Small Subpopulation Research Articles

Distinct molecular properties and functions of small EV subpopulations isolated from human umbilical cord MSCs using tangential flow filtration combined with size exclusion chromatography.

As functional derivatives of mesenchymal stem cells (MSCs), small extracellular vesicles (sEVs) have garnered significant attention and application in regenerative medicine. However, the technical limitations for large-scale isolation of sEVs and their heterogeneous nature have added complexity to their applications. It remains unclear if the heterogeneous sEVs represent different aspects of MSCs functions. Here, we provide a method for the large-scale production of sEVs subpopulations derived from human umbilical cord mesenchymal stem cells (HucMSCs), utilizing tangential flow filtration combined with size exclusion chromatography. The resulting subpopulations, S1-sEVs and S2-sEVs, exhibited stable variations in size, membrane-marked proteins, and carrying cargos, thereby displaying distinct functions both in vitro and in animal disease models. S1-sEVs, that highly expressed CD9, HRS and GPC1, demonstrated a greater immunomodulatory impact, while S2-sEVs with enriched expression of CD63 and FLOT1/2 possessed enhanced capacities in promoting cell proliferation and angiogenesis. These discrepancies are attributed to the specific proteins and miRNAs they contain. Further investigation revealed that the two distinct sEVs subpopulations corresponded to different biological processes: the ESCRT pathway (S1-sEVs) and the ESCRT-independent pathway represented by lipid rafts (S2-sEVs). Therefore, we propose the potential for large-scale isolation and purification of sEVs subpopulations from HucMSCs with distinct functions. This approach may provide advantages for targeted therapeutic interventions in various MSC indications.

Combination of histological and transcriptomic approaches for cell types annotation in non-model organisms by example of spiny mice <i>Acomys cahirinus</i>

In mammals, cartilage tissue has a low potential for regeneration. Typically, the defect site is replaced by connective tissue. The Acomys cahirinus mouse is a relatively new model for studying tissue regeneration processes, specifically the elastic cartilage of the auricle. To investigate the molecular genetic mechanisms responsible for these processes and gain insight into the cellular and tissue composition of the intact auricle, we utilized the method of single-cell RNA sequencing (scRNA-seq). This method enables quantification of gene expression in the sample and modeling of cell clustering based on expression profiles. This allows for assessment of sample heterogeneity in terms of specific cell populations. Annotation of cell types, particularly in non-model organisms, should be supported by classical morphological studies to allow for more detailed identification of cell populations. This is necessary to separate clusters of cells that are grouped statistically based on similar expression profiles of a group of genes into smaller subpopulations. The objective of this study was to annotate all cell types present in the intact Acomys cahirinus auricle using a combination of transcriptomic approaches and classical histology methods. The study resulted in the annotation of 24 cell clusters based on known marker genes and by comparing genetic and morphological data.

DNA duplication-mediated activation of a two-component regulatory system serves as a bet-hedging strategy for Burkholderia thailandensis.

Transposable elements naturally accumulate within genomes in all kingdoms of life. When present in the same orientation, a pair of homologous elements can act as substrates for DNA recombination reactions that can duplicate and delete intervening sequences - giving rise to genetically heterogenous populations. We showed here that Burkholderia thailandensis strain E264 uses this mechanism to amplify genes encoding a two-component regulatory system and an archaic chaperone usher pilus, priming the cells for rapid biofilm formation. The formation of a small subpopulation of biofilm-ready bacteria serves as a bet- hedging strategy, ensuring overall population survival should conditions change rapidly from those in which planktonic growth is optimal to those in which adherence and biofilm formation is required.

