Synergistic Effect of Human Chorionic Gonadotropin and Granulocyte Colony Stimulating Factor in the Mobilization of HSPCs Improves Overall Survival After PBSCT in a Preclinical Murine Model. Are We Far Enough for Therapy?

Strategies to improve hematopoietic stem and progenitor cell (HSPC) mobilization from the bone marrow can have a pivotal role in addressing iatrogenic bone-marrow insufficiency from chemo(radio)therapy and overcoming peripheral blood stem cell transplantation (PBSCT) limitations such as insufficient mobilization. Granulocyte-colony stimulating factor (G-CSF) represents the standard mobilization strategy for HSPC and has done so for more than three decades since its FDA approval. Its association with non-G-CSF agents is often employed for difficult HSPC mobilization. However, obtaining a synergistic effect between the two classes is limited by different timing and mechanisms of action. Based on our previous in vitro results, we tested the mobilization potential of human chorionic gonadotropin (HCG), alone and in combination with G-CSF in vivo in a murine study. Our results show an improved mobilization capability of the combination, which seems to act synergistically in stimulating hematopoiesis. With the current understanding of the dynamics of HSPCs and their origins in more primitive cells related to the germline, new strategies to employ the mobilization of hematopoietic progenitors using chorionic gonadotropins could soon become clinical practice.

Current trends in luminescence-based assessment of apoptosis.

Apoptosis, the most extensively studied type of cell death, is known to play a crucial role in numerous processes such as elimination of unwanted cells or cellular debris, growth, control of the immune system, and prevention of malignancies. Defective regulation of apoptosis can trigger various diseases and disorders including cancer, neurological conditions, autoimmune diseases and developmental disorders. Knowing the nuances of the cell death type induced by a compound can help decipher which therapy is more effective for specific diseases. The detection of apoptotic cells using classic methods has brought significant contribution over the years, but innovative methods are quickly emerging and allow more in-depth understanding of the mechanisms, aside from a simple quantification. Due to increased sensitivity, time efficiency, pathway specificity and negligible cytotoxicity, these innovative approaches have great potential for both in vitro and in vivo studies. This review aims to shed light on the importance of developing and using novel nanoscale methods as an alternative to the classic apoptosis detection techniques.

Exploring Therapeutic Avenues in Lung Cancer: The Epigenetic Perspective.

Lung cancer, primarily non-small cell lung carcinoma (NSCLC) and small cell lung carcinoma (SCLC), is distinguished by its high prevalence and marked mortality rates. Traditional therapeutic approaches, encompassing chemotherapy, radiation, and targeted therapies, frequently show limited efficacy due to acquired resistance and notable side effects. The objective of this review is to introduce a fresh perspective on the therapeutic strategies for lung cancer, emphasizing interventions targeting the epigenetic alterations often seen in this malignancy. This review presents the most recent advancements in the field, focusing on both past and current clinical trials related to the modulation of methylation patterns using diverse molecular agents. Furthermore, an in-depth analysis of the challenges and advantages of these methylation-modifying drugs will be provided, assessing their efficacy as individual treatments and their potential for synergy when integrated with prevailing therapeutic regimens.

Design and preclinical testing of an anti-CD41 CAR T cell for the treatment of acute megakaryoblastic leukaemia.

Acute megakaryoblastic leukaemia (AMkL) is a rare subtype of acute myeloid leukaemia (AML) representing 5% of all reported cases, and frequently diagnosed in children with Down syndrome. Patients diagnosed with AMkL have low overall survival and have poor outcome to treatment, thus novel therapies such as CAR T cell therapy could represent an alternative in treating AMkL. We investigated the effect of a new CAR T cell which targets CD41, a specific surface antigen for M7-AMkL, against an in vitro model for AMkL, DAMI Luc2 cell line. The performed flow cytometry evaluation highlighted a percentage of 93.8% CAR T cells eGFP-positive and a limited acute effect on lowering the target cell population. However, the interaction between effector and target (E:T) cells, at a low ratio, lowered the cell membrane integrity, and reduced the M7-AMkL cell population after 24 h of co-culture, while the cytotoxic effect was not significant in groups with higher E:T ratio. Our findings suggest that the anti-CD41 CAR T cells are efficient for a limited time spawn and the cytotoxic effect is visible in all experimental groups with low E:T ratio.

