Initiation of DNA replication requires actin dynamics and formin activity.

Nuclear actin regulates transcriptional programmes in a manner dependent on its levels and polymerisation state. This dynamics is determined by the balance of nucleocytoplasmic shuttling, formin- and redox-dependent filament polymerisation. Here, using Xenopus egg extracts and human somatic cells, we show that actin dynamics and formins are essential for DNA replication. In proliferating cells, formin inhibition abolishes nuclear transport and initiation of DNA replication, as well as general transcription. In replicating nuclei from transcriptionally silent Xenopus egg extracts, we identified numerous actin regulators, and disruption of actin dynamics abrogates nuclear transport, preventing NLS (nuclear localisation signal)-cargo release from RanGTP-importin complexes. Nuclear formin activity is further required to promote loading of cyclin-dependent kinase (CDK) and proliferating cell nuclear antigen (PCNA) onto chromatin, as well as initiation and elongation of DNA replication. Therefore, actin dynamics and formins control DNA replication by multiple direct and indirect mechanisms.

Using conformational constraints at position 6 of Angiotensin II to generate compounds with enhanced AT2R selectivity and proteolytic stability.

The Renin-Angiotensin System (RAS) plays a crucial role in numerous pathological conditions. Two of the critical RAS players, the angiotensin receptors AT1R and AT2R, possess differential functional profiles, although they share high sequence similarity. Although the main focus has been placed on AT1R, several epidemiological studies have evidenced that activation of AT2R could operate as a multimodal therapeutic target for different diseases. Thus, the development of selective AT2R ligands could have a high clinical potential for different therapeutic directions. Furthermore, they could serve as a powerful tool to interrogate the molecular mechanisms that are mediated by AT2R. Based on our recently established high affinity and AT2R selective compound [Y]6-AII we developed several analogues through modifying aminoacids located at positions 6 and 7 with various conformationally constrained analogues to enhance both the selectivity and stability. We report the development of high-affinity AT2R binders, which displayed high selectivity for AT2R versus AT1R. Furthermore, all analogues presented enhanced stability in human plasma with respect to the parent hormone Angiotensin II as also [Y]6-AII.

Chemical Profiling, Bioactivity Evaluation and the Discovery of a Novel Biopigment Produced by Penicillium purpurogenum CBS 113139.

Biobased pigments are environmentally friendly alternatives to synthetic variants with an increased market demand. Production of pigments via fermentation is a promising process, yet optimization of the production yield and rate is crucial. Herein, we evaluated the potential of Penicillium purpurogenum to produce biobased pigments. Optimum sugar concentration was 30 g/L and optimum C:N ratio was 36:1 resulting in the production of 4.1-4.5 AU (namely Pigment Complex A). Supplementation with ammonium nitrate resulted in the production of 4.1-4.9 AU (namely Pigment Complex B). Pigments showed excellent pH stability. The major biopigments in Pigment Complex A were N-threonyl-rubropunctamin or the acid form of PP-R (red pigment), N-GABA-PP-V (violet pigment), PP-O (orange pigment) and monascorubrin. In Pigment Complex B, a novel biopigment annotated as N-GLA-PP-V was identified. Its basic structure contains a polyketide azaphilone with the same carboxyl-monascorubramine base structure as PP-V (violet pigment) and γ-carboxyglutamic acid (GLA). The pigments were not cytotoxic up to 250 µg/mL.

Synthetic Analogues of Aminoadamantane as Influenza Viral Inhibitors-In Vitro, In Silico and QSAR Studies.

A series of nineteen amino acid analogues of amantadine (Amt) and rimantadine (Rim) were synthesized and their antiviral activity was evaluated against influenza virus A (H3N2). Among these analogues, the conjugation of rimantadine with glycine illustrated high antiviral activity combined with low cytotoxicity. Moreover, this compound presented a profoundly high stability after in vitro incubation in human plasma for 24 h. Its thermal stability was established using differential and gravimetric thermal analysis. The crystal structure of glycyl-rimantadine revealed that it crystallizes in the orthorhombic Pbca space group. The structure-activity relationship for this class of compounds was established, with CoMFA (Comparative Molecular Field Analysis) 3D-Quantitative Structure Activity Relationships (3D-QSAR) studies predicting the activities of synthetic molecules. In addition, molecular docking studies were conducted, revealing the structural requirements for the activity of the synthetic molecules.

