Search Results (171)

Diketopiperazines (DKPs) are biologically important cyclic dipeptides widespread in nature, associated primarily with microorganisms. This is the case for the 2,5-DKP derivative cyclo(L-Leu-L-Pro) (cLP), also known as gancidin W or PPDHMP, identified from a variety of bacteria and fungi, and occasionally found in food products. The present review retraces the discovery of cLP, its identification in living species, its chemical syntheses, and its biochemical properties. In bacteria, cLP is often associated with other DKPs to serve as a defense element against other microorganisms and/or as a regulator of bacterial growth. cLP plays a role in quorum-sensing and functions as an anticariogenic and antifungal agent. The antimicrobial mechanism of action and molecular targets of cLP are evoked. The interest in cLP for combatting certain parasitic diseases, such as malaria, and cancers is discussed. The capacity of cLP to interact with CD151 and to down-regulate the expression of this tetraspanin can be exploited to reduce tumor dissemination and metastases. The review sheds light on the pharmacology and specific properties of cyclo(L-Leu-L-Pro), which can be useful for the development of a novel therapeutic approach for different human pathologies. It is also of interest to help define the bioactivity and mechanisms of action of closely related DKP-based natural products. Full article

The cellular membrane is a dynamic structure composed of lipids and proteins organized into specialized domains that facilitate interactions between extracellular molecules and the intracellular environment. Tetraspanins are a family of transmembrane proteins involved in diverse cellular processes, including membrane stabilization and fusion, endocytosis, extracellular vesicle formation, and the organization of proteins and lipids at specific membrane sites known as Tetraspanin-Enriched Microdomains (TEMs). These lipid–protein interactions play a critical role in the replicative cycle of Orthoflavivirus, including dengue, Zika, and West Nile, by facilitating viral entry, replication, assembly, and egress. In addition, tetraspanins also regulate the biogenesis and function of extracellular vesicles, contributing to viral dissemination, persistent infection, and immune evasion. This review summarizes the current knowledge on the structural and functional aspects of tetraspanins, their interplay with lipids, and their emerging roles in the Orthoflavivirus replicative cycle. We also discuss how these insights may inform the development of antiviral strategies targeting membrane organization and virus–host interactions. Full article

Background: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of immature T cells, driven by dysregulated transcriptional networks and oncogenic signaling pathways. Here, we present the first comprehensive analysis of the expression and regulation of TSPAN32, a tetraspanin implicated in lymphocyte homeostasis, in T-ALL. Methods: Transcriptomic data from the Leukemia MILE study (GSE13159) were analyzed to assess TSPAN32 expression across leukemic subtypes. Gene Set Enrichment Analysis (GSEA) was performed to explore biological pathways associated with TSPAN32-correlated genes. For mechanistic validation, HPB-ALL cells were used as a model, with NOTCH signaling inhibited by γ-secretase inhibitor (GSI) treatment and TAL1–LMO1 overexpression induced through doxycycline-inducible lentiviral vectors. Gene expression changes were quantified by RT-qPCR. Results: TSPAN32 was frequently downregulated in T-ALL compared to healthy bone marrow, although expression was retained in a subset of cases. GSEA revealed that TSPAN32-correlated genes were inversely associated with cell cycle–related programs, consistent with its established role as a negative regulator of T cell proliferation. Mechanistically, TAL1–LMO1 overexpression strongly induced TSPAN32, while GSI-mediated NOTCH inhibition partially reactivated its expression. Interestingly, GSI treatment also increased TAL1 levels despite downregulating LMO1. Conversely, TAL1–LMO1 overexpression suppressed NOTCH1 and NOTCH3, highlighting a reciprocal regulatory interplay between NOTCH and TAL1/LMO1 oncogenic circuits that shapes TSPAN32 expression dynamics in T-ALL. Conclusions: This study identifies TSPAN32 as a novel transcriptional target under the influence of key leukemogenic pathways and suggests its potential role as a modulator of leukemic T cell proliferation, with implications for therapeutic strategies targeting TAL1 and NOTCH signaling. Full article