Insights into the Human Microbiome and Its Connections with Prostate Cancer.

The human microbiome represents the diversity of microorganisms that live together at different organ sites, influencing various physiological processes and leading to pathological conditions, even carcinogenesis, in case of a chronic imbalance. Additionally, the link between organ-specific microbiota and cancer has attracted the interest of numerous studies and projects. In this review article, we address the important aspects regarding the role of gut, prostate, urinary and reproductive system, skin, and oral cavity colonizing microorganisms in prostate cancer development. Various bacteria, fungi, virus species, and other relevant agents with major implications in cancer occurrence and progression are also described. Some of them are assessed based on their values of prognostic or diagnostic biomarkers, while others are presented for their anti-cancer properties.

Direct, Rapid Detection of Pathogens from Urine Samples.

The problem of rapidly detecting pathogens directly from clinical samples poses significant analytical challenges. Addressing this issue in relation to urinary tract infections, we propose an effective protocol and related immunomagnetic test kits enabling versatile screening for the presence of pathogenic bacteria in unprocessed urine samples. To achieve this, the components of a typical immunomagnetic separation protocol were optimized towards the sensitive assessment of the aggregates formed out of immunomagnetically tagged target pathogens collected from clinical samples. Specifically, a dedicated immunomagnetic material was developed via the functionalization of standardized, micron-sized magnetic beads with generic antibodies against gram-specific bacterial constituents with mannan binding lectin. As such, we demonstrate efficient procedures for achieving the enhanced, specific, and pathogen-mediated cluster formation of these tailored affinity-coated magnetic beads in complex samples. We further show how cluster analysis, in conjunction with the use of nonspecific, inexpensive fluorescent dye, allows for a straightforward optical assessment of the bacterial load directly from urine samples. The optimized sensing protocol and related kits provide, in less than 60 min, qualitative (positive/negative) information on the bacterial load with 85% specificity and 96% sensitivity, which is appropriate to empower clinical microscopy with a new analytic dimension. The procedure is prone to automation, can be conveniently used in clinical microbiology laboratories and, since it preserves the viability of the captured bacteria, can be interfaced with downstream analyses and antimicrobial susceptibility testing. Moreover, the study emphasizes a suite of practical validation assays that are useful for bringing the tool-box of immunomagnetic materials outside the academic laboratory and into real-life applications.

New Insights into the Multivariate Analysis of SER Spectra Collected on Blood Samples for Prostate Cancer Detection: Towards a Better Understanding of the Role Played by Different Biomolecules on Cancer Screening: A Preliminary Study.

It is possible to obtain diagnostically relevant data on the changes in biochemical elements brought on by cancer via the use of multivariate analysis of vibrational spectra recorded on biological fluids. Prostate cancer and control groups included in this research generated almost similar SERS spectra, which means that the values of peak intensities present in SERS spectra can only give unspecific and limited information for distinguishing between the two groups. Our diagnostic algorithm for prostate cancer (PCa) differentiation was built using principal component analysis and linear discriminant analysis (PCA-LDA) analysis of spectral data, which has been widely used in spectral data management in many studies and has shown promising results so far. In order to fully utilize the entire SERS spectrum and automatically determine the most meaningful spectral features that can be used to differentiate PCa from healthy patients, we perform a multivariate analysis on both the entire and specific spectral intervals. Using the PCA-LDA model, the prostate cancer and control groups are clearly distinguished in our investigation. The separability of the following two data sets is also evaluated using two alternative discrimination techniques: principal least squares discriminant analysis (PLS-DA) and principal component analysis-support vector machine (PCA-SVM).

Real Time SPR Assessment of the Structural Changes of Adaptive Dynamic Constitutional Frameworks as a New Route for Sensing.

