Discovery of Protein-Protein Interaction Inhibitors by Integrating Protein Engineering and Chemical Screening Platforms.

Protein-protein interactions (PPIs) govern intracellular life, and identification of PPI inhibitors is challenging. Roadblocks in assay development stemming from weak binding affinities of natural PPIs impede progress in this field. We postulated that enhancing binding affinity of natural PPIs via protein engineering will aid assay development and hit discovery. This proof-of-principle study targets PPI between linear ubiquitin chains and NEMO UBAN domain, which activates NF-κB signaling. Using phage display, we generated ubiquitin variants that bind to the functional UBAN epitope with high affinity, act as competitive inhibitors, and structurally maintain the existing PPI interface. When utilized in assay development, variants enable generation of robust cell-based assays for chemical screening. Top compounds identified using this approach directly bind to UBAN and dampen NF-κB signaling. This study illustrates advantages of integrating protein engineering and chemical screening in hit identification, a development that we anticipate will have wide application in drug discovery.

Functional and cell surface characteristics of periodontal ligament cells (PDLCs) on RGD-synthetic polypeptide conjugate coatings.

BACKGROUND AND OBJECTIVE: Periodontal ligament cells (PDLCs) are an important source for periodontal tissue healing and regeneration. Proper cell adhesion is a key for survival of anchorage-dependent cells and also initiates further intracellular signals for essential cellular functions. We aimed to test 3 different synthetic conjugates with integrin-binding RGD sequence (SAK-c[RGDfC], AK-c[RGDfC], and SAK-opn on the adhesion of human PDLCs and subsequent events including proliferation, migration, behavior of cell surface molecules, and osteogenic differentiation. MATERIALS AND METHODS: Synthetic peptides were synthesized by solid-phase technique and attached to branched chain polymeric polypeptides via thioether linkage. Simple adsorption method was used to coat tissue culture plastic or electric arrays. PDLCs were isolated from 24 surgically extracted human third molars. Cell adhesion and proliferation were measured with real-time impedimetric xCELLigence SP system. Cell migration assay was performed with Ibidi® Culture inserts. Cell surface antigens were detected using flow cytometry analysis. Osteogenic differentiation was assessed with alkaline phosphatase (ALP) assay and Alizarin Red S staining, and real-time qPCR was performed to analyze the osteoblast-related gene expression. Osteogenic differentiation and adipogenic differentiation of PDLCs were monitored by real-time Electrical Cell-Substrate Impedance Spectroscopy (ECIS). RESULTS: Primary outcome of this study relies on that all three synthetic RGD peptides improved PDLC adhesion (P < .05). When animal serum is absent in culture medium, SAK-c[RGDfC] and AK-c[RGDfC] elevated cell adhesion (P < .05). Cell migration was enhanced by SAK-c[RGDfC] and AK-c[RGDfC] (P < .05). After 1-week treatment, all synthetic peptides elevated CD105 (1.7- to 2.2-fold) and CD146 (1.3- to 1.5-fold) markers and caused different integrin patterns. ALP activity (1.4-fold) and ARS (1.8- and 2.0-fold) were increased by SAK-c[RGDfC] and AK-c[RGDfC] in absence of osteogenic supplements, and all the peptides supported the mineralization under osteogenic condition (P < .05). RT-qPCR revealed the upregulation of bone sialoprotein (5.0- to 7.8-fold), osteocalcin (2.3- to 2.7-fold), and ALP (1.9- to 2.3-fold) gene expression in osteogenesis-induced PDLCs. ECIS monitoring showed that higher impedance was generated by the osteogenic induction compared with the adipogenic or the non-induced (P < .05). CONCLUSIONS: Our study demonstrates that SAK-c[RGDfC] and AK-c[RGDfC] improved adhesion and migration of PDLCs and supported osteogenic differentiation of PDLCs. These cyclic RGD peptides proved to be applicable biocompatible material in regenerative medicine.

The combination of hawthorn extract and camphor significantly increases blood pressure: A meta-analysis and systematic review.

