2 groups and each half served as its own control. In Group 1, only gross pulpal remnants were removed with pliers. Root canal walls in the corresponding 102 halves (Group 2) were instrumented with Gates-Glidden burs and treated with sequential use of 15% EDTA solution for 3 minutes and 2.5% NaOCl solution for 3 minutes. Finally, all teeth were washed with distilled water. Each specimen was placed individually in each well of a 24-well cell culture plate. After the assembly was sterilized with ethylene-oxide, the root canal of each specimen was inoculated with 20 microL of C. albicans (1-1.5 x 10(6) cfu/mL) that was kept in place for 24 hours for initial attachment. Then, 2 mL of SDB was added to each well and the assembly was placed in an incubator at 37 degrees C for 10 days. Following the incubation period, the specimens were washed, fixed, dehydrated, and processed for scanning electron microscopy. RESULTS: C. albicans was present on the root canal surfaces of all specimens; however, the colonization pattern was different. In the untreated group, the main growth pattern was a dense mass of yeast cells forming biofilm layers while hyphal structures were not common. On the other hand, pseudohyphae invaded all root canal surfaces in Group 2 and yeast cells were occasionally observed. CONCLUSION: The treatment procedures of root canal dentin have a strong influence on the colonization pattern of C. albicans. This fact should be considered when planning and evaluating in vitro Candida adhesion and/or penetration studies.