The burden of increasing cancer incidence among the population, and, in particular, of prostate cancer in men living in highly developed countries, brings with it, on one hand, the need for new devices that allow a faster and earlier diagnosis, ideally in a non-invasive way and with low consumption of expensive reagents, and on the other the need for the assessment of new in vitro models that allow a more reliable assessment of cancer features, including its microenvironment and sensibility to different drugs. At the crossroads of these features, microfluidic devices are found. These, taking advantage of the chemical-physical properties of cells and human samples, have demonstrated great sensitivity and sensibility at an on-chip scale. Many fields of biomedical sciences have tried to exploit all their potentialities: from the detection of antigens in the early phases of the disease (when they are very low concentrated, but the treatment is more effective) to isolation and characterization of circulating tumor cells. However, is in the building of in vitro 3D models to better assess and comprehend the fundamental dynamics occurring in the tumor microenvironment and metastasis that 3D bioprinting techniques come into play. The aim of the present review is to describe the potential of these two different cutting-edge technologies for the detection and treatment of prostate cancer, in the perspective of a possible future combination of them that allows scientists to fill the gaps present in the field to improve patient care and treatment.

Vaginal atrophy consists of the loss of elasticity and thinning of the vaginal tissues and mucous membranes. This is a condition that affects women especially during menopause, following the hormonal decline resulting from the cessation of ovarian activity. The objective of the present study was evaluated the effects of Purasomes IRC100+ on the proliferation and migration of VK2/E6E7 vaginal epithelial cells and on fibroblasts by in vitro experiments. We tested three different concentrations (0.5%, 1% and 2%). We found that cell viability was highest at 2% Purasomes IRC100+ concentration for both cell lines. The scratch-wound assay was also done. Compared with the cell scratch at 0 h, cells in all groups migrated after 24 h, the scratch was significantly reduced in treated cells compared to untreated cells. In particular, the cell migration rates were 77% and 82%, respectively for VK2/E6E7 cells and fibroblasts treated with 2% of Purasomes IRC100+. Finally, this product promotes vaginal epithelial cell proliferation and therefore it could improve vaginal atrophy and vaginal wound healing. ### Competing Interest Statement The authors have declared no competing interest.

In this study, we compared the effectiveness of two commercial products contain biomimetic and natural exosomes on skin aging used two cells line, keratinocytes and fibroblasts. MTT assay and scratch-wound assay were employed to evaluate their in vitro effects. The treatment with product containing natural exosomes purified from bovine colostrum and loaded with growth factors and cytokines purified from bovine colostrum (AMPLEX PLUS technology), was able to significantly enhance cell proliferation of fibroblasts and keratinocytes at 24 hours compared to cells treatment with product containing biomimetic exosomes and compared to untreated cells (control). Product containing natural exosomes was also able to close the wound scratch after 24 hours. The obtained results suggest that the product containing exosomes derived from bovine colostrum has excellent structural and functional stability offer great potential as natural product for skin aging damage and wound healing. ### Competing Interest Statement The authors have declared no competing interest.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by cognitive decline and memory loss. A critical aspect of AD pathology is represented by oxidative stress, which significantly contributes to neuronal damage and death. Microglia and astrocytes, the primary glial cells in the brain, are crucial for managing oxidative stress and supporting neuronal function. Carnosine is an endogenous dipeptide possessing a multimodal mechanism of action that includes antioxidant, anti-inflammatory, and anti-aggregant activities. The present study investigated the effects of Aβ1-42 oligomers (oAβ), small aggregates associated with the neurodegeneration observed in AD, on primary rat mixed glia cultures composed of both microglia and astrocytes, focusing on the ability of these detrimental species to induce oxidative stress. We assessed intracellular reactive oxygen species (ROS) and nitric oxide (NO) levels as markers of oxidative stress. Exposure to oAβ significantly elevated both ROS and NO intracellular levels compared to control cells. However, this effect was completely inhibited by the pre-treatment of mixed cultures with carnosine, resulting in ROS and NO levels similar to those observed in untreated (control) cells. Single-cell analysis of cellular responses to oAβ revealed heterogeneous ROS production, resulting in two distinct clusters of cells, one of which was very responsive to the treatment. The presence of carnosine counteracted the overproduction of ROS, also leading to a single, homogeneous cluster, similar to that observed in the case of control cells. Interestingly, unlike ROS response, single-cell analysis of NO production did not show any distinct clusters. Overall, our findings demonstrated the ability of carnosine to mitigate Aβ-induced oxidative stress in mixed glia cells, by rescuing ROS and NO intracellular levels, as well as to normalize the heterogeneous response to the treatment measured in terms of clusters’ formation. The present study suggests a therapeutic potential of carnosine in pathologies characterized by oxidative stress including AD.