Identification of Cancer Stem Cell (CSC)-Associated Genes, Prognostic Value, and Candidate Drugs as Modulators of CSC-Associated Signaling in Carcinomas Through a Multiomics Data Analysis Approach

Background: Cancer stem cells (CSCs) are a small subpopulation of cancer cells that have the potential for self-renewal and a strong proliferative capacity, and sustain tumorigenesis capabilities. This ability of CSCs to escape immune responses makes the CSCs a primary source of functionally altered, immune-resistant, chemoresistant, aggressive tumor cells. These characteristics determine the potential advantage of targeting CSCs for the treatment of solid tumors. Method: First, we downloaded different gene expression datasets of CSCs from the NCBI-GEO (National Center for Biotechnology Information–Gene Expression Omnibus) database and identified common genes by using a suitable Venn tool. Subsequently, we explored the prognostic significance of the particular genes in particular cancers and analyzed the expression of these genes at the protein level in human solid tumors by using KM plotter (Kaplan-Meier plotter) and an HPA (The Human Protein Atlas) database, respectively. Finally, using a comparative toxicogenomic database, we selected several important drugs or chemicals. Result: From this study, we identified APOC1 as a common upregulated gene in breast cancer and SLC44A5 and CAV2 as common up- and downregulated genes in lung cancer. In ovarian cancer, PRRG4 is a commonly upregulated gene, and ADCY7, AKAP12, TPM2, and FLNC are commonly downregulated genes. These genes also show prognostic significance in respective cancers. Several drugs that are capable of targeting the expression or signaling network of designated genes of CSC were also identified, which may contribute in CSC-targeted cancer therapy. Conclusion: Our study suggests a need for more in-depth experimental investigations to determine the actual functional activity and the mechanism of action of these CSC-associated genes.

Characterizing iconic gesture during narratives in chronic traumatic brain injury recovery.

It is known that co-speech hand gestures increase and supplement speech in individuals with language impairment after brain injury, e.g., post-stroke aphasia. Traumatic Brain Injury (TBI) provides a unique avenue to evaluate gestures as TBI often presents with both anomia (word-finding impairments) and cognitive impairments, resulting in a cognitive-communicative disorder. However, there is a great need for evaluation of gestures in TBI during typical spontaneous speech and across the recovery trajectory (from sub-acute to chronic stages). In a large population (N = 54) of persons with moderate-severe TBI, who were examined at 3 months post-TBI whilst telling a procedural narrative ("how to make a sandwich"), we examined three aims: (1) characterize the extent to which adults with moderate-severe TBI produce iconic gestures; (2) identify the extent to which language impairment relates to iconic gesturing in TBI; and (3) characterize the extent to which iconic gesturing changes across TBI recovery. In a subpopulation (Group 1, N = 14) who were examined at three- and 24-months (sub-acute and substantially chronic), and in a smaller subpopulation (Group 2, N = 6) who had data for five timepoints (three-, six-, nine-, 12-, and 24-months), we used paired tests to examine and characterize longitudinal changes in iconic gesturing. The large group analysis suggested that individuals with TBI use iconic gesture during narrative, which take several different iconic forms (e.g., enacting use of an object), and that a minority employed gestures that supplemented (added to, disambiguated, or replaced) speech. The subpopulation analyses suggested that participants did not produce iconic gestures significantly differently across the 2-year recovery timeframe. Case examination of a participant with moderate-severe aphasia suggested a relationship between language impairment and gesture, with this individual producing the highest proportion of supplemental gesturing of the entire group. This finding aligns with research from the post-stroke aphasia field. Broadly, this study significantly extends prior research on the relationship between gesturing, language, and brain injury.

Immunophenotypic Profile of Adult Glioblastoma IDH-Wildtype Microenvironment: A Cohort Study.