Protein phosphatase 2A controls the order and dynamics of cell-cycle transitions.

Bistability of the Cdk1-Wee1-Cdc25 mitotic control network underlies the switch-like transitions between interphase and mitosis. Here, we show by mathematical modeling and experiments in Xenopus egg extracts that protein phosphatase 2A (PP2A), which can dephosphorylate Cdk1 substrates, is essential for this bistability. PP2A inhibition in early interphase abolishes the switch-like response of the system to Cdk1 activity, promoting mitotic onset even with very low levels of Cyclin, Cdk1, and Cdc25, while simultaneously inhibiting DNA replication. Furthermore, even if replication has already initiated, it cannot continue in mitosis. Exclusivity of S and M phases does not depend on bistability only, since partial PP2A inhibition prevents replication without inducing mitotic onset. In these conditions, interphase-level mitotic kinases inhibit Cyclin E-Cdk2 chromatin loading, blocking initiation complex formation. Therefore, by counteracting both Cdk1 activation and activity of mitotic kinases, PP2A ensures robust separation of S phase and mitosis and dynamic transitions between the two states.

Exploiting the beneficial effects of Salvia officinalis L. extracts in human health and assessing their activity as potent functional regulators of food microbiota.

Salvia officinalis L. has attracted scientific and industrial interest due to its pharmacological properties. However, its detailed phytochemical profile and its correlation with beneficial effects in the human microbiome and oxidative stress remained elusive. To unveil this, S. officinalis was collected from the region of Epirus and its molecular identity was verified with DNA barcoding. Phytochemical profile for both aqueous and ethanol-based extracts was determined by high-pressure liquid chromatography-tandem mass spectrometry and 103 phytochemicals were determined. The effect of S. officinalis extracts as functional regulators of food microbiota by stimulating the growth of Lacticaseibacillus rhamnosus strains and by suppressing evolution of pathogenic bacteria was verified. Furthermore, we recorded that both extracts exhibited a significant cellular protection against H2O2-induced DNA damage. Finally, both extracts exhibited strong inhibitory effect towards LDL oxidation. This study provides a comprehensive characterization of S. officinalis on its phytochemical components as also its potential impact in human microbiome and oxidative stress.

Detection of experimentally induced root fractures on digital and conventional radiographs: an in vitro study.

This study compared the diagnostic accuracy of digital radiographic images with conventional radiographs for the detection of experimentally induced root fractures. Horizontal fractures were induced by a mechanical force to the root of 15 single rooted teeth. Vertical fractures were induced in 15 single rooted teeth and 15 molars, respectively, by disk cutting. The teeth were mounted in dried mandibles and were radiographed with the parallel technique using a CCD-based Planmeca imaging system and F-speed films. Seven observers recorded their findings and then 2 pairs of them examined the same images together. Az values expressed the diagnostic accuracy of the imaging systems and the degree of agreement was estimated using the Cohen's kappa statistic. The areas under the ROC curves (Az) in single rooted teeth were 0.61 for conventional radiography and 0.64 for digital radiography. Digital imaging system did not perform significantly better than conventional one (p > 0.01). Digital radiographs (Az: 0.72) provided higher detection accuracy in molars than conventional images (Az: 0.51) (p = 0.0102). The digital images scored higher kappa values with narrower range than conventional. Observers in pairs did not perform significantly better than individually. The results were not affected by the observers but were affected by the cases (p < 0.001). Digital images were equivalent to F-speed films for the detection of root fractures in single rooted teeth. The digital system performed significantly better than the conventional in detecting root fractures in molars.

Dimeric G-quadruplex motifs-induced NFRs determine strong replication origins in vertebrates.