CD9 protein belongs to a family of proteins called tetraspanins, so named for their four-transmembrane-spanning architectures. These proteins are located in domains in the plasmatic membrane, called tetraspanin-enriched microdomains (TEMs). Several proteases and cellular receptors for virus entry cluster into TEMs, suggesting that TEMs are preferred virus entry portals. Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein mediates virus attachment and entry into cells by binding to human angiotensin-converting enzyme 2 (ACE-2). In addition, the secretory, type-I membrane-bound SARS-CoV-2 S protein is synthesized as a precursor (proS) that undergoes posttranslational cleavages by host cell proteases, such as furin and TMPRSS2. Moreover, it has been shown that neuropilin-1 (NRP1), which is known to bind furin-cleaved substrates, potentiates SARS-CoV-2 infectivity. Our results indicate that CD9 facilitates SARS-CoV-2 infection. In addition, we show how knocking out CD9 leads to a decrease in the expression of NRP1, a protein that improves SARS-CoV-2 infection. Furthermore, we show that CD9 colocalizes with ACE-2, NRP1, furin, and TMPRSS2 at the plasma membrane; that the absence of CD9 decreases the expression of these proteins on the plasma membrane CD9-enriched microdomains, and that CD9 interacts with ACE2. In conclusion, our data suggest that CD9 facilitates SARS-CoV-2 infection and that CD9 brings together different host proteins involved in SARS-CoV-2 attachment and entry into host cells, such as ACE2, NRP1, furin, and TMPRSS2. Importantly, the fact that a blocking antibody targeting CD9 can effectively reduce SARS-CoV-2 titers highlights not only the mechanistic role of CD9 in viral entry but also offers translational potential, suggesting that tetraspanin-targeting antibodies could be developed as therapeutic agents against SARS-CoV-2 and possibly other coronaviruses, with meaningful implications for clinical intervention. Full article

(1) Background: Chronic myeloid leukemia (CML) is a myeloproliferative disorder driven by the BCR::ABL oncoprotein. During the chronic phase, Philadelphia chromosome-positive hematopoietic stem cells generate proliferative myeloid cells with various stages of maturation. Despite this expansion, leukemic stem cells (LSCs) retain self-renewal capacity via asymmetric cell divisions, sustaining the stem cell pool. Quiescent LSCs are known to be resistant to tyrosine kinase inhibitors (TKIs), potentially through BCR::ABL-independent signaling pathways. We hypothesize that dysregulation of genes governing asymmetric division in LSCs contributes to disease progression, and that their expression pattern may serve as a prognostic marker during the chronic phase of CML. (2) Methods: Genes related to asymmetric cell division in the context of hematopoietic stem cells were extracted from the PubMed database with the keyword “asymmetric hematopoietic stem cell”. The collected relative gene set was tested on two independent bulk transcriptome cohorts and the results were confirmed by single-cell RNA sequencing. (3) Results: The expression of genes involved in asymmetric hematopoietic stem cell division was found to discriminate disease phases during CML progression in the two independent transcriptome cohorts. Concordance between cohorts was observed on asymmetric molecules downregulated during blast crisis (BC) as compared to the chronic phase (CP). This downregulation during the BC phase was confirmed at single-cell level for SELL, CD63, NUMB, HK2, and LAMP2 genes. Single-cell analysis during the CP found that CD63 is associated with a poor prognosis phenotype, with the opposite prediction revealed by HK2 and NUMB expression. The single-cell trajectory reconstitution analysis in CP samples showed CD63 regulation highlighting a trajectory cluster implicating HSPB1, PIM2, ANXA5, LAMTOR1, CFL1, CD52, RAD52, MEIS1, and PDIA3, known to be implicated in hematopoietic malignancies. (4) Conclusion: Regulation of CD63, a tetraspanin involved in the asymmetric division of hematopoietic stem cells, was found to be associated with poor prognosis during CML progression and could be a potential new therapeutic target. Full article

The expanding distribution and geographic range of mosquitoes have potentially contributed to increased flaviviral dissemination and transmission. Despite the growing burden of flaviviral infections, there are no effective antiviral treatments or vaccines, highlighting the need for novel therapeutic targets. Tetraspanins, a superfamily of transmembrane domain glycoproteins involved in cellular organization, signaling, and protein–protein interactions have been recognized as potential mediators of flaviviral infection and transmission. While their roles in vertebrate hosts have been explored, their involvement in flaviviral replication and dissemination within medically important vectors remains poorly understood. In this study, we investigated the role of arthropod tetraspanins in mosquito cells and extracellular vesicles (EVs) derived from cells infected with Zika virus (ZIKV) and dengue virus (serotype 2; DENV2). Among several of the tetraspanins analyzed, only CD151 was significantly upregulated in both mosquito cells and in EVs derived from ZIKV/DENV2-infected cells. RNAi-mediated silencing of CD151 led to a marked reduction in viral burden, suggesting its crucial role in flavivirus replication. Inhibition of EV biogenesis using GW4869 further demonstrated that EV-mediated viral transmission contributes to flavivirus propagation. Additionally, co-immunoprecipitation and immunofluorescence analyses revealed direct interactions between CD151 and ZIKV NS2B and DENV2 capsid proteins. Overall, our findings highlight the functional importance of mosquito CD151 in the replication and transmission of ZIKV and DENV2. This study provides new insights into the molecular mechanisms of flaviviral infection in mosquitoes and suggests that targeting vector tetraspanins may offer a potential approach to controlling mosquito-borne flaviviruses. Full article