Background: Glioblastoma IDH-wildtype (GBM IDH-wt) is the most aggressive brain tumor in adults and is characterized by an immunosuppressive microenvironment. Different factors shaping its tumor microenvironment (TME) regulate tumor progression and treatment response. The aim of this study was to characterize the main immunosuppressive elements of the GBM IDH-wt TME. Methods: Immunohistochemistry for CD3, CD4, CD8, CD163, programmed death ligand 1 (PD-L1) and programmed death 1 (PD1) was performed on surgical tumor specimens from patients diagnosed with GBM IDH-wt, according to the CNS WHO 2021 criteria. The impact of categorical variables on time-dependent outcomes such as overall survival (OS) and progression-free survival (PFS) has been estimated through the Kaplan-Meier method. Results: We included 30 patients (19 males and 11 females), median age of 59.8 years (range 40.2-69.1 years). All patients underwent surgery followed by temozolomide concurrent with and adjuvant to radiotherapy. MGMT was methylated in 14 patients (47%) and unmethylated in 16 patients (53%). The overall absolute percentages of CD4+ lymphocytes, both intratumoral and perivascular, were significantly more represented than CD8+ lymphocytes in the TME (p = 0.02). A low density of CD4+ lymphocytes (≤10%) was found to be a favorable prognostic factor for GBM outcome (p = 0.02). Patients with MGMT methylated and unmethylated tumors exhibited a distinct TME composition, with a significant higher number of perivascular CD8+ lymphocytes (p = 0.002), intratumoral CD8+ lymphocytes (p = 0.0024) and perivascular CD4+ lymphocytes (p = 0.014) in MGMT unmethylated tumors. PD-L1 expression in tumor cell surface was observed in four tumors (13.3%), and PD1 expression in infiltrating T lymphocytes was observed in nine (30%) tumors, with predominantly perivascular distribution. Conclusions: MGMT methylated and unmethylated tumors exhibit different immune profiles, likely reflecting the different biology of these tumors. The expression of PD-L1 in GBM IDH-wt patients is confined to a small subpopulation. While we found a significant association between low CD4+ lymphocyte density (≤10%) and survival, given the small numbers of our cohort, the prognostic value of CD4+ lymphocyte density will need to be validated in large-scale studies.

Prolonged ventricular activation time as a novel biomarker identifying burn-out phase of hypertrophic cardiomyopathy

Abstract Background Patients with hypertrophic cardiomyopathy (HCM) classically have preserved systolic function and decreased left ventricular end-diastolic volume. However, in a small sub-population, patients paradoxically develop systolic dysfunction, left ventricular dilatation, and ventricular wall thinning, which is known as end-stage hypertrophic cardiomyopathy or "burned-out cardiomyopathy’. Although an electrocardiogram (ECG) is a pivotal tool in HCM, its role in the detection of burned-out HCM has not been investigated. Purpose We assessed the ventricular activation time (VAT), an ECG marker of the duration of ventricular depolarization, as a predictive tool in the detection of the HCM burn-out phase. Methods We prospectively studied consecutive patients diagnosed with HCM via echocardiography at our medical center between 2017 and 2022. The ECG, along with echocardiography, was performed on the same day as the diagnosis. VAT was measured in milliseconds between the onset of the QRS complex to the peak or R wave on the V5 and V6 precordial ECG lead. HCM burn-out phase was supported when the left ventricular ejection fraction (LVEF) was below 50%, measured with echocardiography. Statistical analysis was performed using the SPSS software. Results Forty-five patients with HCM were studied, and their mean age was 51, while 78% (35/45) were male. Echocardiography data upon diagnosis revealed a mean maximum wall thickness of 19 (±3mm) and a mean left ventricular ejection fraction of 65% (±5%). Left ventricular outflow obstruction was present in 31% (14/45). During the study, 24% (11/45) presented atrial fibrillation, 44% (20/45) had an ICD implantation, and 22% (10/45) progressed to the burn-out phase of HCM. HCM patients who presented atrial fibrillation (63±31 vs. 46±14msec, p=0.018) or burn-out phase (65±32 vs. 46±13msec, p=0,009) had a significantly higher VAT than those subsets without. Notably, there was a significant positive relationship between VAT a burn-out phenotype (r=0,385, p=0.009). HCM patients with greater VAT also had a significantly higher risk of presenting a larger left atrial diameter (r=0,295, p=0.048), an increased left ventricular end-diastolic diameter (r=0.306, p=0.041), and a trend of presenting a lower LVEF (r=-0,262, p=0.082). Finally, we revealed a positive relationship between VAT and atrial fibrillation incidence (r=0,352, p=0.018). Conclusion Prolongation of VAT predicts the incidence of burn-out stage in patients with HCM and atrial fibrillation. Therefore, it seems to be a promising, inexpensive ECG marker for HCM progression and severity. Our findings emphasize the pivotal role of the ECG as a useful and inexpensive tool in the diagnostic follow-up of HCM.

