A New Approach for Studying Semithin Sections of Human Pathological Material: Intermicroscopic Correlation Between Light Microscopy and Scanning Electron Microscopy

In order to obtain useful and complete information on the study of pathological material, we observed by scanning electron microscopy (SEM) the same semithin sections observed by light microscopy (LM). For this purpose, the specimen must have, at the same time, chromatic and electron dense characteristics. We thus developed different specimen preparation methods, subjecting the semithin sections to specific polychromatic staining with high atomic number (Z) elements, to monochromatic staining followed by routine contrasting with uranyl acetate and lead citrate, and to specific cytochemical and immunocytochemical procedures. The specimens were examined in sequence by LM, by SEM equipped with secondary electron, backscattered electron, transmitted electron detectors and by scanning transmission electron microscopy (S(T)EM)

Antimicrobial activity of nanoconjugated glycopeptide antibiotics and their effect on Staphylococcus Aureus biofilm

In the era of antimicrobial resistance, the use of nanoconjugated antibiotics is regarded as a promising approach for preventing and fighting infections caused by resistant bacteria, including those exacerbated by the formation of difficult-to-treat bacterial biofilms. Thanks to their biocompatibility and magnetic properties, iron oxide nanoparticles (IONPs) are particularly attractive as antibiotic carriers for the targeting therapy. IONPs can direct conjugated antibiotics to infection sites by the use of an external magnet, facilitating tissue penetration and disturbing biofilm formation. As a consequence of antibiotic localization, a decrease in its administration dosage might be possible, reducing the side effects to non-targeted organs and the risk of antibiotic resistance spread in the commensal microbiota. Here, we prepared nanoformulations of the ‘last-resort’ glycopeptides teicoplanin and vancomycin by conjugating them to IONPs via surface functionalization with (3-aminopropyl) triethoxysilane (APTES). These superparamagnetic NP-TEICO and NP-VANCO were chemically stable and NP-TEICO (better than NP-VANCO) conserved the typical spectrum of antimicrobial activity of glycopeptide antibiotics, being effective against a panel of staphylococci and enterococci, including clinical isolates and resistant strains. By a combination of different methodological approaches, we proved that NP-TEICO and, although to a lesser extent, NP-VANCO were effective in reducing biofilm formation by three methicillin-sensitive or resistant Staphylococcus aureus strains. Moreover, when attracted and concentrated by the action of an external magnet, NP-TEICO exerted a localized inhibitory effect on S. aureus biofilm formation at low antibiotic concentration. Finally, we proved that the conjugation of glycopeptide antibiotics to IONPs reduced their intrinsic cytotoxicity toward a human cell line. Copyright © 2021 Berini, Orlandi, Gamberoni, Martegani, Armenia, Gornati, Bernardini and Marinelli

Relationship between distance run per week, omega-3 index, and arachidonic acid (AA)/eicosapentaenoic acid (EPA) ratio: An Observational Retrospective Study in Non-elite Runners

Background: Tissue availability of polyunsaturated fatty acids (PUFA) depends on several factors, including dietary intake, physical exercise, genetic variation, and metabolic turnover. However, there is limited evidence whether running training activity per se may influence indices associated with PUFA metabolism such as Omega-3 (ω-3) index and arachidonic acid (AA; 20:4ω-6)/eicosapentaenoic acid (EPA; 20:5ω-3) ratio. Objective: To examine the association between kilometers (Km) run per week and changes in ω-3 index and AA/EPA ratio. Methods: We conducted a retrospective, observational, cohort study of 257 non-elite runners (mean age: 40.85 ± 12.17 years) who consumed no fatty acid supplements and provided a blood sample for analysis. The whole blood samples were collected by finger sticks, stored on absorbent filter paper, and then PUFA were quantified by gas chromatography (GC) and ω-3 index and AA/EPA ratio measured. Results: In a multivariate linear regression model, a gradual decrease of the ω-3 index was observed with higher weekly running distance (β = −0.033; 95% CI −0.039 to −0.026; R2 = 0.447; p < 0.0001). We also found a progressive increase of the AA/EPA ratio in subjects who ran greater weekly distances (β = 0.092; 95% CI 0.038 to 0.146; R2 = 0.320; p = 0.001). No other significant associations were observed with other variables, including years of running training and weekly training frequency (hours/week). Finally, as expected, a significant inverse correlation between ω-3 index and AA/EPA ratio (β = −2.614; 95% CI −3.407 to −1.821; R2 = 0.336; p < 0.0001) was detected. Conclusions: These findings suggest that distance running training and its weekly volume may negatively contribute to changes of the ω-3 index and AA/EPA ratio. Further studies with greater sample size will be required to replicate and extend these data