Cross linked gold-dynamic constitutional frameworks (DCFs) are functional materials of potential relevance for biosensing applications, given their adaptivity and high responsivity against various external stimuli (such as pH, temperature) or specific interactions with biomolecules (enzymes or DNA) via internal constitutional dynamics. However, characterization and assessment of their dynamic conformational changes in response to external stimuli has never been reported. This study proves the capability of Surface Plasmon Resonance (SPR) assays to analyse the adaptive structural modulation of a functional matrix encompassing 3D gold-dynamic constitutional frameworks (Au-DCFs) when exposed to pH variations, as external stimuli. We analyse Au-DCFs formed from Au nanoparticles, (AuNP) connected through constitutionally dynamic polymers, dynamers, with multiple functionalities. For increased generality of this proof-of-concept assay, Au-DCFs, involving DCFs designed from 1,3,5-benzene-tricarbaldehyde (BTA) connecting centres and polyethylene glycol (PEG) connectors, are covalently attached to standard SPR sensing chips (Au nanolayers, carboxyl terminated or with carboxymethyl dextran, CMD top-layer) and analysed using state-of-the art SPR instrumentation. The SPR effects of the distance from the Au-DCFs matrix to the Au nanolayer of the sensing chip, as well as of Au-DCFs thickness were investigated. This study reveals the SPR response, augmented by the AuNP, to the conformational change, i.e., shrinkage, of the dynamer and AuNP matrix when decreasing the pH, and provides an unexplored insight into the sensing applicability of SPR real-time analysis of adaptive functional materials.

Electrochemically Synthesized Poly(3-hexylthiophene) Nanowires as Photosensitive Neuronal Interfaces.

Poly(3-hexylthiophene) (P3HT) is a hole-conducting polymer that has been intensively used to develop organic optoelectronic devices (e.g., organic solar cells). Recently, P3HT films and nanoparticles have also been used to restore the photosensitivity of retinal neurons. The template-assisted electrochemical synthesis of polymer nanowires advantageously combines polymerization and polymer nanostructuring into one, relatively simple, procedure. However, obtaining P3HT nanowires through this procedure was rarely investigated. Therefore, this study aimed to investigate the template-assisted electrochemical synthesis of P3HT nanowires doped with tetrabutylammonium hexafluorophosphate (TBAHFP) and their biocompatibility with primary neurons. We show that template-assisted electrochemical synthesis can relatively easily turn 3-hexylthiophene (3HT) into longer (e.g., 17 ± 3 µm) or shorter (e.g., 1.5 ± 0.4 µm) P3HT nanowires with an average diameter of 196 ± 55 nm (determined by the used template). The nanowires produce measurable photocurrents following illumination. Finally, we show that primary cortical neurons can be grown onto P3HT nanowires drop-casted on a glass substrate without relevant changes in their viability and electrophysiological properties, indicating that P3HT nanowires obtained by template-assisted electrochemical synthesis represent a promising neuronal interface for photostimulation.

Hsa-miR-125b Therapeutic Role in Colon Cancer Is Dependent on the Mutation Status of the TP53 Gene.

Colon cancer is the third most common cancer type worldwide and is highly dependent on DNA mutations that progressively appear and accumulate in the normal colon epithelium. Mutations in the TP53 gene appear in approximately half of these patients and have significant implications in disease progression and response to therapy. miR-125b-5p is a controversial microRNA with a dual role in cancer that has been reported to target specifically TP53 in colon adenocarcinomas. Our study investigated the differential therapeutic effect of miR-125b-5p replacement in colon cancer based on the TP53 mutation status of colon cancer cell lines. In TP53 mutated models, miR-125b-5p overexpression slows cancer cells' malignant behavior by inhibiting the invasion/migration and colony formation capacity via direct downregulation of mutated TP53. In TP53 wild type cells, the exogenous modulation of miR-125b-5p did not significantly affect the molecular and phenotypic profile. In conclusion, our data show that miR-125b-5p has an anti-cancer effect only in TP53 mutated colon cancer cells, explaining partially the dual behavior of this microRNA in malignant pathologies.

Azacytidine plus olaparib for relapsed acute myeloid leukaemia, ineligible for intensive chemotherapy, diagnosed with a synchronous malignancy.