Spinal cord injury (SCI) triggers both local and systemic pathological responses that evolve over time and differ with injury severity. Small extracellular vesicles (sEVs), known mediators of intercellular communication, may serve as biomarkers reflecting these complex dynamics. In this study, we investigated whether SCI severity modulates the composition and abundance of circulating plasma-derived sEVs across subacute and chronic phases. Using a graded thoracic contusion model in mice, plasma was collected at defined timepoints post-injury. sEVs were isolated via size-exclusion chromatography and characterized using nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and MACSPlex surface marker profiling. We observed an SCI-dependent increase in sEVs during the subacute (7 days) phase, most notably in moderate injuries (50 kdyne), with overall vesicle counts lower chronically (3 months). CD9 emerged as the predominant tetraspanin sEV marker, while CD63 and CD81 were generally present at low levels across all injury severities and timepoints. Surface sEV analysis revealed dynamic regulation of CD41⁺, CD44⁺, and CD61⁺ in the CD9⁺ sEV subset, suggesting persistent systemic signaling activity. These markers, traditionally associated with platelet function, may also reflect immune or reparative responses following SCI. Our findings highlight the evolving nature of sEV profiles after SCI and support their potential as non-invasive biomarkers for monitoring injury progression. Full article

Extracellular vesicles (EVs), including small EVs (sEVs) such as exosomes, play crucial roles in intercellular communication and disease pathology. Their heterogeneous nature, shaped by cellular origin and activation state, requires precise and multiplexed profiling of surface markers for effective characterization. Despite recent advances, current analytical methods remain complex, costly, or inaccessible for routine laboratory use. Here, we present a simple and cost-effective flow cytometry-based assay for the multiplexed analysis of tetraspanin markers (CD63, CD81, CD9) on fluorescently labeled sEVs. Our method combines metabolic labeling with paraformaldehyde fixation and low-speed centrifugation using a benchtop centrifuge, enabling efficient removal of unbound antibodies and minimizing nonspecific signals while preserving vesicle integrity. Using either metabolically labeled exosomes or bulk sEVs stained with carboxyfluorescein succinimidyl ester (CFSE), we demonstrate robust recovery and accurate, semi-quantitative profiling of tetraspanin expression. The assay reveals substantial variability in tetraspanin distribution across different cell lines and does not require ultracentrifugation or immunocapture. Notably, this versatile and reproducible method supports high sEV recovery and is adaptable to additional protein markers. Its compatibility with standard laboratory equipment makes it a practical and scalable alternative to more complex techniques, expanding access to multiplex sEV analysis for both research and clinical applications. Full article

Extracellular vesicles (EVs) are key mediators of intercellular communication and play a vital role in cancer progression. While EVs in the blood are well-studied, those in local body fluids, such as gastric juice (GJ), remain underinvestigated. Previously, we first characterized GJ-derived EVs and demonstrated their potential for gastric cancer (GC) screening. Here, we conducted a detailed morphological analysis of GJ-EVs using cryo-electron microscopy, identifying both typical and atypical EV subtypes, and categorized their relative abundances. A subsequent comparison of the size distribution of GJ-derived EVs by nanoparticle tracking analysis revealed significant differences between samples obtained from GC patients (n = 40) and healthy subjects (n = 25). Additionally, the mean EV sizes differed significantly according to the presence of the tetraspanin protein CD9. Furthermore, the ratio of CD9-positive to CD9-negative EV samples differed between cancer patients and healthy donors. These data suggest that GJ contains distinct subpopulations of EVs that vary in size and CD9 expression, as well as EVs with certain types of atypical morphology. The identification of discrepancies in EV size and the presence of CD9 between GJ from cancer patients and healthy individuals offers potential avenues for the identification of new GC markers. Full article