Replication of vertebrate genomes is tightly regulated to ensure accurate duplication, but our understanding of the interplay between genetic and epigenetic factors in this regulation remains incomplete. Here, we investigated the involvement of three elements enriched at gene promoters and replication origins: guanine-rich motifs potentially forming G-quadruplexes (pG4s), nucleosome-free regions (NFRs), and the histone variant H2A.Z, in the firing of origins of replication in vertebrates. We show that two pG4s on the same DNA strand (dimeric pG4s) are sufficient to induce the assembly of an efficient minimal replication origin without inducing transcription in avian DT40 cells. Dimeric pG4s in replication origins are associated with formation of an NFR next to precisely-positioned nucleosomes enriched in H2A.Z on this minimal origin and genome-wide. Thus, our data suggest that dimeric pG4s are important for the organization and duplication of vertebrate genomes. It supports the hypothesis that a nucleosome close to an NFR is a shared signal for the formation of replication origins in eukaryotes.

Ultrasound-Assisted Extraction of Taro Leaf Antioxidants Using Natural Deep Eutectic Solvents: An Eco-Friendly Strategy for the Valorization of Crop Residues.

Colocasia esculenta L. leaves are considered a by-product of taro cultivation and are discarded as environmental waste, despite their valuable phenolic composition. Their valorization to obtain value-added substances for medicinal, food, and cosmetic applications is the aim of the current work. An ultrasound-assisted extraction was developed for the environmentally friendly and sustainable isolation of taro leaf antioxidants using natural deep eutectic solvents (NaDESs). Among the utilized solvents, the NaDES based on betaine and ethylene glycol provided the best extraction efficiencies in terms of polyphenolic content and antioxidant activity. Multi-response optimization suggested a solvent-to-solid ratio of 10 mL g-1, a processing time of 60 min, an extraction temperature of 60 °C, and a water content of 33.8% (w/w) as optimal extraction parameters. Leaf extract obtained under these optimum operational parameters demonstrated a strong radical scavenging activity against 2,2-diphenyl-1-picrylhydrazyl (65.80 ± 0.87%), a high ferric reducing antioxidant power (126.62 ± 1.92 µmol TE g-1 sample), and significant protection against oxidative stress-induced DNA damage. The chromatographic characterization of the optimum extract revealed its richness in flavonoids (flavones and flavonols). The outcomes of the present study suggest that the proposed method could serve as a highly efficient and green alternative for the recovery of polyphenols from agricultural wastes.

Histone H3 serine-57 is a CHK1 substrate whose phosphorylation affects DNA repair.

Histone post-translational modifications promote a chromatin environment that controls transcription, DNA replication and repair, but surprisingly few phosphorylations have been documented. We report the discovery of histone H3 serine-57 phosphorylation (H3S57ph) and show that it is implicated in different DNA repair pathways from fungi to vertebrates. We identified CHK1 as a major human H3S57 kinase, and disrupting or constitutively mimicking H3S57ph had opposing effects on rate of recovery from replication stress, 53BP1 chromatin binding, and dependency on RAD52. In fission yeast, mutation of all H3 alleles to S57A abrogated DNA repair by both non-homologous end-joining and homologous recombination, while cells with phospho-mimicking S57D alleles were partly compromised for both repair pathways, presented aberrant Rad52 foci and were strongly sensitised to replication stress. Mechanistically, H3S57ph loosens DNA-histone contacts, increasing nucleosome mobility, and interacts with H3K56. Our results suggest that dynamic phosphorylation of H3S57 is required for DNA repair and recovery from replication stress, opening avenues for investigating the role of this modification in other DNA-related processes.

Incidence of impacted and supernumerary teeth-a radiographic study in a North Greek population.

