Selected Mediators of Inflammation in Patients with Acute Ischemic Stroke.

During a stroke, a series of biochemical and metabolic changes occur which eventually lead to the death of cells by necrosis or apoptosis. This is a multi-stage process involving oxidative stress and an inflammatory response from the first signs of occlusion of a blood vessel until the late stages of regeneration and healing of ischemic tissues. The purpose of the research was to assess the concentration of pro-inflammatory cytokines IL-6 and TNF-α in the blood serum of patients with ischemic stroke (AIS) and to investigate their role as new markers in predicting functional prognosis after thrombolytic therapy. The researches have shown that the concentrations of the measured biomarkers were higher compared to the control group. Serum levels of IL-6 and THF-α before the initiation of intravenous thrombolysis were lower in the subgroup of patients with a favourable functional result (mRS: 0−2 pts) compared to the group of patients with an unfavourable functional result (mRS: 3−6 pts). A positive correlation was found between the concentration of IL-6 and TNF-α in patients with AIS during <4.5 h and on one day after the onset of stroke, which means that the concentration of IL-6 increases with the increase in TNF-α concentration. It has also been shown that higher levels of IL-6 in the acute phase of stroke and on the first and seventh days, and TNF-α during onset, were associated with poorer early and late prognosis in patients treated with intravenous thrombolysis. A relationship was found between the level of IL-6 and TNF-α in the subacute AIS and the severity of the neurological deficit. It has been shown that the investigated biomarkers may be a prognostic factor in the treatment of thrombolytic AIS.

Does experience of the surgeon affect surgical margins in head and neck basal cell carcinoma?

<b>Aim:</b> The aim of our study was to evaluate the impact of surgical experience in a high volume head and neck surgery department on basal cell carcinoma margin status. </br></br> <b>Material and methods:</b> A retrospective analysis of 546 patients surgically treated for primary basal cell carcinoma of the head and neck region was carried out. Resections were performed by 4 specialists with equal experience in head and neck surgery and 4 ENT residents at the same level of surgical training. A margin of 3-5 mm was chosen, according to guidelines. </br></br> <b>Results:</b> The study consisted of 304 males and 242 females, mean age of 69 (range 26-100). Most of the tumors were loca-ted on the nose (165 pts; 30.2%) and auricle (119; 21.7%). The most common histological subtype was nodular (119; 21.7%). Tumor size was up to 20 mm in 394 cases (72%). Positive surgical margins were found in 112 cases (20.5%). There was no difference in terms of positive surgical margins between residents (19/119 cases; 15.9%) and specialists (93/426; 21.8%; p = 0.161). </br></br> <b>Conclusions:</b> The results of our study have shown that adequate surgical training in a dedicated head and neck surgery de-partment is an efficient factor in obtaining free surgical margins in head and neck basal cell carcinoma.

The m6A RNA Modification Quantity and mRNA Expression Level of RNA Methylation-Related Genes in Head and Neck Squamous Cell Carcinoma Cell Lines and Patients.

RNA methylation at the nitrogen sixth of adenosine (m6A, N6-methyladenosine) is the most abundant RNA modification which plays a crucial role in all RNA metabolic aspects. Recently, m6A modification has been assigned to mediate the biological processes of cancer cells, but their significance in HNSCC development is still poorly described. Thus, the main aim of this study was to globally quantify m6A modification by the mass spectrometry approach and determine the mRNA expression level of selected m6A RNA methyltransferase (METTL3), demethylase (FTO), and m6A readers (YTHDF2, YTHDC2) in 45 HNSCC patients and 4 cell lines (FaDu, Detroit 562, A-253 and SCC-15) using qPCR. In the results, we have not observed differences in the global amount of m6A modification and the mRNA level of the selected genes between the cancerous and paired-matched histopathologically unchanged tissues from 45 HNSCC patients. However, we have found a positive correlation between selected RNA methylation machinery genes expression and m6A abundance on total RNA and characterized the transcript level of those genes in the HNSCC cell lines. Moreover, the lack of global m6A differences between cancerous and histopathologically unchanged tissues suggests that m6A alterations in specific RNA sites may specifically influence HNSCC tumorigenesis.

PepComposer: computational design of peptides binding to a given protein surface.

There is a wide interest in designing peptides able to bind to a specific region of a protein with the aim of interfering with a known interaction or as starting point for the design of inhibitors. Here we describe PepComposer, a new pipeline for the computational design of peptides binding to a given protein surface. PepComposer only requires the target protein structure and an approximate definition of the binding site as input. We first retrieve a set of peptide backbone scaffolds from monomeric proteins that harbor the same backbone arrangement as the binding site of the protein of interest. Next, we design optimal sequences for the identified peptide scaffolds. The method is fully automatic and available as a web server at http://biocomputing.it/pepcomposer/webserver.