Assessing the Current Demography and Future Shape of a Minority Sub-Population: the Case of Liverpool UK Jewry

The paper sets out how a small religion-based sub-population based in a UK city, Liverpool Jewry, underpinned its planning for the future in the light of its reducing size and the consequent strain on the community’s infrastructure and resources. This was achieved by carrying out a voluntary census to provide information on the community’s current size (about 1800 individuals living in 900 households) and its age profile, household types and other characteristics. The census questions were designed to provide data that allowed future population projections to be developed. The low number of births in the community necessitated the devising of a novel approach to the fertility assessment, though mortality rates were derived in a traditional way. In particular, the various elements of migration were investigated via historical information and stated preference responses. The analysis facilitated the estimation of levels of future demand for educational, youth, cultural, religious, welfare and burial services, and the community’s ability to continue to provide those services. Whilst the subject of this paper is the Jewish community in the city of Liverpool, the approach set out here could be adopted by other minority groups, whether shrinking, growing or stable, in other localities and in other countries.

Early-Stage Infection-Specific Heterobasidion annosum (Fr.) Bref. Transcripts in H. annosum-Pinus sylvestris L. Pathosystem.

Transcriptomes from stem-inoculated Scots pine saplings were analyzed to identify unique and enriched H. annosum transcripts in the early stages of infection. Comparing different time points since inoculation identified 131 differentially expressed H. annosum genes with p-values of ≤0.01. Our research supports the results of previous studies on the Norway spruce-Heterobasidion annosum s.l. pathosystem, indicating the role of carbohydrate and lignin degradation genes in pathogenesis at different time points post-inoculation and the role of lipid metabolism genes (including but not limited to the delta-12 fatty acid desaturase gene previously reported to be an important factor). The results of this study indicate that the malic enzyme could be a potential gene of interest in the context of H. annosum virulence. During this study, difficulties related to incomplete reference material of the host plant species and a low proportion of H. annosum transcripts in the RNA pool were encountered. In addition, H. annosum transcripts are currently not well annotated. Improvements in sequencing technologies (including sequencing depth) or bioinformatics focusing on small subpopulations of RNA would be welcome.

Targeting Lipid Metabolism in Cancer Stem Cells for Anticancer Treatment.

Cancer stem cells (CSCs), or tumor-initiating cells (TICs), are small subpopulations (0.0001-0.1%) of cancer cells that are crucial for cancer relapse and therapy resistance. The elimination of each CSC is essential for achieving long-term remission. Metabolic reprogramming, particularly lipids, has a significant impact on drug efficacy by influencing drug diffusion, altering membrane permeability, modifying mitochondrial function, and adjusting the lipid composition within CSCs. These changes contribute to the development of chemoresistance in various cancers. The intricate relationship between lipid metabolism and drug resistance in CSCs is an emerging area of research, as different lipid species play essential roles in multiple stages of autophagy. However, the link between autophagy and lipid metabolism in the context of CSC regulation remains unclear. Understanding the interplay between autophagy and lipid reprogramming in CSCs could lead to the development of new approaches for enhancing therapies and reducing tumorigenicity in these cells. In this review, we explore the latest findings on lipid metabolism in CSCs, including the role of key regulatory enzymes, inhibitors, and the contribution of autophagy in maintaining lipid homeostasis. These recent findings may provide critical insights for identifying novel pharmacological targets for effective anticancer treatment.

