During spermiogenesis, mammalian chromatin undergoes replacement of nuclear histones by protamines, resulting in a DNA that is highly condensed in the mature sperm. We have previously demonstrated that a percentage of human spermatozoa exhibit 1) positivity to the guanine-cylosine-specific chromomycin A3 (CMA3) fluorochrome and 2) the presence of endogenous nicks in their DNA. In situ protamination of mature human sperm limits the percentage of sperm positive to CMA3 and exhibiting endogenous nicks. In this study, we report further investigations that aim to clarify the relationship existing between levels of CMA3 stainability and the presence of endogenous nicks in the DNA of mature human spermatozoa. Human spermatozoa from 25 different samples showed values of sensitivity to the CMA3 fluorochrome ranging from 13% to 75%. The same samples showed a percentage of sensitivity to endogenous nick translation ranging from 1% to 38%. A strong correlation (r = 0.86) was evident between these two parameters. Prior staining of sperm with the CMA3 fluorochrome drastically reduced sensitivity to nick translation. In contrast, previously nick-translated sperm stained with CMA3 showed very little difference from samples that had not been pretreated. The presence of nicked sperm in the ejaculate may indicate anomalies during spermiogenesis and be an indicator of male infertility.

a b s t r a c tGold nanoparticles (AuNPs) represent an effective choice for topical drug delivery systems thanks to their small size, general non-toxicity, ease of functionalization and high surface to volume ratio. Even if systemic, methotrexate still plays an important role in psoriasis treatment: its topical use shows insufficient percutaneus penetration owing to limited passive diffusion, high molecular weight and dissociation at physiological pH. The aim of our study was to design a new drug delivery nanocarrier for Methotrexate and to improve its solubility, stability and biodistribution. AuNPs were on purpose prepared with a hydrophilic stabilizing layer, in order to improve the colloidal stability in water. Watersoluble gold nanoparticles functionalized by sodium 3-mercapto-1-propansulfonate (Au-3MPS) were prepared and loaded with methotrexate (MTX). The loading efficiency of MTX on Au-3MPS was assessed in the range 70-80%, with a fast release (80% in one hour). The release was studied up to 24 h reaching the value of 95%. The Au-3MPS@MTX conjugate was fully characterized by spectroscopic techniques (UV-vis, FTIR) and DLS. Preliminary toxicity tests in the presence of keratinocytes monolayers allowed to assess that the used Au-3MPS are not toxic. The conjugate was then topically used on C57BL/6 mouse normal skin in order to trace the absorption behavior. STEM images clearly revealed the distribution of gold nanoparticles inside the cells. In vitro studies showed that Methotrexate conjugated with Au-3MPS is much more efficient than Methotrexate alone. Moreover, DL50, based on MTT analysis, is 20 folds reduced at 48 h, by the presence of nanoparticles conjugation. UV-vis spectra for in vivo tracing of the conjugate on bare mouse skin after 24 h of application, show increased delivery of Methotrexate in the epidermis and dermis using Au-3MPS@MTX conjugate, compared to MTX alone. Moreover we observed absence of the Au-3MPS in the dermis and in the epidermis, suggesting that these layers of the skin do not retain the nanoparticles. Based on our data, we found that the novel Au-3MPS@MTX conjugate is an effective non-toxic carrier for the satisfactory percutaneous absorption of Methotrexate and could help in possible topical treatment of psoriasis.

ConclusionFCM seems to be a promising tool for enhanced specimens' reporting performance, given its simple application and very rapid microscopic image generation (<5 min/specimen). This technique may potentially be used for intraoperative pathological specimens' analysis.

Ex vivo fluorescence confocal microscopy (FCM) is an optical technology that provides fast H&E-like images of freshly excised tissues, and it has been mainly used for "real-time" pathological examination of dermatological malignancies. It has also shown to be a promising tool for fast pathological examination of prostatic tissues. We aim to create an atlas for FCM images of prostatic and periprostatic tissues to facilitate the interpretation of these images. Furthermore, we aimed to evaluate the learning curve of images interpretation of this new technology. Eighty fresh and unprepared biopsies obtained from radical prostatectomy specimens were evaluated using the FCM VivaScope® 2500 M-G4 (Mavig GmbH, Munich, Germany; Caliber I.D.; Rochester NY, USA) by two pathologists. Images of FCM with the corresponding H&E are illustrated to create the atlas. Furthermore, the two pathologists were asked to re-evaluate the 80 specimens after 90 days interval in order to assess the learning curve of images' interpretation of FCM. FCM was able to differentiate between different types of prostatic and periprostatic tissues including benign prostatic glands, benign prostatic hyperplasia, high-grade intraepithelial neoplasm, and prostatic adenocarcinoma. As regards the learning curve, FCM demonstrated a short learning curve. We created an atlas that can serve as the base for urologists and pathologists for learning and interpreting FCM images of prostatic and periprostatic tissues. Furthermore, FCM images is easily interpretable; however, further studies are required to explore the potential applications of this new technology in prostate cancer diagnosis and management.

Background: A microscopic analysis of tissue is the gold standard for cancer detection. Hematoxylin-eosin (HE) for the reporting of prostate biopsy (PB) is conventionally based on fixation, processing, acquisition of glass slides, and analysis with an analog microscope by a local pathologist. Digitalization and real-time remote access to images could enhance the reporting process, and form the basis of artificial intelligence and machine learning. Fluorescence confocal microscopy (FCM), a novel optical technology, enables immediate digital image acquisition in an almost HE-like resolution without requiring conventional processing. Objective: The aim of this study is to assess the diagnostic ability of FCM for prostate cancer (PCa) identification and grading from PB. Design, setting, and participants: This is a prospective, comparative study evaluating FCM and HE for prostate tissue interpretation. PBs were performed (March to June 2019) at a single coordinating unit on consecutive patients with clinical and laboratory indications for assessment. FCM digital images (n = 427) were acquired immediately from PBs (from 54 patients) and stored; corresponding glass slides (n = 427) undergoing the conventional HE processing were digitalized and stored as well. A panel of four international pathologists with diverse background participated in the study and was

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Ex vivo fluorescence confocal microscopy (FCM) is an innovative imaging tool that can be used intraoperatively to obtain real-time images of untreated excised tissue with almost histologic resolution. As inflammatory diseases often share overlapping clinical features, histopathology evaluation is required for dubious cases, delaying definitive diagnoses, and therefore therapy. This study identifies key-features at ex vivo FCM for differential diagnoses of cutaneous inflammatory diseases, in particular, psoriasis, eczema, lichen planus and discoid lupus erythematosus. Retrospective ex vivo FCM and histological evaluations with relevant diagnoses were correlated with prospectively reported histopathologic diagnoses, to evaluate agreement and the level of expertise required for correct diagnoses. We demonstrated that ex vivo FCM enabled the distinction of the main inflammatory features in most cases, providing a substantial concordance to histopathologic diagnoses. Moreover, ex vivo FCM and histological evaluations reached a substantial agreement with histopathologic diagnoses both for all raters and for each operator. After a yet to be defined learning curve, these preliminary results suggest that dermatologists may be able to satisfactorily interpret ex vivo FCM images for correct real-time diagnoses. Despite some limitations mainly related to the equipment of FCM with a single objective lens, our study suggests that ex vivo FCM seems a promising tool in assisting diagnoses of cutaneous inflammatory lesions, with a level of accuracy quite close to that offered by histopathology. This is the first study to investigate ex vivo FCM application in cutaneous inflammatory lesions, and to evaluate the diagnostic capability of this technology.

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