Cocoa polyphenols are absorbed in Caco-2 cell model of intestinal epithelium.

Cocoa is an abundant source of polyphenols, mainly flavan-3-ol monomers and polymers. In the literature, there are contradictory data on the absorption limit of procyanidins in humans. In our study, the Caco-2 cell model of intestinal epithelium was used to determine the absorption and secretion of cocoa flavan-3-ols. Three compounds: (+)-catechin, (-)-epicatechin and procyanidin B2 were detected and quantified at the receiver side of Caco-2 monolayer after 2h transport experiment. The obtained results of apparent permeability coefficient suggest paracellular route of transport of investigated compounds. Additionally, the results suggest that compounds of cocoa powder purified extract are able to affect tight junction functioning.

Phenolic compound profiles and antioxidant capacity of Persea americana Mill. peels and seeds of two varieties.

Avocado processing by the food and cosmetic industries yields a considerable amount of phenolic-rich byproduct such as peels and seeds. Utilization of these byproducts would be favorable from an economic point of view. Methanolic (80%) extracts obtained from lyophilized ground peels and seeds of avocado (Persea americana Mill.) of the Hass and Shepard varieties were characterized for their phenolic compound profiles using the HPLC-PAD technique. The structures of the identified compounds were subsequently unambiguously confirmed by ESI-MS. Compositional analysis revealed that the extracts contained four polyphenolic classes: flavanol monomers, proanthocyanidins, hydroxycinnamic acids, and flavonol glycosides. The presence of 3-O-caffeoylquinic acid, 3-O-p-coumaroylquinic acid, and procyanidin A trimers was identified in seeds of both varieties. Intervarietal differences were apparent in the phenolic compound profiles of peels. Peels of the Shepard variety were devoid of (+)-catechin and procyanidin dimers, which were present in the peels of the Hass variety. Peels of both varieties contained 5-O-caffeoylquinic acid and quercetin derivatives. The differences in the phenolic profiles between varietals were also apparent in the different antioxidant activity of the extracts. The peel extracts had a higher total phenolic compound content and antioxidant activity when compared to the seed extracts. The highest TEAC and ORAC values were apparent in peels of the Haas variety in which they amounted to 0.16 and 0.47 mmol Trolox/g DW, respectively. No significant (p > 0.05) differences were apparent between the TEAC values of seeds of the two varieties but the ORAC values differed significantly (p < 0.05). Overall these findings indicate that both the seeds and peel of avocado can be utilized as a functional food ingredient or as an antioxidant additive.

Antioxidant properties of extracts obtained from raw, dry-roasted, and oil-roasted US peanuts of commercial importance.

Raw, skinless peanut kernels from US commercial production lines were dry- and oil-roasted according to standard industrial practices. Eighty percent (v/v) methanolic extracts from the peanut cultivars were prepared and characterized by RP-HPLC: five predominant compounds were found comprising free p-coumaric acid and potential p-coumaric acid derivatives, as elucidated by DAD-UV spectra with comparisons to those of commercial standards. A Spanish high-oleic peanut possessed the greatest naturally-occurring level of p-coumaric acid and its derivatives, followed by a high-oleic Runner, a normal Runner, and a Virginia peanut. Upon thermal processing, p-coumaric acid was liberated at the expense of its derivatives according to the relationship: oil roasting > dry roasting > raw. A high-oleic Runner exhibited the greatest increase (∼785%) in free p-coumaric acid levels after oil roasting. For many of the samples from the 2007 crop, processing increased the TPC and antioxidant capacities in the order of raw < dry roast < oil roast, but results were cultivar dependent. Oil-roasted peanuts were more effective at scavenging O2(.-) than their dry-roasted counterparts, as determined by a photochemiluminescence assay. Overall findings indicate that although thermal processing altered the composition of peanut kernel antioxidants, TPC values and radical-scavenging activities are preserved. Depending on peanut type, cultivar, and harvest date, enhanced antioxidant capacities can result.

Antioxidant activity of hazelnut skin phenolics.

