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Leishmaniasis is an infectious disease caused by several species of Leishmania parasites that preferentially infect macrophages as host cells. These intracellular parasites can evade the main microbicidal effector mechanisms of phagocytic cells and, in turn, are able to stimulate marked Th2 or regulatory T cell immune responses, which are not protective for the host. The presence of a non-protective immune response, together with the multiplication and spread of Leishmania parasites throughout the tissues, leads to the main clinical forms of leishmaniasis, such as cutaneous and visceral leishmaniasis. Although some clinical forms can be reproduced in experimental hosts such as mice and hamsters, these models do not fully mimic natural infection, which, in fact, impacts experimental vaccine development. For example, BALB/c mice are generally infected with around one million parasites, whereas humans are not infected with more than 1000 parasites, together with vector saliva. This excessive number of parasites in experimental models may affect the efficacy of vaccines in preclinical studies. Indeed, many experimental studies conducted over the past 20 years have shown only partial protection, regardless of the vaccine generation, host species employed, or the use of adjuvants. This review aims to summarize the main aspects associated with Leishmania vaccine development, including parasite diversity, host factors, immune responses, adjuvants, and antigens. Although many elegant studies have been conducted, it is possible that some essential step is still missing for the development of an effective vaccine for human use. Full article

Bone regeneration relies on the coordinated interplay between mechanical and biological cues. Piezoelectric composites, capable of converting mechanical strain into electrical signals, offer a promising approach to stimulate osteogenesis. This study aimed to develop and characterize polycaprolactone (PCL) and barium titanate (BaTiO₃) composite scaffolds fabricated through thermally induced phase separation (TIPS), and to systematically evaluate the effects of polymer concentration and ceramic incorporation on scaffold morphology, porosity, mechanical properties, and cytocompatibility were systematically evaluated. The resulting scaffolds exhibited a highly porous, interconnected architecture, with 9% PCL formulation showing the most uniform morphology and consistent mechanical and biological behavior. Incorporation of BaTiO₃ did not alter pore structure or compromise cytocompatibility but slightly enhanced stiffness and surface uniformity. SEM-based image analysis confirmed homogeneous BaTiO₃ dispersion across all formulations. MTT assays and confocal microscopy demonstrated robust pre-osteoblast adhesion and spreading, particularly on denser composite scaffolds, confirming that the inclusion of BaTiO₃ supports a favorable environment for cell proliferation. Overall, optimizing polymer concentration and ceramic dispersion enables fabrication of structurally coherent, cytocompatible scaffolds. The findings establish structure–property–biology relationships that serve as a baseline for future investigations into the electromechanical behavior of PCL/BaTiO₃ scaffolds and their potential to promote osteogenic differentiation under physiological loading. Full article

Smart agriculture technology is rapidly spreading for its economic benefits and increase in farming efficiency. The management of agricultural activities is fulfilled by a network of connected devices and sensors, using wireless technologies and software to exchange data over the Internet. The electromagnetic fields (EMFs) these systems use increase the background level in farmlands, and the crop plants are exposed to unusual levels of unnatural, polarized, coherent, and variable EM radiation. This combination determines EMF influence on plants. Many studies found effects at different levels of organization—molecular, organismal, and even ecosystem levels—but the underlying mechanisms are still not well understood. In this review paper, we attempted to clarify possible mechanisms on the very basic molecular level involved in the realization of biological effects, discussing the interaction of EMFs with water molecules in living systems, from their effects on biologically significant molecules, membranes, ion channels, and ion transport, oxidative processes in cells, and photosynthesis to the effects on plant growth and development. In conclusion, we discuss the obstacles to defining the conditions for the manifestation of beneficial or adverse effects and setting exposure limits. Full article