Patients with relapsed/refractory acute myeloid leukaemia (AML), ineligible for intensive chemotherapy and allogeneic stem cell transplantation, have a dismal prognosis. For such cases, hypomethylating agents are a viable alternative, but with limited success. Combination chemotherapy using a hypomethylating agent plus another drug would potentially bring forward new alternatives. In the present manuscript, we present the cell and molecular background for a clinical scenario of a 44-year-old patient, diagnosed with high-grade serous ovarian carcinoma, diagnosed, and treated with a synchronous AML. Once the ovarian carcinoma relapsed, maintenance treatment with olaparib was initiated. Concomitantly, the bone marrow aspirate showed 30% myeloid blasts, consistent with a relapse of the underlying haematological disease. Azacytidine 75 mg/m2 treatment was started for seven days. The patient was administered two regimens of azacytidine monotherapy, additional to the olaparib-based maintenance therapy. After the second treatment, the patient presented with leucocytosis and 94% myeloid blasts on the bone marrow smear. Later, the patient unfortunately died. Following this clinical scenario, we reproduced in vitro the combination chemotherapy of azacytidine plus olaparib, to accurately assess the basic mechanisms of leukaemia progression, and resistance to treatment. Combination chemotherapy with drugs that theoretically target both malignancies might potentially be of use. Still, further research, both pre-clinical and clinical, is needed to accurately assess such cases.

A narrative review of central nervous system involvement in acute leukemias.

Acute leukemias (both myeloid and lymphoblastic) are a group of diseases for which each year more successful therapies are implemented. However, in a subset of cases the overall survival (OS) is still exceptionally low due to the infiltration of leukemic cells in the central nervous system (CNS) and the subsequent formation of brain tumors. The CNS involvement is more common in acute lymphocytic leukemia (ALL), than in adult acute myeloid leukemia (AML), although the rates for the second case might be underestimated. The main reasons for CNS invasion are related to the expression of specific adhesion molecules (VLA-4, ICAM-1, VCAM, L-selectin, PECAM-1, CD18, LFA-1, CD58, CD44, CXCL12) by a subpopulation of leukemic cells, called "sticky cells" which have the ability to interact and adhere to endothelial cells. Moreover, the microenvironment becomes hypoxic and together with secretion of VEGF-A by ALL or AML cells the permeability of vasculature in the bone marrow increases, coupled with the disruption of blood brain barrier. There is a single subpopulation of leukemia cells, called leukemia stem cells (LSCs) that is able to resist in the new microenvironment due to its high adaptability. The LCSs enter into the arachnoid, migrate, and intensively proliferate in cerebrospinal fluid (CSF) and consequently infiltrate perivascular spaces and brain parenchyma. Moreover, the CNS is an immune privileged site that also protects leukemic cells from chemotherapy. CD56/NCAM is the most important surface molecule often overexpressed by leukemic stem cells that offers them the ability to infiltrate in the CNS. Although asymptomatic or with unspecific symptoms, CNS leukemia should be assessed in both AML/ALL patients, through a combination of flow cytometry and cytological analysis of CSF. Intrathecal therapy (ITT) is a preventive measure for CNS involvement in AML and ALL, still much research is needed in finding the appropriate target that would dramatically lower CNS involvement in acute leukemia.

2D materials in electrochemical sensors for in vitro or in vivo use.

Individual cells and cell populations are at the present time investigated with a myriad of analytical tools. While most of them are commercially available, some of these analytical tools are just emerging from research laboratories and are in the developmental phase. Electrochemical sensors which allow the monitoring of low molecular weight compounds released (and / or uptaken) by cells are among these emerging tools. Such sensors are increasingly built using 2D materials (e.g. graphene-based materials, transition metal dichalcogenides, etc.) with the aim of conferring better analytical performances to these devices. The present work critically reviews studies published during the last 10 years describing electrochemical sensors made with 2D materials and exploited to monitor small compounds (e.g. H2O2, ·NO, glucose, etc.) in living biological systems. It also discusses the very few 2D material-based electrochemical sensors which are wearable or usable in vivo. Finally, the present work includes a specific section about 2D material biocompatibility, a fundamental requirement for 2D material-based sensor applications in vitro and in vivo. As such, the review provides a critical view on the state of the art of electrochemical sensors made with 2D materials and used at cellular level and it evaluates the possibility that such sensors will be used on / in the human body on a wider scale.