Poly(ε-caprolactone) (PCL) is a hydrolytically degradable biopolyester used in drug delivery to enhance drug solubility and bioavailability, where drugs are typically incorporated physically within the biopolymeric matrix rather than covalently bonded, due to the limited availability of functional groups required for covalent attachment. In pursuit of developing a facile method for the production of a biopolyester-drug covalent conjugate with effective drug loading capacity, this study reports the synthesis of a covalent Silibinin-PCL conjugate (Sil-PCLHyd) through a two-step approach. This involves the controlled hydrolysis of a high molecular weight PCL to increase the concentration of carboxylic end groups, which are subsequently used for the catalyzed esterification with Silibinin. The Sil-PCLHyd is characterized with mass spectrometry, gel permeation chromatography, thermogravimetric analysis, differential scanning calorimetry, and NMR and UV-vis spectroscopies. The cytotoxic effects of Sil-PCLHyd against colorectal adenocarcinoma cells (Caco-2) are measured through the MTT assay. The results of the Sil-PCLHyd characterization revealed a Silibinin loading of ≈9.8 wt.%. The MTT assay demonstrated that Sil-PCLHyd induced cytotoxic effects at concentrations a hundred times lower than those required for free Silibinin. The proposed approach might represent a reliable pathway for the development of biopolyester-based covalent conjugates with a high drug loading capacity.

Skin is the largest multifunctional human organ and possesses a complex multilayered structure with the ability to regenerate and renew. The key role in skin regeneration is played by fibroblasts, also playing an important role in wound healing process. We used different methods to evaluate on human fibroblasts the in vitro effects of a new compound called Nutri Complex 150+ (NC150+), containing a mixture of 20 different biologically active factors (GF20) and exosomes isolated and purified from bovine colostrum. NC150+ was able to significantly enhance cell proliferation/metabolic status of fibroblasts at both 24 and 48 hours compared to untreated (control) cells. NC150+ was also able to enhance the ability of human fibroblasts to close the wound scratch. Our findings demonstrate the ability of NC150+, based on a new technology called AMPLEX plus, to enhance cell proliferation/metabolic status of fibroblasts. The obtained results also suggest how NC150+ could be potentially effective in treating skin injury. ### Competing Interest Statement The authors have declared no competing interest.

Chromosomal instability is a hallmark of colorectal carcinogenesis and produces an accumulation of different forms of aneuploidies or broad copy number aberrations. Colorectal cancer is characterized by gain-type broad copy number aberrations, specifically in Chr20, Chr8q, Chr13 and Chr7, but their roles and mechanisms in cancer progression are not fully understood. It has been suggested that broad copy number gains might contribute to tumor development through the so-called caricature transcriptomic effect. We intend to investigate the impact of broad copy number gains on long non-coding RNAs’ expression in colorectal cancer, given their well-known role in oncogenesis. The influence of such chromosomal aberrations on lncRNAs’ transcriptome profile was investigated by SNP and transcriptome arrays in our series of colorectal cancer samples and cell lines. The correlation between aneuploidies and transcriptomic profiles led us to obtain a class of Over-UpT lncRNAs, which are transcripts upregulated in CRC and further overexpressed in colon tumors bearing specific chromosomal aberrations. The identified lncRNAs can contribute to a wide interaction network to establish the cancer driving effect of gain-type aneuploidies.