Background/Objectives: Peritoneal carcinomatosis is the leading cause of death in advanced ovarian cancer (AOC). Mesothelial cells lining the peritoneum modulate tumor implantation, yet the role of their apoptosis in metastasis development remains unclear. This study investigated the relationship between mesothelial cell apoptosis and metastatic spread in ovarian cancer (OC). Methods: The study included 26 patients with AOC, 11 with early-stage OC (EOC), and 13 healthy controls. Apoptotic activity in parietal peritoneal wall and omental mesothelial cells was assessed using the TUNEL technique. Metastases were classified as pushing or infiltrating. Associations with the peritoneal cancer index (PCI), BRCA mutation, and homologous recombination deficiency (HRD) status were analyzed. Results: Mesothelial cells adjacent to AOC metastases exhibited significantly higher apoptotic activity compared to controls (p < 0.05). Apoptosis was greater near infiltrating metastases than near pushing ones in both parietal (p < 0.01) and omental (p = 0.04) sites. The infiltration pattern was consistent between omental and parietal metastases (R = 0.588, p < 0.01). No significant differences in apoptosis were found between EOC and healthy controls, or in tumor and stromal cells between invasion types. Mesothelial apoptosis was independent of PCI, BRCA mutation, and HRD status. Conclusions: Our study suggests that mesothelial cell apoptosis may be associated with peritoneal spread in OC. Mesothelial cell apoptosis is more pronounced near infiltrative-type lesions, independent of BRCA/HRD status. These findings highlight mesothelial apoptosis as a relevant process in peritoneal dissemination. Further studies are needed to clarify its role in ovarian cancer progression. Full article

The process of bacterial reproduction on surfaces conducive to growth forms colonies, which are defined as physical bodies with functional and environmental effects. This phenomenon can be conceptualized as transforming biological processes into physical phenomena. Large bacterial multicellular aggregates can be conceptualized as physical entities, produced by “colonial organisms”, thereby transforming physics into biology. The formation of colonies requires surfaces, typically hydrogels or liquid–air interfaces, but also hard solid surfaces. Bacterial cell layers also contribute to the production of surfaces. Within a typical 3D-shaped, frequently domed colony, a variety of microcompartments form at the intersections of gradients that diffuse from its aerial and surface limits, leading to cellular functional diversity. This heterogeneity can lead to physical changes and fractures in the colony material, leading to the formation of fluid microchannels. The second primary type of colony is the 2D-shaped form that spreads over larger surfaces and is known as a biofilm. These physical structures possess significant water content, which is retained by a bacterial-excreted exopolymer. Biofilms are structurally organized as multilayer structures that can expand in the space through the lateral slippage of a more fluid overlayer on top of the surface-attached layer. The dissemination of biofilms may entail the integration of additional bacterial colonies, thereby giving rise to complex biofilms. The physical occupancy of microenvironments by colonies created on surfaces of higher organisms or on environmental surfaces exerts a significant influence on fluid mechanics and the functioning of organisms and ecosystems. In addition, colonies also contribute to the pathology of industrial constructions and devices, often leading to microbiologically influenced electrochemical corrosion, which results in material degradation. Full article