Synovial inflammation is recognised as a pathological driver of osteoarthritis (OA), a degenerative joint disease involving cartilage degradation and joint pain. Since extracellular vesicles (EVs) have emerged as key mediators of cellular cross-talk, this study characterised synovial fluid EVs (SFEVs) in OA patients with varying disease severity and determined their functional effects on OA articular chondrocytes. Synovial fluid and articular cartilage were collected from patients undergoing knee surgery. SFEVs were isolated via ultracentrifugation and characterised by nanoparticle tracking analysis, ExoView, and Luminex analysis of protein cargo. Patients were stratified into mild/moderate- and severe-OA groups based on Oxford Knee Score and EQ5D. Chondrocytes were treated with SFEVs, and transcriptomic and secretome responses were analysed using RNA sequencing, Luminex, and ELISA. SFEVs from patients with severe OA were more abundant, smaller and exhibited increased tetraspanin expression. Synovial fluid and SFEVs induced distinct transcriptomic changes in chondrocytes. SFEVs from patients with severe OA promoted a pro-inflammatory and catabolic chondrocyte phenotype, with upregulation of CRTAC1, COL6A3, TNC, and CXCL5, greater secretion of IL-6, MMP1, MMP3 and MMP13, and pro-nociceptive mediators (NGF and Substance P). These findings suggest that SFEVs may contribute to OA progression by exacerbating cartilage damage and promoting pain sensitisation. Full article

ADAM (A Disintegrin and Metalloproteinase) family members are multifunctional transmembrane proteases that govern tumorigenesis and metastasis by cleaving membrane-bound substrates such as growth factors, cytokines, and cell adhesion molecules. Several ADAMs, including ADAM8, ADAM9, ADAM10, ADAM12, and ADAM17, are overexpressed in malignancies and are linked with a poor prognosis. These proteases contribute to tumour growth by regulating cell proliferation, cell fate, invasion, angiogenesis, and immune evasion. ADAM10 and ADAM17, especially, facilitate the shedding of critical developmental and growth factors and their receptors, as well as immuno-regulatory molecules, hence promoting tumour progression, immune escape, and resistance to therapy. Recent work has unveiled multiple regulatory pathways that modulate ADAM functions, which include trafficking, dimerization, and conformational modifications that affect substrate accessibility. These observations have rekindled efforts to produce selective ADAM inhibitors, avoiding the off-target consequences reported with early small molecule inhibitors targeting the enzyme active site, which is conserved also in matrix metalloproteinases (MMPs). Promising approaches tested in preclinical models and, in some cases, clinical settings include more selective small-molecule inhibitors, monoclonal antibodies, and antibody–drug conjugates designed to specifically target ADAMs. In this review, we will discuss the emerging roles of ADAMs in cancer biology, as well as the molecular processes that control their function. We further discuss the therapeutic potential of targeting ADAMs, with a focus on recent advances and future directions in the development of ADAM-specific cancer therapies. Full article

Alzheimer’s disease (AD) is one of the most common progressive neurodegenerative diseases leading to impairments in memory, orientation, and behavior. However, significant work is still needed to fully understand the progression of such disease and develop novel therapeutic agents for AD prevention and treatment. Small extracellular vesicles (sEVs) have received attention in recent years due to their potential therapeutic effects on AD. The aim of this study was to determine the potential effect of sEVs in an in vitro model of AD. sEVs were isolated from human Wharton’s jelly mesenchymal stem cells (MSCs) by asymmetric depth filtration, a method developed recently by us. AD was modeled in vitro using cells obtained from the hippocampi of newborn 5xFAD transgenic mice carrying mutations involved in familial AD. After isolation, sEVs underwent detailed characterization that included scanning electron microscopy, nanoparticle tracking analysis, confocal microscopy, Western blotting, and Luminex assay. When added to 5xFAD hippocampal cells, sEVs were nontoxic, colocalized with neurons and astrocytes, decreased the level of Aβ peptide, and increased the synaptic density. These results support the possibility that sEVs can improve brain cell function during aging, decrease the risk of AD, and potentially be used for AD therapeutics. Full article