Schizophrenia-associated changes in neuronal subpopulations in the human midbrain.

Dysfunctional GABAergic and dopaminergic neurons are thought to exist in the ventral midbrain of patients with schizophrenia, yet transcriptional changes underpinning these abnormalities have not yet been localized to specific neuronal subsets. In the ventral midbrain, control over dopaminergic activity is maintained by both excitatory (glutamate) and inhibitory (GABA) input neurons. To further elucidate neuron pathology at the single-cell level, we characterized the transcriptional diversity of distinct NEUN+ populations in the human ventral midbrain and then tested for schizophrenia-associated changes in neuronal subset proportions and gene activity changes within neuronal subsets. Combining single nucleus RNA-sequencing with fluorescence-activated sorting of NEUN+ nuclei, we analysed 31,669 nuclei. Initially, we detected 18 transcriptionally distinct neuronal populations in the human ventral midbrain, including 2 "mixed" populations. The presence of neuronal populations in the midbrain was orthogonally validated with immunohistochemical stainings. "Mixed" populations contained nuclei expressing transcripts for vesicular glutamate transporter 2 (SLC17A6) and Glutamate Decarboxylase 2 (GAD2), but these transcripts were not typically co-expressed by the same nucleus. Upon more fine-grained subclustering of the 2 "mixed" populations, 16 additional subpopulations were identified that were transcriptionally classified as excitatory or inhibitory. In the midbrains of individuals with schizophrenia, we observed potential differences in the proportions of two (sub)populations of excitatory neurons, two subpopulations of inhibitory neurons, one "mixed" subpopulation, and one subpopulation of TH-expressing neurons. This may suggest that transcriptional changes associated with schizophrenia broadly affect excitatory, inhibitory, and dopamine neurons. We detected 99 genes differentially expressed in schizophrenia compared to controls within neuronal subpopulations identified from the 2 "mixed" populations, with the majority (67) of changes within small GABAergic neuronal subpopulations. Overall, single-nucleus transcriptomic analyses profiled a high diversity of GABAergic neurons in the human ventral midbrain, identified putative shifts in the proportion of neuronal subpopulations, and suggested dysfunction of specific GABAergic subpopulations in schizophrenia, providing directions for future research.

Evaluation of Gremlin-1 as a therapeutic target in metabolic dysfunction-associated steatohepatitis

Gremlin-1 has been implicated in liver fibrosis in metabolic dysfunction-associated steatohepatitis (MASH) via inhibition of bone morphogenetic protein (BMP) signalling and has thereby been identified as a potential therapeutic target. Using rat in vivo and human in vitro and ex vivo model systems of MASH fibrosis, we show that neutralisation of Gremlin-1 activity with monoclonal therapeutic antibodies does not reduce liver inflammation or liver fibrosis. Still, Gremlin-1 was upregulated in human and rat MASH fibrosis, but expression was restricted to a small subpopulation of COL3A1/THY1+ myofibroblasts. Lentiviral overexpression of Gremlin-1 in LX-2 cells and primary hepatic stellate cells led to changes in BMP-related gene expression, which did not translate to increased fibrogenesis. Furthermore, we show that Gremlin-1 binds to heparin with high affinity, which prevents Gremlin-1 from entering systemic circulation, prohibiting Gremlin-1-mediated organ crosstalk. Overall, our findings suggest a redundant role for Gremlin-1 in the pathogenesis of liver fibrosis, which is unamenable to therapeutic targeting.

Evaluation of Gremlin-1 as a therapeutic target in metabolic dysfunction-associated steatohepatitis.