BACKGROUND: A fixed combination of hawthorn and camphor (Korodin Herz-Kreislauf-Tropfen®) has been used in the therapy of hypotension for decades. Although its efficacy was evaluated in clinical trials, these studies have not been critically assessed in meta-analyses. PURPOSE: To systematically evaluate the efficacy of a fix combination of camphor and hawthorn extract (Korodin®) on blood pressure and cognition compared to placebo, in a meta-analysis based on randomized controlled trials (RCTs). STUDY DESIGN: The meta-analysis was carried out following the PRISMA guidelines, using the PICO format, and it was registered in the PROSPERO register. METHODS: The Cochrane Central Register of Controlled Trials, PubMed, Embase, and Web of Science databases were searched for relevant studies. Placebo-controlled clinical studies involving adult patients receiving a fix combination of hawthorn extract and camphor were included. No language or publication year restrictions were applied. RESULTS: Four randomized trials including a total of 221 patients were pooled for statistical analysis. According to the present meta-analysis, the fixed combination of hawthorn and camphor significantly increases systolic and diastolic blood pressure compared to placebo (p-values: 0.017 and 0.049, respectively) and had a beneficial, but not statistically significant effect on the cognitive performance in the connect-the-numbers test (p-value: 0.071). CONCLUSION: Korodin® is an effective and presumably safe complementary therapy for the treatment of hypotension. Its blood pressure increasing effect is confirmed; however, the evidence supporting its use is very limited. The optimum dose and duration of treatment is still unclear. The comprehensive evaluation of efficacy and safety is required in further, high-quality clinical studies, involving larger patient populations and comparable endpoints.

The Efficacy of Saffron in the Treatment of Mild to Moderate Depression: A Meta-analysis.

Herbal products, especially Hypericum perforatum extracts, have been widely used as first-line treatments for mild to moderate depression. Recently, several randomized, controlled clinical trials have been conducted to evaluate the efficacy of another plant, saffron (Crocus sativus), in mild to moderate depression. We have carried out a literature review of currently available published randomized, controlled clinical trials to give an up-to-date evaluation of the efficacy of saffron in mild to moderate depression, compared to placebo or routinely used antidepressants. The meta-analysis is reported according to the preferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines using the PICO (patients, intervention, comparison, outcome) format and was conducted using the statistical programs Comprehensive Meta-analysis and RevMan. PubMed, Embase, the Cochrane Central Register of Controlled Trials, and Web of Science databases were searched for relevant studies. Only placebo or active controlled, randomized clinical studies involving patients suffering from mild to moderate depression and using pharmacological doses of saffron per os were included. Hedges' g was used to calculate effect sizes. Risk of bias was assessed using the Cochrane Collaboration tool, and heterogeneity was tested by both performing the Cochran's Q test and calculating Higgins' I2 indicator. Eleven randomized trials were included in the qualitative analysis, and nine were pooled for statistical analysis. According to the present meta-analysis, saffron has a significant effect on the severity of depression. Available data from randomized, controlled clinical trials support that saffron is significantly more effective than placebo (g = 0.891; 95% CI: 0.369 - 1.412, p = 0.001), and non-inferior to tested antidepressant drugs (g = - 0.246; 95% CI: - 0.495 - 0.004, p = 0.053).

Diverse effect of BMP-2 homodimer on mesenchymal progenitors of different origin.

Bone morphogenetic protein-2 (BMP-2), is a potential factor to enhance osseointegration of dental implants. However, the appropriate cellular system to investigate the osteogenic effect of BMP-2 in vitro in a standardized manner still needs to be defined. The aim of this study was to examine the effect of BMP-2 on the cell proliferation and osteogenic differentiation of human osteogenic progenitors of various origins: dental pulp stem cells (DPSC), human osteosarcoma cell line (Saos-2) and human embryonic palatal mesenchymal cell line (HEPM). For induction of osteogenic differentiation, cell culture medium was supplemented with BMP-2 homodimer alone or in combination with conventionally used differentiation inducing agents. Differentiation was monitored for 6-18 days. To assess differentiation, proliferation rate, alkaline phosphatase activity, calcium deposition and the expression level of osteogenic differentiation marker genes (Runx2, BMP-2) were measured. BMP-2 inhibited cell proliferation in a concentration and time-dependent manner. In a concentration which caused maximal cell proliferation, BMP-2 did not induce osteogenic differentiation in any of the tested systems. However, it had a synergistic effect with the osteoinductive medium in both DPSC and Saos-2, but not in HEPM cells. We also found that the differentiation process was faster in Saos-2 than in DPSCs. Osteogenic differentiation could not be induced in the osteoblast progenitor HEPM cells. Our data suggest that in a concentration that inhibits proliferation the differentiation inducing effect of BMP-2 is evident only in the presence of permissive osteoinductive components. ß-glycerophosphate, was identified interacting with BMP-2 in a synergistic manner.