AbstractTo understand how oncogenes affect genome organization, it is essential to visualize fundamental processes such as DNA replication and transcription at high resolution in intact cells. At the same time, it is important to determine the progression of the cell along the cell cycle, as cell cycle regulation is crucial for the control of cell proliferation and oncogenesis. Here, we present a super-resolution imaging-based method to analyze single cell nuclei sorted according to specific phases of the cell cycle. The sorting is based on the evaluation of the number and the intensity of pixels in the replication foci image and the colocalization analysis is based on image cross-correlation spectroscopy (ICCS). We evaluate the colocalization between replication and transcription, at different cell cycle phases, in a model of PML-RARα oncogene activation. We find that colocalization between replication and transcription is higher in cells in early S phase compared to cells in middle and late S phase. When we turn on the PML-RARα oncogene, this colocalization pattern is preserved but we detect an increase of colocalization between replication and transcription in the early S phase which points to an effect of the PML-RARα oncogene on the coordination between replication and transcription.

The balance between fundamental processes, such as DNA replication, transcription, cell cycle progression and DNA damage response (DDR), finely regulates cell functioning. The impairment of one or more of these mechanisms can lead to genome instability, a common feature in rapidly proliferating tumour cells, which can result from different conditions generating DNA damage. In particular, oncogene-induced replication stress can trigger genomic instability in cancer, hampering replication progression and, consequently, accelerating mutation rates and promoting early-stage tumorigenesis.

Carnosine is an endogenous dipeptide characterized by a multimodal mechanism of action. However, its clinical potential is limited by serum and cytosolic carnosinases, which significantly reduce its bioavailability. Based on that, different research groups have worked on the development of new strategies able not only to prevent its rapid metabolization but also to improve its distribution and specific targeting. In the present study, the development and in vitro characterization of new liposomal formulations loaded with carnosine are described. Nanoliposomes, produced through Thin-Layer Hydration followed by Extrusion method, were first investigated for their physicochemical stability. Photon correlation spectroscopy and electrophoretic light scattering, assessing the stability of the formulations, showed a strong homogeneity-oriented tendency for up to two months. Particle size, polydispersity index, and zeta potential were determined through dynamic light scattering and electrophoretic light scattering, demonstrating an almost neutral charge of the formulation and an effective encapsulation of carnosine. The morphology assessment performed via scanning electron microscopy showed good conformity and polydispersity. Differential scanning calorimetry measurements suggest the ability of carnosine to stabilize the large unilamellar vesicles. Lastly, the newly developed carnosine-loaded liposomal formulations also showed a good safety profile in human microglia.

The journal retracts the article, “Fluoxetine Ecofriendly Nanoemulsion Enhances Wound Healing in Diabetic Rats: In Vivo Efficacy Assessment” [...]

Abstract Inflammation represents a complex biological response of the body to dangerous stimuli such as pathogens and involves different types of cells including immune cells (e.g., astrocytes and microglia), blood vessels, and molecular mediators. The main function of inflammation is to eradicate the causes responsible for cell injury, remove dead cells and damaged tissues, and allow the beginning of tissue repair. Carnosine is a dipeptide composed by β‐alanine and l ‐histidine possessing multiple functions that include anti‐aggregant, antioxidant, and anti‐inflammatory activities. Carnosine has also been shown to accelerate the wound‐healing process. Since numerous diseases are characterized by systemic and/or central inflammation such as cardiovascular diseases, type 2 diabetes mellitus, and neurodegenerative disorders, this molecule could be of great interest. Key Concepts Carnosine is a naturally occurring molecule characterized by a multimodal mechanism of action. The inflammatory process can be either short‐lived (acute; hours or days) or long‐lasting (chronic; months or years). Numerous systemic and neurodegenerative diseases show common pathogenic mechanisms including oxidative stress, inflammation and aggregation phenomena such as diabetes and Alzheimer disease. The versatile pharmacodynamic profile of carnosine including its anti‐inflammatory potential suggests a promising therapeutic potential in different diseases. Inflammation represents a complex biological response of the body to dangerous stimuli and involves different cell types such as immune cells (eg., astrocytes and microglia), blood vessels, and molecular mediators.