OBJECTIVES: The aim of the present study was to investigate the prevalence of impacted and supernumerary teeth, apart from third molars. STUDY DESIGN: This was a retrospective study of 1.239 panoramic radiographs taken of patients who presented to the Department of Dentoalveolar Surgery, Implantology and Radiology at the School of Dentistry of the Aristotle University of Thessaloniki, Greece between 1991 and 1999. The panoramic radiographs and dental records were reviewed in order to determine whether there were impacted or supernumerary teeth. Observations were also made on the space in dentition, corresponding to the position of each impacted tooth, the lack of space for tooth eruption, transmigration, retained primary teeth or prosthetic restoration. RESULTS: A total of 170 (13.7%) patients presented with at least one impacted tooth. None of them had an impacted incisor. Impacted canines were the most prevalent dental anomaly (8.8%), followed by impacted premolars (2.2%). Supernumerary teeth (1.8%) and impacted molars (1%) were the least common anomalies. Among the 225 impacted teeth, the most frequently affected teeth were the canines (59.6%), followed by premolars (19.1%), and supernumerary teeth (15.1%), while the incidence of impacted molars was substantially lower (6.2%). CONCLUSIONS: The most frequently impacted teeth were the maxillary canine, the second mandibular premolar and the second mandibular molar. The majority of the supernumerary teeth consisted of mesiodens. There was space in the dentition of each impacted tooth in 29.3% of the cases examined; there was a retained primary tooth in 25.1%, and a prosthetic restoration had been constructed in 24%. Insufficient space for the eruption of the impacted tooth and transmigration was observed in 17.3% and 4.2% of the cases, respectively.

Evaluation of the osteogenic potential in experimental defects, with and without bone marrow, in the rabbit tibia: a pilot study.

PURPOSE: The influence of bone marrow in the osteogenic potential of bone has not been evaluated in the rabbit tibia model. Previous studies employed this model extensively to test the osteogenic capabilities of bone grafts. The primary aim of the present study was to assess the role of bone marrow in the healing of experimental defects in the rabbit tibia model. MATERIALS AND METHODS: Ten New Zealand rabbits were divided into two groups of five each. In the first experimental test group (PoP), the marrow cavity was emptied completely under a 6-mm defect in the rabbit tibia metaphysis; the marrow was replaced by plaster of Paris, and a round piece of lyophilized collagen membrane was placed in contact with the endosteum and rested on the plaster. In the second experimental group (control), similar artificial defects were made, but the marrow was left intact. After 8 weeks, the animals were sacrificed and prepared for histologic and histomorphometric analysis. RESULTS: There was a statistically significant difference (P = .016) in newly formed bone area between the control group (mean, 68.08% +/- 12.09%) and the PoP group (mean, 54.02% +/- 13.93%). Similarly, there was a statistically significant difference in new bone density (P < .001) between the control group (mean, 95.99% +/- 1.74%) and the PoP group (mean, 75.37% +/- 13.27%). CONCLUSIONS: The current study confirms the significant effect that bone marrow has in bone regeneration and also the true regenerative capabilities of the osseous walls of the defects. The proposed experimental model may be a more reliable method of investigating and comparing the potential of different graft materials and methods.

The role of temperature in the action of mepivacaine.

The role of temperature in the action of local anesthetics was studied in 20 healthy young volunteers with plain 3% mepivacaine injected periapically twice in their maxillary first premolar, the first time with the solution at a temperature of 20 degrees C and the second time at 4 degrees C. The pulpal response was measured with a pulp tester every minute. The onset of pulp anesthesia was found to be of no statistical difference between 20 degrees C and 4 degrees C. On the other hand, mepivacaine at a temperature of 4 degrees C was found to have a statistically significant longer duration of action. Our conclusion is that the drop in temperature of mepivacaine from 20 degrees C to 4 degrees C provides a longer duration of pulpal anesthesia.

The cell proliferation antigen Ki-67 organises heterochromatin.

Antigen Ki-67 is a nuclear protein expressed in proliferating mammalian cells. It is widely used in cancer histopathology but its functions remain unclear. Here, we show that Ki-67 controls heterochromatin organisation. Altering Ki-67 expression levels did not significantly affect cell proliferation in vivo. Ki-67 mutant mice developed normally and cells lacking Ki-67 proliferated efficiently. Conversely, upregulation of Ki-67 expression in differentiated tissues did not prevent cell cycle arrest. Ki-67 interactors included proteins involved in nucleolar processes and chromatin regulators. Ki-67 depletion disrupted nucleologenesis but did not inhibit pre-rRNA processing. In contrast, it altered gene expression. Ki-67 silencing also had wide-ranging effects on chromatin organisation, disrupting heterochromatin compaction and long-range genomic interactions. Trimethylation of histone H3K9 and H4K20 was relocalised within the nucleus. Finally, overexpression of human or Xenopus Ki-67 induced ectopic heterochromatin formation. Altogether, our results suggest that Ki-67 expression in proliferating cells spatially organises heterochromatin, thereby controlling gene expression.

