Evaluation of Controlled Ovarian Stimulation Protocols in Patients with Normal and Low Ovarian Reserve: Analyses of miRNAs and Selected Target Genes Involved in the Proliferation of Human Cumulus Cells and Oocyte Quality

The oocyte and the surrounding cumulus cells (CCs) are deeply linked by a complex bidirectional cross-talk. In this light, the molecular analysis of the CCs is nowadays considered to be precious in providing information on oocyte quality. It is now clear that miRNAs play a key role in several ovarian functions, such as folliculogenesis, steroidogenesis, and ovulation. Thus, in this study, specific miRNAs, together with their target genes, were selected and investigated in CCs to assess the response of patients with normal (NR) and low (LR) ovarian reserve to two different controlled ovarian stimulation (COS) protocols, based on rFSH and hMG. Moreover, a Fourier transform infrared microspectroscopy (FTIRM) analysis was performed to evaluate DNA conformational changes in CCs and to relate them with the two COS protocols. The results evidenced a modulation of the expression of miRNAs and related target genes involved in CCs’ proliferation, in vasculogenesis, angiogenesis, genomic integrity, and oocyte quality, with different effects according to the ovarian reserve of patients. Moreover, the COS protocols determined differences in DNA conformation and the methylation state. In particular, the results clearly showed that treatment with rFSH is the most appropriate in NR patients with normal ovarian reserve, while treatment with hMG appears to be the most suitable in LR patients with low ovarian reserve.     

Exploring the Molecular Mechanisms Underlying the in vitro Anticancer Effects of Multitarget-Directed Hydrazone Ruthenium(II)–Arene Complexes

The molecular targets and the modes of action behind the cytotoxicity of two structurally established N,O- or N,N-hydrazone ruthenium(II)–arene complexes were explored in human breast adenocarcinoma cells (MCF-7) and paralleled in non-cancerous and cisplatin-resistant counterparts (MCF-10A and MCF-7CR respectively). Both complexes, [Ru(hmb)(L1)Cl] ( 1 , L1=4-((2-(2,4-dinitrophenyl)hydrazono)(phenyl)methyl)-3-methyl-1-phenyl-1H-pyrazol-5-olate) and [Ru(cym)(L2)Cl] ( 2 , L2=1-((3-methyl-5-oxo-1-phenyl-1H-pyrazol-4(5H)-ylidene)(phenyl)methyl)-2-(pyridin-2-yl)hydrazin-1-ide), reversibly interact with moderate-to-high affinity with a number of molecular targets in cell-free assays, namely serum albumin, DNA, the 20S proteasome and hydroxymethylglutaryl-CoA reductase. Most interestingly, only 2 readily crosses the cell membrane and preserves its binding/modulatory ability toward the targets of interest upon rapid cellular internalization. The resulting action at multiple levels of the cancer cascade is likely the cause for the selective sensitization of tumour cells to p27-mediated apoptotic death, and for the ability of 2 to overcome the drug resistance problem.     

Self-Assembled Particles Combining SARS-CoV-2 RBD Protein and RBD DNA Vaccine Induce Synergistic Enhancement of the Humoral Response in Mice

Despite the fact that a range of vaccines against COVID-19 have already been created and are used for mass vaccination, the development of effective, safe, technological, and affordable vaccines continues. We have designed a vaccine that combines the recombinant protein and DNA vaccine approaches in a self-assembled particle. The receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 was conjugated to polyglucin:spermidine and mixed with DNA vaccine (pVAXrbd), which led to the formation of particles of combined coronavirus vaccine (CCV-RBD) that contain the DNA vaccine inside and RBD protein on the surface. CCV-RBD particles were characterized with gel filtration, electron microscopy, and biolayer interferometry. To investigate the immunogenicity of the combined vaccine and its components, mice were immunized with the DNA vaccine pVAXrbd or RBD protein as well as CCV-RBD particles. The highest antigen-specific IgG and neutralizing activity were induced by CCV-RBD, and the level of antibodies induced by DNA or RBD alone was significantly lower. The cellular immune response was detected only in the case of DNA or CCV-RBD vaccination. These results demonstrate that a combination of DNA vaccine and RBD protein in one construct synergistically increases the humoral response to RBD protein in mice.     