Spectral imaging is a fluorescence microscopy technique with several applications, including imaging of environment-sensitive probes, spectal unmixing and identification of fluorescent species. The spectral phasor approach was introduced to provide a simple and intuitive framework for the analysis of spectral imaging data, inspired by the phasor analysis of lifetime (Fereidouni et al., Opt. Expr. 2012; Digman et al., Biophys. J. 2008). Here, we show a spectral phasor analysis of multi-color images acquired with a Leica SP8 confocal microscope.

Fluorescence correlation spectroscopy (FCS) is a powerful technique to study molecular dynamics and interactions in biological systems. FCS is typically performed on dedicated modules where the excitation laser is focused on a point of the specimen, the fluorescence intensity is acquired, and the autocorrelation function (ACF) is generated by the dedicated software. Performing FCS in cells is more challenging due to the complexity of the cellular environment. In this respect, it has been shown that analysis of the data in short temporal segments can be very useful to increase the accuracy of FCS measurements inside cells (Di Bona et al, Biophys J 2019; Kohler, Hur et al Biophys J 2022).

Exosomes have been studied as a potential therapeutic option for improving skin texture. The mode of action of exosomes for skin texture likely involves several mechanisms, including stimulation of collagen production, promotion of skin cell proliferation; reduction of oxidative stress; regulation of inflammatory and antioxidant responses; extracellular matrix remodeling, which is important for maintaining skin structure and function. Aim of this study is to compare the in vitro effects of some commercial products that use new technologies containing plant derived nanovesicles from Rosa damascena callus, Centella asiatica callus, Euphorbia supina stem and exosomes from bovine colostrum passively loaded with growth factors and cytokines purified from bovine colostrum, for facial repair, regeneration and rejuvenation. Of all the products tested, only product containing exosomes purified from colostrum gave very encouraging results in terms of effects on proliferation, cell viability and wound repair. The other products tested gave results comparable to the untreated samples. ### Competing Interest Statement The authors have declared no competing interest.

Oncogenes activation affects genome organization causing a disruption of cell mechanisms such as DNA replication and transcription in intact cells. To visualize and quantify the alterations of their spatiotemporal organization, we present an imaging-based method to analyze replication foci labelled with 5-ethynyl-2′-deoxyuridine (EdU) and sort the cells according to specific phases of the cell cycle. The method is based on the analysis of single optical sections acquired with confocal microscopy or super-resolved-stimulated-emission-depletion (STED) microscopy.