Gremlin-1 has been implicated in liver fibrosis in metabolic dysfunction-associated steatohepatitis (MASH) via inhibition of bone morphogenetic protein (BMP) signalling and has thereby been identified as a potential therapeutic target. Using rat in vivo and human in vitro and ex vivo model systems of MASH fibrosis, we show that neutralisation of Gremlin-1 activity with monoclonal therapeutic antibodies does not reduce liver inflammation or liver fibrosis. Still, Gremlin-1 was upregulated in human and rat MASH fibrosis, but expression was restricted to a small subpopulation of COL3A1/THY1+ myofibroblasts. Lentiviral overexpression of Gremlin-1 in LX-2 cells and primary hepatic stellate cells led to changes in BMP-related gene expression, which did not translate to increased fibrogenesis. Furthermore, we show that Gremlin-1 binds to heparin with high affinity, which prevents Gremlin-1 from entering systemic circulation, prohibiting Gremlin-1-mediated organ crosstalk. Overall, our findings suggest a redundant role for Gremlin-1 in the pathogenesis of liver fibrosis, which is unamenable to therapeutic targeting.

The Potential of Epigallocatechin Gallate in Targeting Cancer Stem Cells: A Comprehensive Review.

The dreadful scenario of cancer prevails due to the presence of cancer stem cells (CSCs), which contribute to tumor growth, metastasis, invasion, resistance to chemo- and radiotherapy, and recurrence. CSCs are a small subpopulation of cells within the tumor that are characterized by self-renewal capability and have the potential to manifest heterogeneous lineages of cancer cells that constitute the tumor. The major bioactive green tea polyphenol (-)-epigallocatechin gallate (EGCG) has been fruitful in downgrading cancer stemness signaling and CSC biomarkers in cancer progression. EGCG has been evidenced to maneuver extrinsic and intrinsic apoptotic pathways in order to decrease the viability of CSCs. Cancer stemness is intricately related to epithelial-mesenchymal transition (EMT), metastasis and therapy resistance, and EGCG has been evidenced to regress all these CSC-related effects. By inhibiting CSC characteristics EGCG has also been evidenced to sensitize the tumor cells to radiotherapy and chemotherapy. However, the use of EGCG in in vitro and in vivo cancer models raises concern about its bioavailability, stability and efficacy against spheroids raised from parental cells. Therefore, novel nano formulations of EGCG and adjuvant therapy of EGCG with other phytochemicals or drugs or small molecules may have a better prospect in targeting CSCs. However, extensive clinical research is still awaited to elucidate a full proof impact of EGCG in cancer therapy.

Significance of relative and absolute content of blood lymphocyte subopopulations for differential diagnosis of viral and bacterial meningitis in children

The search for markers of early etiological verification of meningitis is an urgent task for clinical laboratory diagnostics.Objective of the study is to determine the significance of relative and absolute content of blood lymphocyte subpopulations for early differential diagnosis of viral and bacterial meningitis in children.Materials and methods. There was performed a clinical and laboratory examination of 61 children, 37 of them were diagnosed with viral meningitis, and 24 ones – with bacterial purulent meningitis. The control group included 15 healthy children. The relative and absolute content of the main subpopulations (T-lymphocytes: T-helpers (Th), cytotoxic T-lymphocytes (CTL), NK, B-cells) and small subpopulations (NKT, DNT- and DPT-lymphocytes, CD3+CD8br, CD3+CD8dim, CD3-CD8+NK) of lymphocytes was assessed by flow cytometry. Statistical analysis of the data was carried out.Results. The decrease of relative and absolute content of all studied subpopulations was identified during an acute period of bacterial purulent meningitis, in comparison with the control group, with the exception of B-lymphocytes, the number of them was increased. Absolute content of all subpopulations was reduced in case of viral meningitis compared with the control group, the relative content did not have any significant differences, with the exception of an increase in the relative content of B-lymphocytes. Calculation of diagnostic sensitivity and specificity revealed the effectiveness of identification of both relative and absolute content of lymphocyte subpopulations for differential diagnosis of viral or bacterial meningitis. Identification of B-lymphocytes, NKT, CD3+CD8dim in the blood had the highest diagnostic efficiency (area under the ROC curve (AUC) ranging from 0,9 to 1).Conclusion. Assessment of relative and absolute content of blood lymphocyte subpopulations is an effective tool for laboratory differential diagnosis of viral and bacterial meningitis and can be used as an early differential diagnostic criterion, especially in case of impossibility to analyze the composition of cerebrospinal fluid.