Cloning and characterization of mouse UBPy, a deubiquitinating enzyme that interacts with the Ras guanine nucleotide exchange factor CDC25(Mm)/Ras-GRF1

We used yeast "two-hybrid" screening to isolate cDNA-encoding proteins interacting with the N-terminal domain of the Ras nucleotide exchange factor CDC25(Mm). Three independent overlapping clones were isolated from a mouse embryo cDNA library. The full-length cDNA was cloned by RACE-polymerase chain reaction. It encodes a large protein (1080 amino acids) highly homologous to the human deubiquitinating enzyme hUBPy and contains a well conserved domain typical of ubiquitin isopeptidases. Therefore we called this new protein mouse UBPy (mUBPy). Northern blot analysis revealed a 4-kilobase mRNA present in several mouse tissues and highly expressed in testis; a good level of expression was also found in brain, where CDC25(Mm) is exclusively expressed. Using a glutathione S-transferase fusion protein, we demonstrated an "in vitro" interaction between mUBPy and the N-terminal half (amino acids 1-625) of CDC25(Mm). In addition "in vivo" interaction was demonstrated after cotransfection in mammalian cells. We also showed that CDC25Mm, expressed in HEK293 cells, is ubiquitinated and that the coexpression of mUBPy decreases its ubiquitination. In addition the half-life of CDC25Mm protein was considerably increased in the presence of mUBPy. The specific function of the human homolog hUBPy is not defined, although its expression was correlated with cell proliferation. Our results suggest that mUBPy may play a role in controlling degradation of CDC25(Mm), thus regulating the level of this Ras-guanine nucleotide exchange factor

Identification of residual tumor with intraoperative contrast-enhanced ultrasound during glioblastoma resection

Objective: The purpose of this study was to assess the capability of contrast-enhanced ultrasound (CEUS) to identify residual tumor mass during glioblastoma multiforme (GBM) surgery, to increase the extent of resection. Methods: The authors prospectively evaluated 10 patients who underwent surgery for GBM removal with navigated ultrasound guidance. Navigated B-mode and CEUS were performed prior to resection, during resection, and after complete tumor resection. Areas suspected for residual tumors on B-mode and CEUS studies were localized within the surgical field with navigated ultrasound and samples were sent separately for histopathological analysis to confirm tumor presence. Results: In all cases tumor remnants were visualized as hyperechoic areas on B-mode, highlighted as CEUS-positive areas, and confirmed as tumoral areas on histopathological analysis. In 1 case only, CEUS partially failed to demonstrate residual tumor because the residual hyperechoic area was devascularized prior to ultrasound contrast agent injection. In all cases CEUS enhanced B-mode findings. Conclusions: As has already been shown in other neoplastic lesions in other organs, CEUS is extremely specific in the identification of residual tumor. The ability of CEUS to distinguish between tumor and artifacts or normal brain on B-mode is based on its capacity to show the vascularization degree and not the echogenicity of the tissues. Therefore, CEUS can play a decisive role in the process of maximizing GBM resection