The impact of dietary lipid sources on nutrient metabolism and reproductive development is a critical focus in aquaculture broodstock nutrition. Previous studies have demonstrated that fish oil supplementation modulates the expression of genes involved in steroid hormone synthesis, glucose, and lipid metabolism promoting ovarian development in female Scatophagus argus (spotted scat). However, the effects of fish oil on hepatic function at the protein level remain poorly characterized. In this study, female S. argus were fed diets containing 8% fish oil (FO, experimental group) or 8% soybean oil (SO, control group) for 60 days. Comparative proteomic analysis of liver tissue identified significant differential protein expression between groups. The FO group exhibited upregulation of lipid metabolism-related proteins, including COMM domain-containing protein 1 (Commd1), tetraspanin 8 (Tspan8), myoglobin (Mb), transmembrane protein 41B (Tmem41b), stromal cell-derived factor 2-like protein 1 (Sdf2l1), and peroxisomal biogenesis factor 5 (Pex5). Additionally, glucose metabolism-associated proteins, such as Sdf2l1 and non-POU domain-containing octamer-binding protein (Nono), were elevated in the FO group. Moreover, proteins linked to inflammation and antioxidant responses, including G protein-coupled receptor 108 (Gpr108), protein tyrosine phosphatase non-receptor type 2 (Ptpn2), Pex5, p120 catenin (Ctnnd1), tripartite motif-containing protein 16 (Trim16), and aquaporin 11 (Aqp11), were elevated in the FO group, while proteins involved in oxidative stress, such as reactive oxygen species modulator 1 (Romo1), cathepsin A (Ctsa), and Cullin 4A (Cul4a), were downregulated. These proteomic findings align with prior transcriptomic data, indicating that dietary fish oil enhances hepatic lipid metabolism, mitigates oxidative stress, and strengthens antioxidant capacity. Furthermore, these hepatic adaptations may synergistically support ovarian maturation in S. argus. This study provides novel proteomic-level evidence supporting the role of fish oil in modulating hepatic lipid and energy metabolism, thereby elucidating the role of fish oil in optimizing hepatic energy metabolism and redox homeostasis to influence reproductive processes, advancing our understanding of n-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA) in teleost liver physiology. Full article

The plant pathogenic fungus Botrytis cinerea causes significant losses in agricultural production and it is rather difficult to control due to its broad host range and environmental persistence. The management of gray mold disease is still mainly based on the use of chemicals, which could have harmful effects not only due to impacts on the environment and human health, but also because they favor the development of fungicide-resistant strains. In this scenario, the strategy of RNA interference (RNAi) is being widely considered, and Spray-Induced Gene Silencing (SIGS) is gaining interest as a versatile, sustainable, effective, and environmentally friendly alternative to the use of chemicals in the protection of crops. The SIGS approach was evaluated to control B. cinerea infection on lettuce plants. In vitro-synthesized dsRNA molecules (BcBmp1-, BcBmp3-, and BcPls1-dsRNAs) were used naked, or complexed to small layered double hydroxide (sLDH) clay nanosheets. Therefore, treatments were applied by pressure spraying whole lettuce plants lately inoculated with B. cinerea. All sprayed dsRNAs proved effective in reducing disease symptoms with a notable reduction compared to controls. The effectiveness of SIGS in reducing disease caused by B. cinerea was high overall and increased significantly with the use of sLDH clay nanosheets. The sLDH clay nanosheet–dsRNA complexes showed better plant protection over time compared to the use of naked dsRNA and this was particularly evident at 27 days post-inoculation. RNAi-based biocontrol could be an excellent alternative to chemical fungicides, and several RNAi-based products are expected to be approved soon, although they will face several challenges before reaching the market. Full article

Tetraspanin 32 (TSPAN32), a member of the tetraspanin superfamily, is one of several tumor-suppressing subtransferable fragments located in the imprinted gene domain of chromosome 11p15.5, a critical tumor-suppressor gene region. Although the biology of TSPAN32 remains largely unexplored, accumulating evidence suggests its involvement in hematopoietic functions. In this study, we performed a comprehensive analysis of the expression patterns and regulatory roles of TSPAN32. Notably, TSPAN32 is highly expressed in immune cells, particularly in natural killer (NK) cells and CD8+ T cells. The observed downregulation of TSPAN32 during immune cell activation highlights its potential role as a regulator of immune cell activation and metabolic adaptations, which are crucial for effective immune responses against pathogens and tumors. Moreover, the modulation of biological processes following TSPAN32 knockout further supports its critical role in regulating immune cell physiology and responses. These findings not only shed light on the biology of TSPAN32 but also provide the basis for exploring its diagnostic, prognostic, and therapeutic potential in autoimmune and inflammatory disorders, as well as in hematopoietic cancers. Full article