Concentration of organic compounds in natural waters with solid-phase dispersion based on advesicle modified silica prior to liquid chromatography.

The ability of vesicle-coated silica to aid the extraction of organic compounds from water prior to liquid chromatographic analysis is presented for the first time. The method is based on the formation of silica supported cationic multi-lamellar vesicles of gemini surfactants inherently ensuring the presence of hydrophilic and hydrophobic sites for the partitioning of analytes bearing different properties. Method development is illustrated by studying the adsolubilization of UV absorbing chemicals from swimming pool water. Due to the requirement for external energy input (intense shearing) a method based on solid-phase dispersion (SPD) was applied producing better results than off-line solid-phase extraction (SPE). Meticulous investigation of the experimental parameters was conducted in order to elucidate the mechanisms behind the proposed extraction pattern. Analyte recoveries were quantitative under the optimum experimental conditions offering recoveries higher than 96% with RSD values below 5%.

Multivariate chemometrics for the forensic discrimination of blue ball-point pen inks based on their Vis spectra.

Fifty blue ball-point pen inks of five different brands were examined on the basis of the Vis spectrum of their ethanolic solutions with a view to achieving good discrimination between them. Samples were dissolved in absolute ethanol and their absorbance values in the range of 400-750 nm, after appropriate transformations, were used as variables in the multivariate statistical techniques of cluster analysis (CA), principal component analysis (PCA) and discriminant analysis (DA). These techniques were used successively so that an effective and meaningful discriminant model was calculated in the final step. The initial 351 variables (log10 transformed ink absorption values at each wavelength) were subjected to a K-means CA over the objects (samples) and only 20 variables were retained. Principal component analysis was used to detect any outliers (four samples were removed) and the remaining samples were re-subjected to PCA to decide how many variables to enter into DA and whether original variables or components should be used. It was found that the first three principal components (in accordance with the Kaiser criterion) were good descriptors of the 20 original variables (96.97% of the data variance was explained) and their use as latent variables in DA lead to low average variable redundancy (33.6%) in the discriminant model. The calculated model had a Wilks' lambda of 8.98 x 10(-5) and was statistically significant at the P = 0.05 level. The post hoc classification of the training dataset was 100% correct. From the DA results and the component loadings it was found that discrimination was achieved on the basis of differences in the shape of the absorption bands as well as their relative intensities. The method was therefore deemed appropriate for supporting exclusionary forensic purposes.

Effect of autogenous harvest site location on the outcome of ridge augmentation for implant dehiscences.

Treatment planning for the placement of an implant in a site with a thin crestal ridge should address the probability that a buccal dehiscence will result. The aim of the present investigation was to perform guided bone regeneration (GBR) around implants with buccal dehiscences and evaluate the outcomes of using autogenous bone grafts harvested from three different intraoral sites. Forty-six Osseotite implants, 4 mm in diameter, were placed in thin crestal ridges, resulting in an uncovered implant surface from the buccal aspect. The lengths of the buccal dehiscences ranged from 3 to 7 mm as measured from the implant cervix to the most apical extent of the uncovered threads. A standard GBR technique was carried out to augment the bone defect around the buccal implant surface immediately after implant placement. The cases were divided into three groups according to receipt of an autogenous bone graft from the ramus, tuberosity, or mandibular symphysis. In all cases, e-PTFE membranes were used to cover the grafted areas. Grafted sites were exposed after 6 months, membranes were removed, and residual distance between the implant cervix and most uncovered thread was recorded. All grafting materials were able to produce a certain degree of bone regeneration. In terms of bone change level, the three groups were not equal. The mandibular symphysis group exhibited the highest mean bone growth level, followed by the ramus group. The tuberosity produced the poorest result. Mandibular and ramus autogenous bone grafts represent the best choice in materials for GBR procedures around implants, while tuberosity bone grafts can be used as an alternative.