Carnosine is an endogenous dipeptide composed of β-alanine and L-histidine, possessing a multimodal pharmacodynamic profile that includes anti-inflammatory and anti-oxidant activities. Carnosine has also shown its ability to modulate cell proliferation, cell cycle arrest, apoptosis, and even glycolytic energy metabolism, all processes playing a key role in the context of cancer. Cancer is one of the most dreaded diseases of the 20th and 21st centuries. Among the different types of cancer, breast cancer represents the most common non-skin cancer among women, accounting for an estimated 15% of all cancer-related deaths in women. The main aim of the present review was to provide an overview of studies on the anti-cancer activity of carnosine, and in particular its activity against breast cancer. We also highlighted the possible advantages and limitations involved in the use of this dipeptide. The first part of the review entailed a brief description of carnosine’s biological activities and the pathophysiology of cancer, with a focus on breast cancer. The second part of the review described the anti-tumoral activity of carnosine, for which numerous studies have been carried out, especially at the preclinical level, showing promising results. However, only a few studies have investigated the therapeutic potential of this dipeptide for breast cancer prevention or treatment. In this context, carnosine has shown to be able to decrease the size of cancer cells and their viability. It also reduces the levels of vascular endothelial growth factor (VEGF), cyclin D1, NAD+, and ATP, as well as cytochrome c oxidase activity in vitro. When tested in mice with induced breast cancer, carnosine proved to be non-toxic to healthy cells and exhibited chemopreventive activity by reducing tumor growth. Some evidence has also been reported at the clinical level. A randomized phase III prospective placebo-controlled trial showed the ability of Zn–carnosine to prevent dysphagia in breast cancer patients undergoing adjuvant radiotherapy. Despite this evidence, more preclinical and clinical studies are needed to better understand carnosine’s anti-tumoral activity, especially in the context of breast cancer.

Carnosine (beta-alanyl-L-histidine) is an endogenous dipeptide synthesized via the activity of the ATP-dependent enzyme carnosine synthetase 1 and can be found at a very high concentration in tissues with a high metabolic rate, including muscles (up to 20 mM) and brain (up to 5 mM). Because of its well-demonstrated multimodal pharmacodynamic profile, which includes anti-aggregant, antioxidant, and anti-inflammatory activities, as well as its ability to modulate the energy metabolism status in immune cells, this dipeptide has been investigated in numerous experimental models of diseases, including Alzheimer’s disease, and at a clinical level. The main limit for the therapeutic use of carnosine is related to its rapid hydrolysis exerted by carnosinases, especially at the plasma level, reason why the development of new strategies, including the chemical modification of carnosine or its vehiculation into innovative drug delivery systems (DDS), aiming at increasing its bioavailability and/or at facilitating the site-specific transport to different tissues, is of utmost importance. In the present review, after a description of carnosine structure, biological activities, administration routes, and metabolism, we focused on different DDS, including vesicular systems and metallic nanoparticles, as well as on possible chemical derivatization strategies related to carnosine. In particular, a basic description of the DDS employed or the derivatization/conjugation applied to obtain carnosine formulations, followed by the possible mechanism of action, is given. To the best of our knowledge, this is the first review that includes all the new formulations of carnosine (DDS and derivatives), allowing a decrease or complete prevention of the hydrolysis of this dipeptide exerted by carnosinases, the simultaneous blood–brain barrier crossing, the maintenance or enhancement of carnosine biological activity, and the site-specific transport to different tissues, which then offers perspectives for the development of new drugs.

In point-scanning microscopy, optical sectioning is achieved using a small aperture placed in front of the detector, i.e. the detection pinhole, which rejects the out-of-focus background. The maximum level of optical sectioning is theoretically obtained for the minimum size of the pinhole aperture, but this is normally prevented by the dramatic reduction of the detected signal when the pinhole is closed, leading to a compromise between axial resolution and signal-to-noise ratio. We have recently demonstrated that, instead of closing the pinhole, one can reach a similar level of optical sectioning by tuning the pinhole size in a confocal microscope and by analyzing the resulting image series. The method, consisting in the application of the separation of photons by lifetime tuning (SPLIT) algorithm to series of images acquired with tunable pinhole size, is called SPLIT-pinhole (SPLIT-PIN). Here, we share and describe a SPLIT-PIN software for the processing of series of images acquired at tunable pinhole size, which generates images with reduced out-of-focus background. The software can be used on series of at least two images acquired on available commercial microscopes equipped with a tunable pinhole, including confocal and stimulated emission depletion (STED) microscopes. We demonstrate applicability on different types of imaging modalities: (1) confocal imaging of DNA in a non-adherent cell line; (2) removal of out-of-focus background in super-resolved STED microscopy; (3) imaging of live intestinal organoids stained with a membrane dye.