Alteration of consciousness via diverse photo-acoustic stimulatory patterns. Phenomenology and effect on salivary flow rate, alpha-amylase and total protein levels.

Long-term photo-acoustic stimulation is used for the induction of altered states of consciousness for both therapeutic and experimental purposes. Long-term photo-acoustic stimulation also leads to changes in the composition of saliva which have a key contribution to the efficiency of this technique in easing mucosal symptoms of oral psychosomatic patients. The aim of this study is to find out whether there is any cumulative effect of repeated stimulation and whether there are any detectable differences between diverse stimulatory patterns of long lasting photo-acoustic stimulation on the phenomenology of the appearing trance state and on salivary secretion. There was significant cumulative effect in relation with the appearance of day dreaming as phenomenological parameter, and in relation with protein output and amylase/protein ratio as salivary parameter. Pattern specific effect was detectable in relation with salivary flow rate only. Although our results clearly indicate the existence of certain cumulative and stimulation-pattern specific effects of repeated photo-acoustic stimulation, the absolute values of all these effects were relatively small in this study. Therefore, in spite of their theoretical importance there are no direct clinical consequences of these findings. However, our data do not exclude at all the possibility that repeated stimulation with other stimulatory parameters may lead to more pronounced effects. Further studies are needed to make clear conclusion in this respect.

[The role of neoadjuvant therapy in the treatment of locally advanced squamous cell cancer of the cervical oesophagus]. / A neoadjuváns kezelés szerepe a nyaki szakaszon elhelyezkedo elorehaladott nyelocsolaphámrákok sebészi kezelésében.

INTRODUCTION: Cervical oesophagus represents a critical location for squamous cell carcinoma, which usually requires extensive surgery (pharyngo-laryngo-oesophagectomy). In the last decade, neoadjuvant chemo-radiotherapy was reported to be beneficial in the treatment of locally advanced squamous cell oesophageal cancer. METHODS: Between November 1997 and January 2012, 55 patients with locally advanced (T3-4) squamous cell oesophageal cancer received preoperative chemo-radiotherapy, where the tumour was localized in the upper third. Patients received preoperative irradiation of 3960 cGy in 180 cGy fractions and simultaneously Cisplatin and 5-FU chemotherapy. Restaging was carried out after four weeks and patients considered operable were underwent surgery. RESULTS: In patients with cervical oesophageal cancer 35 of 55 (64%) underwent oesophageal resection or pharyngo-laryngectomy. In 16 out of 35 resected specimens (46%) complete histopathological remission (pCR) was observed. Perioperative mortality and anastomotic leaks were the same: 5/35 (14%). R0 resection rate was 82% and the 2- and 5 years survival rates were 41% and 18%. In 19 cases a larynx preserving pharyngo-oesophagectomy was performed and a free jejunal graft was used for reconstruction after a pharyngo-laryngectomy in 11 cases. CONCLUSION: The high rate of pCR (46%) confirmed that upper third oesophageal cancer has superior sensitivity to multimodal treatment. In 30 cases neoadjuvant chemo-radiotherapy was able to achieve tumour regression and render pharyngo-laryngo-oesophagectomy unnecessary.

Inhibition of quorum-sensing signals by essential oils.

The role of quorum sensing (QS) is well known in microbial pathogenicity and antibiotic resistance. QS is responsible for motility, swarming, and biofilm production based on the signal molecules, e.g., acylated homoserine lactones (AHLs) produced by micro-organisms above certain population density. The inhibition of QS may reduce pathogenicity, antibiotic resistance and biofilm formation in systemic and local infections. The homoserine lactones and other transmitters contribute to antibiotic resistance and pathogenicity of several bacteria; consequently the inhibition of QS signals reduces the problem of resistance and virulence. Due to the increasing number of persistent non-treatable infections, there is an urgent need to develop new strategies to combat infections that destabilize bacterial communities in the host. The effect of essential oils on bacterial growth and QS were evaluated using the sensor strain Chromobacterium violaceum CV026 and N-acyl homoserine lactone (AHL) producing Escherichia coli ATTC 31298 and the grapevine colonizing Ezf 10-17 strains. Of the tested oils, rose, geranium, lavender and rosemary oils were the most potent QS inhibitors. Eucalyptus and citrus oils moderately reduced pigment production by CV026, whereas the chamomile, orange and juniper oils were ineffective.