Background/Objectives: This study aimed to evaluate the prognostic effects of tumor spread through air spaces (STAS) and other clinical and pathological risk factors on disease-free survival (DFS) in patients with non-small cell lung cancer (NSCLC) who underwent curative lobectomy and had tumors measuring 4 cm or less. Methods: NSCLC patients who underwent surgery between March 2015 and May 2024 and had at least 12 months of follow-up were retrospectively analyzed. Patients with tumors measuring 4 cm or less who underwent R0 resection, lobectomy, and STAS assessment on intraoperative frozen sections were included in the study. Clinicopathological features of all patients were restaged according to the 9th edition of the TNM staging system. The Kaplan–Meier method, log-rank test, and univariate Cox regression analysis were used to determine the factors affecting DFS. Results: 88 patients were included in the study. The median age of the patients was 61 years, 77.3% were male, and 72.7% had adenocarcinoma histology. According to TNM 9, 23.9% of the cases were staged T1b, 18.2% T1c, and 58.0% T2a. STAS positivity was detected in 45 patients (51.1%). The rates of lymphovascular invasion (LVI) (40.0% vs. 18.6%; p = 0.028) and visceral pleural invasion (VPI) (57.8% vs. 27.9%; p = 0.005) were significantly higher in the STAS-positive group than in the STAS-negative group. Recurrence was observed in a total of 31 patients (35.2%) during a median follow-up period of 68.1 months. In Kaplan–Meier analysis, the median DFS was not reached for the entire cohort. The estimated median DFS in STAS-positive patients was 52.7 months, while the median was not reached in the STAS-negative group (p = 0.001). The median DFS was 52.3 months in those with lymph node positivity, while the median was not reached in those with lymph node negativity (p = 0.031). According to TNM 9, the difference in DFS between stage IA/IB and stage IIAB groups was not statistically significant (p = 0.080). In univariate Cox analysis, STAS positivity (HR = 3.79; 95% CI: 1.69–8.51; p = 0.001), lymph node positivity (HR = 2.58; 95% CI: 1.05–6.31; p = 0.038) and VPI (HR = 2.28; 95% CI: 1.07–4.86; p = 0.032) were found to be significant prognostic factors adversely affecting DFS. Age, gender, histological type, tumor location, T stage, LVI, perineural invasion (PNI), and adjuvant chemotherapy had no significant effect on DFS. Conclusions: STAS is a strong negative prognostic indicator for recurrence in patients with operated NSCLC with tumor size ≤ 4 cm. It is believed that STAS should be integrated into risk-based staging and adjuvant treatment decision-making processes in early-stage NSCLC, particularly when evaluated in conjunction with VPI and lymph node positivity. Full article

The continuing spread of SARS-CoV-2 and the associated morbidity and mortality, especially in vulnerable populations, highlight the need for the development of antiviral therapeutics. Reverse genetics systems and reporter viruses are valuable for antiviral screening by simplifying methods to detect and quantify virus infections. This study aimed to generate wild-type and Nluc reporter full-length SARS-CoV-2 molecular clones and viruses as tools for high-throughput antiviral assays. The large SARS-CoV-2 genome (~30 kb) makes cDNA cloning and virus rescue technically challenging, so we opted to use cDNA chemical synthesis services to generate full-length wild-type and reporter Delta and Omicron clones. Clone-derived Delta and Omicron wild-type and reporter viruses were successfully rescued and showed replication kinetics comparable to patient-derived isolates. Nluc reporter viruses displayed stable luciferase expression that correlated with viral titres, supporting their reliability as replication substitutes. Antiviral assays measuring replication inhibition by Remdesivir, Molnupiravir, and Nirmatrelvir, based on Nluc expression, yielded IC₅₀ values and selectivity indices consistent with published ranges. Finally, Delta Nluc viruses replicated in primary human bronchial epithelial cells, demonstrating the application of clone-derived viruses in physiologically relevant models. The SARS-CoV-2 cDNA clones and Nluc reporter viruses derived from DNA synthesis services provide a rapid, scalable reverse genetics platform for generating new viruses and developing assays to rapidly assess antiviral compounds against current and emerging SARS-CoV-2 variants or coronaviruses that may emerge in the future. Full article