Diversification of Pseudomonas aeruginosa Biofilm Populations under Repeated Phage Exposures Decreases the Efficacy of the Treatment.

Phage therapy has been proposed as a therapeutic alternative to antibiotics for the treatment of chronic, biofilm-related P. aeruginosa infections. To gain a deeper insight into the complex biofilm-phage interactions, we investigated in the present study the effect of three successive exposures to lytic phages of biofilms formed by the reference strains PAO1 and PA14 as well as of two sequential clinical P. aeruginosa isolates from the sputum of a patient with cystic fibrosis (CF). The Calgary device was employed as a biofilm model and the efficacy of phage treatment was evaluated by measurements of the biomass stained with crystal violet (CV) and of the cell density of the biofilm bacterial population (CFU/mL) after each of the three phage exposures. The genetic alterations of P. aeruginosa isolates from biofilms exposed to phages were investigated by whole-genome sequencing. We show here that the anti-biofilm efficacy of the phage treatment decreased rapidly with repeated applications of lytic phages on P. aeruginosa strains with different genetic backgrounds. Although we observed the maintenance of a small subpopulation of sensitive cells after repeated phage treatments, a fast recruitment of mechanisms involved in the persistence of biofilms to the phage attack occurred, mainly by mutations causing alterations of the phage receptors. However, mutations causing phage-tolerant phenotypes such as alginate-hyperproducing mutants were also observed. In conclusion, a decreased anti-biofilm effect occurred after repeated exposure to lytic phages of P. aeruginosa biofilms due to the recruitment of different resistance and tolerance mechanisms.

Unveiling the Dynamic Interplay between Cancer Stem Cells and the Tumor Microenvironment in Melanoma: Implications for Novel Therapeutic Strategies.

Cutaneous melanoma still represents a significant health burden worldwide, being responsible for the majority of skin cancer deaths. Key advances in therapeutic strategies have significantly improved patient outcomes; however, most patients experience drug resistance and tumor relapse. Cancer stem cells (CSCs) are a small subpopulation of cells in different tumors, including melanoma, endowed with distinctive capacities of self-renewal and differentiation into bulk tumor cells. Melanoma CSCs are characterized by the expression of specific biomarkers and intracellular pathways; moreover, they play a pivotal role in tumor onset, progression and drug resistance. In recent years, great efforts have been made to dissect the molecular mechanisms underlying the protumor activities of melanoma CSCs to provide the basis for novel CSC-targeted therapies. Herein, we highlight the intricate crosstalk between melanoma CSCs and bystander cells in the tumor microenvironment (TME), including immune cells, endothelial cells and cancer-associated fibroblasts (CAFs), and its role in melanoma progression. Specifically, we discuss the peculiar capacities of melanoma CSCs to escape the host immune surveillance, to recruit immunosuppressive cells and to educate immune cells toward an immunosuppressive and protumor phenotype. We also address currently investigated CSC-targeted strategies that could pave the way for new promising therapeutic approaches for melanoma care.