Simultaneous PKC and cAMP activation induces differentiation of human dental pulp stem cells into functionally active neurons.

The plasticity of dental pulp stem cells (DPSCs) has been demonstrated by several studies showing that they appear to self-maintain through several passages, giving rise to a variety of cells. The aim of the present study was to differentiate DPSCs to mature neuronal cells showing functional evidence of voltage gated ion channel activities in vitro. First, DPSC cultures were seeded on poly-l-lysine coated surfaces and pretreated for 48h with a medium containing basic fibroblast growth factor and the demethylating agent 5-azacytidine. Then neural induction was performed by the simultaneous activation of protein kinase C and the cyclic adenosine monophosphate pathway. Finally, maturation of the induced cells was achieved by continuous treatment with neurotrophin-3, dibutyryl cyclic AMP, and other supplementary components. Non-induced DPSCs already expressed vimentin, nestin, N-tubulin, neurogenin-2 and neurofilament-M. The inductive treatment resulted in decreased vimentin, nestin, N-tubulin and increased neurogenin-2, neuron-specific enolase, neurofilament-M and glial fibrillary acidic protein expression. By the end of the maturation period, all investigated genes were expressed at higher levels than in undifferentiated controls except vimentin and nestin. Patch clamp analysis revealed the functional activity of both voltage-dependent sodium and potassium channels in the differentiated cells. Our results demonstrate that although most surviving cells show neuronal morphology and express neuronal markers, there is a functional heterogeneity among the differentiated cells obtained by the in vitro differentiation protocol described herein. Nevertheless, this study clearly indicates that the dental pulp contains a cell population that is capable of neural commitment by our three step neuroinductive protocol.

Comparison of dialysis and clinical characteristics of patients with frequent and occasional hemodialysis-associated hypotension.

BACKGROUND: Symptomatic dialysis hypotension (DH) continues to be a common problem. By comparing patients prone and resistant to DH, several dialysis session and patient related characteristics have been identified that confer susceptibility to DH. Less is known, however, about the comparison of patients with frequent and only occasional DH. The aim of the study was to compare clinical and dialysis-session- (complicated by hypotension) related data between those with frequent (fDH) and those with occasional dialysis hypotension (oDH). METHODS: Nine hundred and fifty-eight patients at 11 dialysis units were followed for 10 months and characteristics of patients with fDH (> or = 10 hypotensive events necessitating medical intervention) (n = 96) were compared to that of patients with oDH (1 or 2 events/10 months) (n = 130). Significant and independent predictors of fDH were obtained by multivariate logistic regression. RESULTS: Significant differences between fDH vs. oDH patients were older age (64.4 vs. 56.9 years, p < 0.001), more females (66 vs. 46%, p < 0.005) in fDH. More fDH patients had diabetes (27 vs. 15%, p < 0.05) and less had glomerulonephritis (15 vs. 35%, p < 0.001) as the cause for ESRD. Coronary artery disease (68 vs. 50%, p < 0.01) and long-acting nitrate treatment (51 vs. 30%, p < 0.001) was more frequent while treatment with ACEI (33 vs. 48%, p < 0.05) or Ca-channel blockers (40 vs. 53%, p < 0.05) were less frequent in patients with fDH. Patients with fDH had higher serum phosphorus levels (1.99 vs. 1.79 mmol, p < 0.005). Dialysis session related data were similar but the hypotensive episode occurred earlier during dialysis in fDH (136 vs. 156 min, p < 0.01). In multivariate analysis, significant independent predictors of fDH were older age (OR = 1.04 [1.02-1.07]), lack of glomerulonephritis as renal diagnosis (2.63 [1.18-5.87]), high phosphorus levels (5.0 [2.45-10.0]), lack of use of Ca-channel blockers (2.09 [1.12-3.91]), and the use of nitrates (2.38 [1.24-4.55]). CONCLUSION: Features of the dialysis sessions complicated by DH seem to be similar between patients with fDH and oDH, while patient characteristics such as older age, renal diagnosis other than glomerulonephritis, higher serum phosphorus levels, use of nitrates, and lack of use of calcium channel blockers are significantly and independently associated with fDH.