Oocyte-granulosa cell complexes (OGCs) cultivation is crucial for advancing reproductive biotechnology but remains incomplete and needs further optimization. Insulin-like growth factor-I (IGF-I) regulates granulosa cell proliferation and apoptosis, and numerous studies have confirmed its role in promoting ovarian follicle development. Porcine follicular fluid (PFF) contains factors beneficial for oocyte growth, which may enhance oocyte development. To investigate whether IGF-I and PFF improve the in vitro culture efficiency of porcine OGCs, we cultured OGCs with IGF-I (0, 10, 50, 100 ng/mL) and PFF (from 3 to 6 mm follicles) at concentrations of 0, 2.5%, 5%, 10%, respectively. The results revealed that 50 and 100 ng/mL IGF-I significantly increased the antrum formation rate of OGCs (from 61.11 ± 7.35% to 88.89 ± 7.35%) and diameter growth of oocytes (from 108.77 ± 0.27 µm to 114.94 ± 0.58 and 113.29 ± 0.50 µm, respectively). However, only the 50 ng/mL group, but not the 100 ng/mL group, significantly improved the maturation rate (38.13 ± 3.77% vs. 25.00 ± 3.27%, p < 0.05) of oocytes. Additionally, 50 ng/mL IGF-I downregulated BAX (a pro-apoptotic gene) and upregulated BCL-2 (an anti-apoptotic factor) in granulosa cells, ultimately reducing apoptosis. In contrast, none of the PFF doses used in this study induced the formation of enclosed antrum-like structures in OGCs, nor did they significantly enhance their in vitro development. Our findings demonstrate that 50 ng/mL IGF-I effectively promotes the in vitro growth of porcine early antral follicle-derived OGCs by reducing apoptosis, whereas tested PFF concentrations had no beneficial effects and induced abnormal granulosa cell growth. How PFF modulates the adherent and spreading growth of granulosa cells has not been fully elucidated and requires further clarification. Full article

In human and non-human primates, filoviruses, e.g., Ebolaviruses, cause severe hemorrhagic fever for which there are few therapeutic options. While there are licensed vaccines and therapeutics for Ebola virus disease, there is no approved vaccine or treatment for other Ebola diseases. There is a need for broad-spectrum antivirals to treat Ebola virus (EBOV), Sudan virus (SUDV), and Marburg virus (MARV). We have previously demonstrated that probenecid, an FDA-approved drug with a safety profile spanning over 7 decades, is safe and effective in preventing the replication of influenza A viruses, SARS-CoV-2, and other RNA respiratory viruses, such as HMPV and RSV, both in vitro and in vivo. In this study, probenecid was shown to inhibit the replication of infectious EBOV, SUDV, and MARV in Vero E6 cells, with IC50 Values of 3 μM, 8 μM, and 13 μM, respectively. It also reduced plaque size in infected Vero cell lawns, suggesting reduced virus spread. These studies show that probenecid is an effective, broad-spectrum, host-directed antiviral drug. Full article

Neutrophil extracellular traps (NETs)—webs of DNA and granular proteins expelled by neutrophils—have been implicated in hepatocellular carcinoma (HCC) progression. NETs promote tumor angiogenesis, facilitate invasion/metastasis, and enable immune evasion. Recent data suggest that perioperative factors, including anesthetic techniques, may modulate NET formation (NETosis), thus potentially influencing oncologic outcomes. We conducted a literature review of experimental and clinical studies on NETosis pathophysiology and involvement in HCC and how anesthetic techniques may modulate NET formation and, implicitly, cancer outcomes. NET biomarkers such as citrullinated histone H3 (CitH3), cell-free DNA (cfDNA), and myeloperoxidase–DNA complexes (MPO-DNA) are elevated in HCC patients and correlate with tumor spread, showing diagnostic and prognostic potential. Perioperative anesthetic choices may influence NET activity and immune function. Regional anesthesia and local anesthetics (e.g., lidocaine infusion) attenuate the surgical stress response and preserve anti-tumor immunity. Notably, lidocaine may modulate NET formation and, in a few studies published so far, was shown to reduce postoperative NET markers and other pro-metastatic factors (MMP-9, VEGF) in cancer surgery. In conclusion, NETosis is a process that is strongly implicated in HCC biology. Data published so far suggest that the clinical significance of NETosis may lie in its potential as a marker for disease evaluation and progression, including during the perioperative period. Preliminary results suggest that lidocaine may have a role in decreasing NETosis. Future large randomized trials are needed to exactly quantify these effects. Targeting NETs may be another way to influence HCC outcomes. Full article

