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.

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.

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.

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.

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.

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.

Pseudopodial and ß-arrestin-interacting proteomes from migrating breast cancer cells upon PAR2 activation.

Metastatic cancer cells form pseudopodia (PD) to facilitate their migration. The proteinase-activated receptor-2 (PAR-2) transduces migratory signals from proteases, and it forms protein complexes with ß-arrestin and other signalling molecules that are enriched in pseudopodia. More generally, however, pseudopodial regulation is poorly understood. Here, we purified the pseudopodial proteomes of breast cancer cells after activation of the endogenous PAR-2 and we combined gel-based approaches with label-free high-resolution mass spectrometry to identify proteins that accumulate at the pseudopodia upon PAR-2-mediated migration. We identified >410 proteins in the cell body and >380 in the pseudopodia upon PAR2 activation, of which 93 were enriched in the pseudopodia. One of the pathways strongly enriched in the PD was the clathrin-mediated endocytosis signalling pathway, highlighting the importance of the scaffolding function of ß-arrestin in PAR-2 signalling via its endocytosis. We therefore immunoprecipitated ß-arrestins, and with mass spectrometry we identified 418 novel putative interactors. These data revealed novel ß-arrestin functions that specifically control PAR-2-regulated signalling in migrating breast cancer cells but also showed that some ß-arrestin functions are universal between GPCRs and cell types. In conclusion, this study reveals novel proteins and signalling pathways potentially important for migration of breast cancer cells.

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.

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.

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.

Bone regeneration around implants using spherical and granular forms of bioactive glass particles.

PURPOSE: It has been reported that previous Biogran (3i Implant Innovations, Inc., Palm Beach Gardens, FL) can be converted in vitro into hydroxyapatite (Biogran II) to accelerate new bone formation. The purpose of this study was to evaluate the bone regeneration around implants placed in critical-sized defects in rabbit tibia using granular and spherical forms of Biogran II in regards to implant contact, bone-to-graft contact, bone graft area, and total bone volume. MATERIALS AND METHODS: Twelve adult New Zealand rabbits were used, offering 24 surgical sites (1 in each tibia), where 6-mm round defects were created allowing the homocentric insertion of a screw type experimental implant with Osseotite (3i Implant Innovations, Inc.) surface. Half of the defects (group A) were filled up with spherical and half (group B) with granular forms of Biogran II. Ossix (3i Implant Innovations, Inc.) membranes covered the surgical sites. RESULTS: The histological evaluation after 8 weeks showed new bone formation in both groups, without any statistically significant differences in regards to bone-to-implant contact, bone-to-graft contact, bone graft area, and bone volume. Both dissolution of the outer shell and inner silica gel of the particles were observed mostly in spherical particles. In addition, new bone formation within the protected pouch interconnected with the surrounding new bone was observed exclusively in spherical particles of Biogran II. CONCLUSION: Faster dissolution of both outer and inner portions of spherical particles of Biogran II led to better integration with the surrounding new bone during an 8-week period of healing.

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 role of temperature in the action of local anesthetics: a double study in vitro and in vivo.

The purpose of this study was the examination of the role of temperature in the action of lidocaine via electrophysiological recordings on the sciatic nerve of the rat in vitro and in vivo. 20 Male Wistar rats were used in each type of experiment. In vitro, lidocaine shows no statistically significant difference regarding the onset of anesthesia but at the temperature of 25 degrees C it is significantly more potent on the establishment of anesthesia compared to the temperature of 36.5 degrees C. In vivo, lidocaine at the temperature of 4 degrees C is statistically significantly more effective in the establishment and the duration of anesthesia related to the temperature of 20 degrees C.