Phenolic compounds were extracted from hazelnut skin using 80% (v/v) aqueous acetone or methanol. The crude extracts were applied onto a Sephadex LH-20 column for two fractionations (Fr. I and Fr. II). Fr. I consisting of low-molecular-weight phenolics was eluted by ethanol, whereas Fr. II consisting of tannins was obtained using acetone/water (1:1, v/v) as the mobile phase. UV spectra of phenolic compounds present in the crude extracts and their fractions exhibited a maximum absorbance at 282 nm. The crude extracts and their fractions were examined for phenolic and condensed tannin contents as well as total antioxidant activity (TAA), antiradical activity against the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, and reducing power. Results of these assays showed higher values when Fr. II containing tannins was tested, followed by crude extract, and Fr. I. Both 80% acetone and methanol were capable of extracting phenolics, but 80% acetone was a more effective solvent for the extraction of condensed tannins (p < 0.05). These results suggest that hazelnut skin can be considered as a value-added byproduct for use as dietary antioxidants.

Functional analysis of MmeI from methanol utilizer Methylophilus methylotrophus, a subtype IIC restriction-modification enzyme related to type I enzymes.

MmeI from Methylophilus methylotrophus belongs to the type II restriction-modification enzymes. It recognizes an asymmetric DNA sequence, 5'-TCCRAC-3' (R indicates G or A), and cuts both strands at fixed positions downstream of the specific site. This particular feature has been exploited in transcript profiling of complex genomes (using serial analysis of gene expression technology). We have shown previously that the endonucleolytic activity of MmeI is strongly dependent on the presence of S-adenosyl-l-methionine (J. Nakonieczna, J. W. Zmijewski, B. Banecki, and A. J. Podhajska, Mol. Biotechnol. 37:127-135, 2007), which puts MmeI in subtype IIG. The same cofactor is used by MmeI as a methyl group donor for modification of an adenine in the upper strand of the recognition site to N(6)-methyladenine. Both enzymatic activities reside in a single polypeptide (919 amino acids [aa]), which puts MmeI also in subtype IIC of the restriction-modification systems. Based on a molecular model, generated with the use of bioinformatic tools and validated by site-directed mutagenesis, we were able to localize three functional domains in the structure of the MmeI enzyme: (i) the N-terminal portion containing the endonucleolytic domain with the catalytic Mg2+-binding motif D(70)-X(9)-EXK(82), characteristic for the PD-(D/E)XK superfamily of nucleases; (ii) a central portion (aa 310 to 610) containing nine sequence motifs conserved among N(6)-adenine gamma-class DNA methyltransferases; (iii) the C-terminal portion (aa 610 to 919) containing a putative target recognition domain. Interestingly, all three domains showed highest similarity to the corresponding elements of type I enzymes rather than to classical type II enzymes. We have found that MmeI variants deficient in restriction activity (D70A, E80A, and K82A) can bind and methylate specific nucleotide sequence. This suggests that domains of MmeI responsible for DNA restriction and modification can act independently. Moreover, we have shown that a single amino acid residue substitution within the putative target recognition domain (S807A) resulted in a MmeI variant with a higher endonucleolytic activity than the wild-type enzyme.

HsdR subunit of the type I restriction-modification enzyme EcoR124I: biophysical characterisation and structural modelling.

Type I restriction-modification (RM) systems are large, multifunctional enzymes composed of three different subunits. HsdS and HsdM form a complex in which HsdS recognizes the target DNA sequence, and HsdM carries out methylation of adenosine residues. The HsdR subunit, when associated with the HsdS-HsdM complex, translocates DNA in an ATP-dependent process and cleaves unmethylated DNA at a distance of several thousand base-pairs from the recognition site. The molecular mechanism by which these enzymes translocate the DNA is not fully understood, in part because of the absence of crystal structures. To date, crystal structures have been determined for the individual HsdS and HsdM subunits and models have been built for the HsdM-HsdS complex with the DNA. However, no structure is available for the HsdR subunit. In this work, the gene coding for the HsdR subunit of EcoR124I was re-sequenced, which showed that there was an error in the published sequence. This changed the position of the stop codon and altered the last 17 amino acid residues of the protein sequence. An improved purification procedure was developed to enable HsdR to be purified efficiently for biophysical and structural analysis. Analytical ultracentrifugation shows that HsdR is monomeric in solution, and the frictional ratio of 1.21 indicates that the subunit is globular and fairly compact. Small angle neutron-scattering of the HsdR subunit indicates a radius of gyration of 3.4 nm and a maximum dimension of 10 nm. We constructed a model of the HsdR using protein fold-recognition and homology modelling to model individual domains, and small-angle neutron scattering data as restraints to combine them into a single molecule. The model reveals an ellipsoidal shape of the enzymatic core comprising the N-terminal and central domains, and suggests conformational heterogeneity of the C-terminal region implicated in binding of HsdR to the HsdS-HsdM complex.