Background/Objectives: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with a high rate of recurrence and a dismal prognosis. Studies have shown that pancreatic cancer stem cells (PCSCs) are a subpopulation that contributes to tumor progression, resistance to therapeutics, and metastasis, making them a key subpopulation to target for treatment. Gedunin (GD), a natural compound derived from Azadirachta indica (neem), has shown anticancer properties in pancreatic cancer cells, but its effects on PCSCs remains unclear. This study evaluated the effects of GD in pancreatic cancer stem cells, highlighting its impacts on tumor growth and progression and focusing on its impact on the sonic hedgehog (Shh) signaling pathway. Methods: Functional assays were performed to assess the effect of GD on the sphere-forming ability, colony formation, and self-renewal of PCSCs. Athymic mice xenograft models were utilized to evaluate the tumor suppression effect of GD in vivo. Furthermore, the anticancer effect of GD on PCSCs was assessed using both in vitro and in vivo limiting dilution assay. GD-induced changes in Shh signaling and key stem cell marker expressions in PCSCs were evaluated. Results: GD effectively inhibited tumor growth in xenograft models and reduced the percentage of PCSCs. GD was effective in decreasing PCSCs’ proliferative, self-renewal, and colony-forming capacity. GD decreased the protein expression levels of key Shh signaling markers Gli1 and Shh, stem cell markers SOX2, Nanog, and Oct4, metastasis-related proteins MMP-2, MMP-3, and MMP-9, and EMT markers Tgf1, Slug, Snail, and Twist in both PDAC cells and PCSCs. We demonstrated a significant decrease in the spheroid formation and self-renewal capacity of the (ALDH+) PCSC population following GD treatment in HPAC cells, indicating its potential antagonistic effects on PCSCs. GD was highly effective in reducing tumor volume, stemness, and metastasis in both early and late chemotherapy. In vivo limiting dilution assay using CD133+/LGR5+ PCSC xenografts demonstrated that GD reduces tumor growth, metastasis, and stemness associated with PCSCs by downregulating the expression of Shh and Gli1. GD treatment also reduced micrometastatic lesions in the lung, liver, and brain, as identified using H&E staining. Conclusions: The findings highlight GD’s potential as a promising therapeutic candidate for PDAC, with the ability to target both bulk tumor cells and PCSCs. By simultaneously suppressing tumor growth, stemness, and metastatic spread, GD may contribute to more effective treatment strategies and improved patient outcomes. Full article

West Nile virus (WNV) is an arthropod-borne flavivirus first identified in 1937. Over time, WNV has spread globally and is now endemic in Italy. Although most human WNV infections are asymptomatic (80%), less than 1% progress to a neuroinvasive disease with high mortality rates. This case involves a 45-year-old woman with post-surgical hypoparathyroidism and Sjögren’s syndrome who developed severe encephalomyelitis linked to WNV, leading to ventilator-associated pneumonia and death. Neuropathological findings revealed a bilaterally cribriform thalamus and reddish punctate lesions near the dentate nucleus of the cerebellum. The trachea and bronchial hilum branches contained whitish foamy liquid. The left lung showed multiple brownish-violet areas, with whitish regions at dissection. The heart appeared unremarkable. A detailed neuropathological examination focused on areas involved in motor control pathways. Tissue samples were stained with hematoxylin and eosin and trichrome techniques, and immunohistochemistry was performed using CD68, CD3, and CD20. A significant damage was observed in the lenticular nucleus and motor thalamus, with prominent concentric vascular calcifications. The cerebellar cortex showed near-total depletion of Purkinje cells. In the spinal cord, CD68 and CD3 positivity was noted in the lateral funiculi, anterior horns, and Clarke’s column. Lung findings showed pulmonary edema, chronic emphysema, and bronchopneumonia. The observed CD3 and CD68 positivity confirms that WNV spreads trans-synaptically along motor control pathways. We speculate on the potential molecular mechanisms by which hypoparathyroidism and Sjögren’s syndrome may have played a role in the neuroinvasive progression of the disease. Full article