CD44 Expression in Oro-Pharyngeal Carcinoma Tissues and Cell Lines

Expression of CD44, a transmembrane hyaluronan-binding glycoprotein, is variably considered to have prognostic significance for different cancers, including oral squamous cell carcinoma. Although unclear at present, tissue-specific expression of particular isoforms of CD44 might underlie the different outcomes in currently available studies. We mined public transcriptomics databases for gene expression data on CD44, and analyzed normal, immortalized and tumour-derived human cell lines for splice variants of CD44 at both the transcript and protein levels. Bioinformatics readouts, from a total of more than 15,000 analyses, implied an increased CD44 expression in head and neck cancer, including increased expression levels relative to many normal and tumor tissue types. Also, meta-analysis of over 260 cell lines and over 4,000 tissue specimens of diverse origins indicated lower CD44 expression levels in cell lines compared to tissue. With minor exceptions, reverse transcribed polymerase chain reaction identified expression of the four main isoforms of CD44 in normal oral keratinocytes, transformed lines termed DT and HaCaT, and a series of paired primary and metastasis-derived cell lines from oral or pharyngeal carcinomas termed HN4/HN12, HN22/HN8 and HN30/HN31. Immunocytochemistry, Western blotting and flow cytometric assessments all confirmed the isoform expression pattern at the protein level. Overall, bioinformatic processing of large numbers of global gene expression analyses demonstrated elevated CD44 expression in head and neck cancer relative to other cancer types, and that the application of standard cell culture protocols might decrease CD44 expression. Additionally, the results show that the many variant CD44 exons are not fundamentally deregulated in a diverse range of cultured normal and transformed keratinocyte lines

Identification of proteins cross-reactive to phosphotyrosine antibodies and of a tyrosine kinase activity in boar spermatozoa

We used two different anti-phosphotyrosine antibodies to identify phosphotyrosine-containing proteins in a germ cell, the boar spermatozoon. Ejaculated spermatozoa presented three major polypeptides, of Mr 43,000, 40,000 and 36,000, respectively, that were immunorecognized on Western blots. These proteins were selectively enriched in the Triton X-100-soluble fraction and were released neither after an A23187-induced acrosome reaction nor after sperm homogenization. These findings suggest the presence of the three proteins in plasma membrane regions not involved in the acrosomal vesiculation. When epididymal boar spermatozoa were investigated, Western blot analysis of the detergent-soluble fractions from caput sperm did not reveal any detectable 43,000, 40,000 and 36,000 Mr proteins cross-reactive with phosphotyrosine antibodies, whereas the detergent-soluble fractions from cauda sperm yielded very strong immunoreactive signals. Labelling of freshly ejaculated spermatozoa with [32P]orthophosphate yielded a wide range of labelled phosphoproteins, but we failed to identify specific tyrosine phosphorylation under the experimental conditions employed. Tyrosine phosphorylation occurred when specific synthetic polymers of tyrosine, commonly used for studying tyrosine protein kinases, were assayed as substrates against both the Triton-soluble and Triton-insoluble sperm fractions. This is the first immunological and biochemical report on the presence of phosphotyrosine-containing proteins and protein kinase activities that phosphorylate tyrosine residues in a mammalian mature spermatozoon

DansylAlanylLysylChlromethylketone as fluorescent probe for localization of acrosin in boar and human spermatozoa

The localization of acrosin (EC 3.4.21.10) activity in mammalian spermatozoa was investigated by use of the fluorescent site-directed acrosin inhibitor, dansylalanyllysylchloromethyl ketone (DALCK). Fluorescence microscope preparations revealed, after the spermatozoa were subjected to a specific treatment, that acrosin activity is confined specifically to the inner acrosomal membrane (IAM). Spectrofluorometric and fluorescence polarization investigations verified that the fluorescent probe, once it is specifically bound to the treated spermatozoa, lies in a very hydrophobic environment and shows a remarkable reduction of rotational freedom. These results are compatible with the hypothesis that, under the experimental conditions used, active acrosin is tightly bound to the IAM and that the "specificity site" of the acrosin-active center is probably of a highly hydrophobic nature