A Novel Xeno-Free Method to Isolate Human Endometrial Mesenchymal Stromal Cells (E-MSCs) in Good Manufacturing Practice (GMP) Conditions

The cyclic regeneration of human endometrium is guaranteed by the proliferative capacity of endometrial mesenchymal stromal cells (E-MSCs). Due to this, the autologous infusion of E-MSCs has been proposed to support endometrial growth in a wide range of gynecological diseases. We aimed to compare two different endometrial sampling methods, surgical curettage and vacuum aspiration biopsy random assay (VABRA), and to validate a novel xeno-free method to culture human E-MSCs. Six E-MSCs cell samples were isolated after mechanical tissue homogenization and cultured using human platelet lysate. E-MSCs were characterized for the colony formation capacity, proliferative potential, and multilineage differentiation. The expression of mesenchymal and stemness markers were tested by FACS analysis and real-time PCR, respectively. Chromosomal alterations were evaluated by karyotype analysis, whereas tumorigenic capacity and invasiveness were tested by soft agar assay. Both endometrial sampling techniques allowed efficient isolation and expansion of E-MSCs using a xeno-free method, preserving their mesenchymal and stemness phenotype, proliferative potential, and limited multi-lineage differentiation ability during the culture. No chromosomal alterations and invasive/tumorigenic capacity were observed. Herein, we report the first evidence of efficient E-MSCs isolation and culture in Good Manufacturing Practice compliance conditions, suggesting VABRA endometrial sampling as alternative to surgical curettage.     

Regenerative Neurology and Regenerative Cardiology: Shared Hurdles and Achievements

From the first success in cultivation of cells in vitro, it became clear that developing cell and/or tissue specific cultures would open a myriad of new opportunities for medical research. Expertise in various in vitro models has been developing over decades, so nowadays we benefit from highly specific in vitro systems imitating every organ of the human body. Moreover, obtaining sufficient number of standardized cells allows for cell transplantation approach with the goal of improving the regeneration of injured/disease affected tissue. However, different cell types bring different needs and place various types of hurdles on the path of regenerative neurology and regenerative cardiology. In this review, written by European experts gathered in Cost European action dedicated to neurology and cardiology-Bioneca, we present the experience acquired by working on two rather different organs: the brain and the heart. When taken into account that diseases of these two organs, mostly ischemic in their nature (stroke and heart infarction), bring by far the largest burden of the medical systems around Europe, it is not surprising that in vitro models of nervous and heart muscle tissue were in the focus of biomedical research in the last decades. In this review we describe and discuss hurdles which still impair further progress of regenerative neurology and cardiology and we detect those ones which are common to both fields and some, which are field-specific. With the goal to elucidate strategies which might be shared between regenerative neurology and cardiology we discuss methodological solutions which can help each of the fields to accelerate their development.     

T-Cell Receptor Repertoire Sequencing and Its Applications: Focus on Infectious Diseases and Cancer

The immune system is a dynamic feature of each individual and a footprint of our unique internal and external exposures. Indeed, the type and level of exposure to physical and biological agents shape the development and behavior of this complex and diffuse system. Many pathological conditions depend on how our immune system responds or does not respond to a pathogen or a disease or on how the regulation of immunity is altered by the disease itself. T-cells are important players in adaptive immunity and, together with B-cells, define specificity and monitor the internal and external signals that our organism perceives through its specific receptors, TCRs and BCRs, respectively. Today, high-throughput sequencing (HTS) applied to the TCR repertoire has opened a window of opportunity to disclose T-cell repertoire development and behavior down to the clonal level. Although TCR repertoire sequencing is easily accessible today, it is important to deeply understand the available technologies for choosing the best fit for the specific experimental needs and questions. Here, we provide an updated overview of TCR repertoire sequencing strategies, providers and applications to infectious diseases and cancer to guide researchers’ choice through the multitude of available options. The possibility of extending the TCR repertoire to HLA characterization will be of pivotal importance in the near future to understand how specific HLA genes shape T-cell responses in different pathological contexts and will add a level of comprehension that was unthinkable just a few years ago.     

Enhancing the Amyloid-β Anti-Aggregation Properties of Curcumin via Arene-Ruthenium(II) Derivatization

Alzheimer’s disease (AD) is a fatal neurodegenerative disorder associated with severe dementia, progressive cognitive decline, and irreversible memory loss. Although its etiopathogenesis is still unclear, the aggregation of amyloid-β (Aβ) peptides into supramolecular structures and their accumulation in the central nervous system play a critical role in the onset and progression of the disease. On such a premise, the inhibition of the early stages of Aβ aggregation is a potential prevention strategy for the treatment of AD. Since several natural occurring compounds, as well as metal-based molecules, showed promising inhibitory activities toward Aβ aggregation, we herein characterized the interaction of an organoruthenium derivative of curcumin with Aβ(1–40) and Aβ(1–42) peptides, and we evaluated its ability to inhibit the oligomerization/fibrillogenesis processes by combining in silico and in vitro methods. In general, besides being less toxic to neuronal cells, the derivative preserved the amyloid binding ability of the parent compound in terms of equilibrium dissociation constants but (most notably) was more effective both in retarding the formation and limiting the size of amyloid aggregates by virtue of a higher hindering effect on the amyloid–amyloid elongation surface. Additionally, the complex protected neuronal cells from amyloid toxicity.     