Gauging Airbnb review sentiments and critical key-topics by small area estimation

In literature, several researchers have discovered that the reviews written about Airbnb accommodation tend to be extremely positive than those published on other famous platforms, consequently, many negative experiences remain untracked. Leaving negative experiences underrepresented hampers hosts’ ability to improve their services. To overcome this gap, we employ Small Area Estimation to quantify negative sentiment in Airbnb reviews and the relative critical topics that characterize them. Our methodology involves a two-step process: first, we employ sentiment analysis and topic modeling to identify negative sentiment and critical issues, followed by the application of a mixed effect random forest model to provide a granular analysis of Airbnb reviews in small sub-populations in the context of small area estimation. We focus on domains of the city of Rome defined by geographical areas and the presence of hosts and Superhosts. Our findings reveal nuanced sentiment variations and critical topic proportions that traditional methods often overlook.

Orai1 and Orai3 act through distinct signalling axes to promote stemness and tumorigenicity of breast cancer stem cells

BackgroundOne of major challenges in breast tumor therapy is the existence of breast cancer stem cells (BCSCs). BCSCs are a small subpopulation of tumor cells that exhibit characteristics of stem cells. BCSCs are responsible for progression, recurrence, chemoresistance and metastasis of breast cancer. Ca2+ signalling plays an important role in diverse processes in cancer development. However, the role of Ca2+ signalling in BCSCs is still poorly understood.MethodsA highly effective 3D soft fibrin gel system was used to enrich BCSC-like cells from ER+ breast cancer lines MCF7 and MDA-MB-415. We then investigated the role of two Ca2+-permeable ion channels Orai1 and Orai3 in the growth and stemness of BCSC-like cells in vitro, and tumorigenicity in female NOD/SCID mice in vivo.ResultsOrai1 RNA silencing and pharmacological inhibition reduced the growth of BCSC-like cells in tumor spheroids, decreased the expression levels of BCSC markers, and reduced the growth of tumor xenografts in NOD/SCID mice. Orai3 RNA silencing also had similar inhibitory effect on the growth and stemness of BCSC-like cells in vitro, and tumor xenograft growth in vivo. Mechanistically, Orai1 and SPCA2 mediate store-operated Ca2+ entry. Knockdown of Orai1 or SPCA2 inhibited glycolysis pathway, whereas knockdown of Orai3 or STIM1 had no effect on glycolysis.ConclusionWe found that Orai1 interacts with SPCA2 to mediate store-independent Ca2+ entry, subsequently promoting the growth and tumorigenicity of BCSC-like cells via glycolysis pathway. In contrast, Orai3 and STIM1 mediate store-operated Ca2+ entry, promoting the growth and tumorigenicity of BCSC-like cells via a glycolysis-independent pathway. Together, our study uncovered a well-orchestrated mechanism through which two Ca2+ entry pathways act through distinct signalling axes to finely control the growth and tumorigenicity of BCSCs.

Identification of Rocaglate Acyl Sulfamides as Selective Inhibitors of Glioblastoma Stem Cells.

Glioblastoma (GBM) is the most aggressive and frequently occurring type of malignant brain tumor in adults. The initiation, progression, and recurrence of malignant tumors are known to be driven by a small subpopulation of cells known as tumor-initiating cells or cancer stem cells (CSCs). GBM CSCs play a pivotal role in orchestrating drug resistance and tumor relapse. As a prospective avenue for GBM intervention, the targeted suppression of GBM CSCs holds considerable promise. In this study, we found that rocaglates, compounds which are known to inhibit translation via targeting of the DEAD-box helicase eIF4A, exert a robust, dose-dependent cytotoxic impact on GBM CSCs with minimal killing of nonstem GBM cells. Subsequent optimization identified novel rocaglate derivatives (rocaglate acyl sulfamides or Roc ASFs) that selectively inhibit GBM CSCs with nanomolar EC50 values. Furthermore, comparative evaluation of a lead CSC-optimized Roc ASF across diverse mechanistic and target profiling assays revealed suppressed translation inhibition relative to that of other CSC-selective rocaglates, with enhanced targeting of the DEAD-box helicase DDX3X, a recently identified secondary target of rocaglates. Overall, these findings suggest a promising therapeutic strategy for targeting GBM CSCs.