MicroRNA-155-5p Plays a Critical Role in Transient Leukemia of Down Syndrome by Targeting Tumor Necrosis Factor Receptor Superfamily Members.

BACKGROUND/AIMS: Down syndrome associated disorders are caused by a complex genetic context where trisomy 21 is a central component in relation to other changes involving epigenetic regulators and signaling molecules. This unique genetic context is responsible for the predisposition of people with Down syndrome to acute leukemia. Although, the research in this field has discovered some important pathogenic keys, the exact mechanism of this predisposition is not known. METHODS: In this study we applied functional enrichment analysis to evaluate the interactions between genes localized on chromosome 21, genes already identify as having a key role in acute leukemia of Down syndrome, miRNAs and signaling pathways implicated in cancer and cell development and found that miR-155 has a high impact in genes present on chromosome 21. Forward, we performed next generation sequencing on DNA samples from a cohort of patients diagnosed with acute leukemia of Down syndrome and in vitro functional assay using a CMK-86 cell line, transfected with either mimic or inhibitor of the microRNA-155-5p. RESULTS: Our results show that the epigenetic alteration of the TNF superfamily receptors in Down syndrome, which can be correlated to microRNA-155-5p aberrant activity, may play an important role in cell signaling and thus be linked to acute myeloid leukemia. CONCLUSION: Some genes, already shown to be mutated in AML-DS, are potential targets for miR-155. Our results show that the epigenetic alteration of the TNF superfamily receptors in Down syndrome may play an important role in cell signaling and thus be linked to acute myeloid leukemia.

PSA Based Biomarkers, Imagistic Techniques and Combined Tests for a Better Diagnostic of Localized Prostate Cancer.

Prostate cancer represents the most encountered urinary malignancy in males over 50 years old, and the second most diagnosed after lung cancer globally. Digital rectal examination and prostatic specific antigen were the long-time standard tools for diagnosis but with a significant risk of overdiagnosis and overtreatment. Magnetic resonance imaging recently entered the diagnosis process, but to this date, there is no specific biomarker that accurately indicates whether to proceed with the prostate biopsy. Research in this area has gone towards this direction, and recently, serum, urine, imagistic, tissue biomarkers, and Risk Calculators promise to help better diagnose and stratify prostate cancer. In order to eliminate the comorbidities that appear along with the diagnosis and treatment of this disease, there is a constant need to implement new diagnostic strategies. Important uro-oncology associations recommend the use of novel biomarkers in the grey area of prostate cancer, to better distinguish the next step in the diagnostic process. Although it is not that simple, they should be integrated according to the clinical policies, and it should be considered that statistical significance does not always equal clinical significance. In this review, we analyzed the contribution of prostate-specific antigen (PSA)-based biomarkers (PHI, PHID, 4Kscore, STHLM3), imagistic techniques (mp-MRI and mp-US), and combined tests in the early diagnosis process of localized prostate cancer.

Development of a Curcumin-Loaded Polymeric Microparticulate Oral Drug Delivery System for Colon Targeting by Quality-by-Design Approach.

The purpose of this study was to apply the quality-by-design (QbD) approach for the development of colon-targeted curcumin-loaded polymeric microparticles (Col-CUR-MPs). The proportion of the enterosoluble polymer (Eudragit® FS) in the polymeric matrix, curcumin concentration, and the concentration of the polymer mixture (Eudragit® FS-polycaprolactone) were identified as potential risk factors for the quality of the final product following risk assessment. The influence of these variables on the critical quality attributes (CQAs) of Col-CUR-MPs was investigated. Therefore, a central composite face experimental design was used in order to determine the functional relationships between variables and product CQAs. The obtained regression model and contour plots were used to establish the design space. Finally, the model was validated by preparing two microparticulate formulations, one corresponding to the robust setpoint from within the design space and one outside the established design space, and calculating the percentage bias between the experimental and predicted values. The in vivo study, which was conducted on a fluorescein-loaded formulation that corresponded to the robust setpoint determined by QbD and that contained a mixture of polycaprolactone and Eudragit® FS (60:40, w/w), confirmed the colon-targeting qualities of this formulation.