Hexagonal Voronoi pattern detected in the microstructural design of the echinoid skeleton

Repeated polygonal patterns are pervasive in natural forms and structures. These patterns provide inherent structural stability while optimizing strength-per-weight and minimizing construction costs. In echinoids (sea urchins), a visible regularity can be found in the endoskeleton, consisting of a lightweight and resistant micro-trabecular meshwork (stereom). This foam-like structure follows an intrinsic geometrical pattern that has never been investigated. This study aims to analyse and describe it by focusing on the boss of tubercles—spine attachment sites subject to strong mechanical stresses—in the common sea urchin Paracentrotus lividus. The boss microstructure was identified as a Voronoi construction characterized by 82% concordance to the computed Voronoi models, a prevalence of hexagonal polygons, and a regularly organized seed distribution. This pattern is interpreted as an evolutionary solution for the construction of the echinoid skeleton using a lightweight microstructural design that optimizes the trabecular arrangement, maximizes the structural strength and minimizes the metabolic costs of secreting calcitic stereom. Hence, this identification is particularly valuable to improve the understanding of the mechanical function of the stereom as well as to effectively model and reconstruct similar structures in view of future applications in biomimetic technologies and designs.     

Monitoring Somatic Genetic Alterations in Circulating Cell-Free DNA/RNA of Patients with “Oncogene-Addicted” Advanced Lung Adenocarcinoma: A Real-World Clinical Study

Liquid biopsy has advantages over tissue biopsy, but also some technical limitations that hinder its wide use in clinical applications. In this study, we aimed to evaluate the usefulness of liquid biopsy for the clinical management of patients with advanced-stage oncogene-addicted non-small-cell lung adenocarcinomas. The investigation was conducted on a series of cases—641 plasma samples from 57 patients—collected in a prospective consecutive manner, which allowed us to assess the benefits and limitations of the approach in a real-world clinical context. Thirteen samples were collected at diagnosis, and the additional samples during the periodic follow-up visits. At diagnosis, we detected mutations in ctDNA in 10 of the 13 cases (77%). During follow-up, 36 patients progressed. In this subset of patients, molecular analyses of plasma DNA/RNA at progression revealed the appearance of mutations in 29 patients (80.6%). Mutations in ctDNA/RNA were typically detected an average of 80 days earlier than disease progression assessed by RECIST or clinical evaluations. Among the cases positive for mutations, we observed 13 de novo mutations, responsible for the development of resistance to therapy. This study allowed us to highlight the advantages and disadvantages of liquid biopsy, which led to suggesting algorithms for the use of liquid biopsy analyses at diagnosis and during monitoring of therapy response.     

Design,Characterization, and Antibacterial Performance of MAPLE-Deposited Coatings of Magnesium Phosphate-Containing Silver Nanoparticles in Biocompatible Concentrations

Bone disorders and traumas represent a common type of healthcare emergency affecting men and women worldwide. Since most of these diseases imply surgery, frequently complicated by exogenous or endogenous infections, there is an acute need for improving their therapeutic approaches, particularly in clinical conditions requiring orthopedic implants. Various biomaterials have been investigated in the last decades for their potential to increase bone regeneration and prevent orthopedic infections. The present study aimed to develop a series of MAPLE-deposited coatings composed of magnesium phosphate (Mg₃(PO₄)₂) and silver nanoparticles (AgNPs) designed to ensure osteoblast proliferation and anti-infective properties simultaneously. Mg₃(PO₄)₂ and AgNPs were obtained through the cooling bath reaction and chemical reduction, respectively, and then characterized through X-ray Diffraction (XRD), Transmission Electron Microscopy (TEM), and Selected Area Electron Diffraction (SAED). Subsequently, the obtained coatings were evaluated by Infrared Microscopy (IRM), Fourier-Transform Infrared Spectroscopy (FT-IR), and Scanning Electron Microscopy (SEM). Their biological properties show that the proposed composite coatings exhibit well-balanced biocompatibility and antibacterial activity, promoting osteoblasts viability and proliferation and inhibiting the adherence and growth of Staphylococcus aureus and Pseudomonas aeruginosa, two of the most important agents of orthopedic implant-associated infections.