Background: Malignant ovarian tumours are most often detected at an advanced stage, when peritoneal dissemination across abdominal organs is already present. Metastasis in ovarian cancer arises from complex interactions between cancer cells and diverse components of the tumour microenvironment (TME), including extracellular matrix elements, fibroblasts, adipocytes, mesenchymal cells and leukocytes. This dynamic niche drives tumour progression, invasiveness and immunosuppression through cytokine- and chemokine-mediated signalling. A deeper understanding of these interactions may enable targeted modulation of the TME and help limit metastatic spread. Methods: In this study, using immunoenzymatic assays and a computational digital twin—a mechanistic, ODE-based in silico model that replicates key cellular and microenvironmental processes—we investigated whether and how caffeic acid phenethyl ester (CAPE) influences TME activation, cytokine and growth factor levels, and extracellular matrix remodelling. Results: Our findings show that CAPE modulates both pro- and antitumourigenic signalling pathways across immune, stromal and hypoxia-related axes, suggesting its potential to reshape the ovarian cancer microenvironment and improve therapeutic outcomes in this challenging malignancy. Conclusions: Taken together, these results indicate that CAPE may serve as a multifaceted modulator capable of simultaneously targeting tumour cells and their microenvironment, offering a promising avenue for enhancing therapeutic strategies in ovarian cancer. Full article

Calcium phosphate cements (CPCs) and biomaterials, such as mesoporous bioactive glass (MBG), are critical for bone tissue engineering. This study aimed to 3D-print CPC scaffolds modified with MBG to enhance their osteogenic potential and regenerative ability. MBG powder was synthesized and characterized using transmission electron microscopy (TEM), X-ray diffraction (XRD), and nitrogen adsorption–desorption techniques. A commercial CPC ink (hydroxyapatite/α-tricalcium phosphate) was mixed with 5% MBG (w/w; CPC/MBG), and, after rheological assessment, the mixture was used to obtain scaffolds via 3D printing. These scaffolds were then tested for chemical, morphological, and mechanical properties, as well as ion release analysis. Unmodified CPC 3D-printed scaffolds served as controls. Biological experiments, including cell viability, DNA content, cell adhesion/spreading, and osteogenic gene expression, were performed by seeding alveolar bone-derived mesenchymal stem cells onto the scaffolds. Statistics were performed using Student’s t-test and ANOVA with post hoc tests (α = 5%). MBG characterization showed a typical mesoporous structure with aligned microchannels and an amorphous structure. Both formulations released calcium and phosphate ions; however, CPC/MBG also released silicon. Cell viability, adhesion/spreading, and DNA content were significantly greater in CPC/MBG scaffolds compared to CPC (p < 0.05) after 3 and 7 days of culture. Furthermore, CPC/MBG supported increased expression of key osteogenic genes, including collagen (COL1A1), osteocalcin (OCN), and Runt-related transcription factor 2 (RUNX2), after 14 days (p < 0.05). The combination of CPC ink with MBG particles effectively enhances the biocompatibility and osteogenic potential of the scaffold, making it an innovative bioceramic ink formulation for 3D printing personalized scaffolds for bone regeneration. Full article

Single exosomes are detected via surface-enhanced Raman scattering (SERS) due to electromagnetic field accumulation on a specially designed flexible metasurface. This metasurface is a modulated silver nanofilm deposited on a thin, flexible plastic substrate. An explicit Equation for calculating the local electric field is given. The field reaches extremely high values under plasmon resonance conditions and fills the depressions of the metasurface. The thin, flexible metasurface can be incorporated into automated Lab-On-Chip analytical systems and used for spectroscopic studies of exosomes. We propose a method to distinguish individual exosomes from the HEK293T cell line on the metasurface and then obtain and assign their SERS spectra. An important advantage of the plasmonic metasurface presented in this work is its spatial complementarity to exosomes and other vesicle-like objects. The plasmonic metasurface is fabricated using holographic lithography and further investigated using a correlation approach combining atomic force microscopy, scanning spreading resistance microscopy, and surface-enhanced spectroscopy. Full article