Spontaneous and Induced Animal Models for Cancer Research.

Considering the complexity of the current framework in oncology, the relevance of animal models in biomedical research is critical in light of the capacity to produce valuable data with clinical translation. The laboratory mouse is the most common animal model used in cancer research due to its high adaptation to different environments, genetic variability, and physiological similarities with humans. Beginning with spontaneous mutations arising in mice colonies that allow for pursuing studies of specific pathological conditions, this area of in vivo research has significantly evolved, now capable of generating humanized mice models encompassing the human immune system in biological correlation with human tumor xenografts. Moreover, the era of genetic engineering, especially of the hijacking CRISPR/Cas9 technique, offers powerful tools in designing and developing various mouse strains. Within this article, we will cover the principal mouse models used in oncology research, beginning with behavioral science of animals vs. humans, and continuing on with genetically engineered mice, microsurgical-induced cancer models, and avatar mouse models for personalized cancer therapy. Moreover, the area of spontaneous large animal models for cancer research will be briefly presented.

Transforming growth factor ß-mediated micromechanics modulates disease progression in primary myelofibrosis.

Primary myelofibrosis (PMF) is a Ph-negative myeloproliferative neoplasm (MPN), characterized by advanced bone marrow fibrosis and extramedullary haematopoiesis. The bone marrow fibrosis results from excessive proliferation of fibroblasts that are influenced by several cytokines in the microenvironment, of which transforming growth factor-ß (TGF-ß) is the most important. Micromechanics related to the niche has not yet been elucidated. In this study, we hypothesized that mechanical stress modulates TGF-ß signalling leading to further activation and subsequent proliferation and invasion of bone marrow fibroblasts, thus showing the important role of micromechanics in the development and progression of PMF, both in the bone marrow and in extramedullary sites. Using three PMF-derived fibroblast cell lines and transforming growth factor-ß receptor (TGFBR) 1 and 2 knock-down PMF-derived fibroblasts, we showed that mechanical stress does stimulate the collagen synthesis by the fibroblasts in patients with myelofibrosis, through the TGFBR1, which however seems to be activated through alternative pathways, other than TGFBR2.

Adipocyte-Based Cell Therapy in Oncology: The Role of Cancer-Associated Adipocytes and Their Reinterpretation as Delivery Platforms.

Cancer-associated adipocytes have functional roles in tumor development through secreted adipocyte-derived factors and exosomes and also through metabolic symbiosis, where the malignant cells take up the lactate, fatty acids and glutamine produced by the neighboring adipocytes. Recent research has demonstrated the value of adipocytes as cell-based delivery platforms for drugs (or prodrugs), nucleic acids or loaded nanoparticles for cancer therapy. This strategy takes advantage of the biocompatibility of the delivery system, its ability to locate the tumor site and also the predisposition of cancer cells to come in functional contact with the adipocytes from the tumor microenvironment for metabolic sustenance. Also, their exosomal content can be used in the context of cancer stem cell reprogramming or as a delivery vehicle for different cargos, like non-coding nucleic acids. Moreover, the process of adipocytes isolation, processing and charging is quite straightforward, with minimal economical expenses. The present review comprehensively presents the role of adipocytes in cancer (in the context of obese and non-obese individuals), the main methods for isolation and characterization and also the current therapeutic applications of these cells as delivery platforms in the oncology sector.

One-Pot Production of RNA in High Yield and Purity Through Cleaving Tandem Transcripts.

There is an increasing demand for efficient and robust production of short RNA molecules in both pharmaceutics and research. A standard method is in vitro transcription by T7 RNA polymerase. This method is sequence-dependent on efficiency and is limited to products longer than ~12 nucleotides. Additionally, the native initiation sequence is required to achieve high yields, putting a strain on sequence variability. Deviations from this sequence can lead to side products, requiring laborious purification, further decreasing yield. We here present transcribing tandem repeats of the target RNA sequence followed by site-specific cleavage to obtain RNA in high purity and yield. This approach makes use of a plasmid DNA template and RNase H-directed cleavage of the transcript. The method is simpler and faster than previous protocols, as it can be performed as one pot synthesis and provides at the same time higher yields of RNA.