Search Results (315)

Objectives: In the era of precision oncology, the management of lung cancer depends fundamentally on the acquisition of sufficient neoplastic material for both definitive histological subtyping and comprehensive molecular profiling. This study aimed to investigate molecular testing adequacy rates for small lung biopsy specimens obtained via minimally invasive procedures at three high-volume oncology centers. Recognizing that a significant subset of specimens remains insufficient for analysis, we evaluated the utility of cell pellets derived from residual fixative media as a supplemental resource for ancillary molecular testing. Methods: Over a six-month period, specimen handling workflows for small biopsies were assessed across three high-volume oncology centers. The pre-analytic molecular adequacy of formalin-fixed paraffin-embedded (FFPE) tissue sections from patients diagnosed with non-small cell lung cancer (NSCLC) was evaluated. During the final two months of the study, in cases where the primary FFPE tissue was deemed inadequate for molecular profiling, the residual fixative solution was recovered and processed to generate supplemental cell pellets. Results: Using adequacy thresholds of >200 tumor cells per section and a tumor cell fraction (TCF) of ≥10% or ≥5% (depending on specific assay requirements), the overall adequacy rates for FFPE samples were 80.6% (2986/3705) and 88.9% (3293/3705), respectively. During the final two months, 18.9% (154/816) of cases exhibited inadequate FFPE sections. However, of these cases, 56% (86/154) yielded adequate cell pellets based on cellularity evaluation and DNA quantification. These results indicate that cell pellets collected from the fixative medium of thoracic small biopsies are a valuable supplemental material for ancillary testing. Conclusions: This multi-center investigation demonstrates that a notable subset of NSCLC specimens obtained via minimally invasive biopsy remains insufficient for molecular analysis. Cell pellet samples obtained from residual fixative media serve as a critical supplemental resource, effectively increasing the success rate of molecular adequacy in clinical practice. Full article

Background/Objectives: Endodontic sealers can interact with periapical tissues through extrusion, yet the molecular mechanisms underlying their biological effects remain poorly defined. This study investigated how commonly used sealers influence mitogen-activated protein kinase (MAPK) signaling, cell viability, and osteogenic-associated responses in MC3T3-E1 pre-osteoblasts. Methods: Four commercial sealers, Calcium-silicate-based Bioceramic Sealer (EndoSequence^® BC Sealer, BC), Zinc oxide eugenol sealer (Kerr Pulp Canal Sealer, ZOE), Sealapex™, and AH26^®, were applied as standardized pellets, allowed to set, and cultured with MC3T3-E1 cells. Calcium deposition was assessed by Alizarin Red S (ARS) staining, and MAPK activation was evaluated by Western blotting. Due to excessive solubility (Sealapex™) or poor cell survival (AH26^®), mechanistic analyses were performed only for BC and ZOE. Osteogenic-associated gene expression was measured by qRT-PCR, and the functional role of MAPK signaling was assessed using ERK, JNK, and p38 inhibitors. Results: BC and Sealapex™ produced robust ARS staining, while ZOE and AH26^® produced minimal mineral-associated staining. Both BC and ZOE activated ERK, JNK, and p38, with ZOE inducing higher phosphorylation. However, BC maintained greater cell viability and increased Runx2 and Osx expression, whereas ZOE impaired early cell attachment and viability. MAPK inhibition in BC-treated cultures reduced osteogenic-associated gene expression and ARS staining, indicating MAPK involvement in BC-mediated responses. Conclusions: BC and ZOE elicit distinct MAPK activation patterns and cellular responses. Under the conditions tested, BC promoted a more favorable osteogenic-associated response, whereas ZOE compromised early cell viability. These mechanistic insights may help explain clinical differences in periapical tissue responses to sealer extrusion. Full article

Bloodstream infection (BSI) is a serious clinical condition associated with high morbidity and mortality, requiring rapid identification of causative agents and timely antimicrobial susceptibility testing (AST). This study evaluated accelerated bacterial identification from positive blood culture samples using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) combined with two rapid processing approaches: a serum separation tube-based centrifugation method (SST method) and shortened cultivation on solid media. Rapid identification was followed by accelerated AST, performed either from a bacterial cell pellet (SST method) and from early-grown bacterial biomass (shortened cultivation protocol). The results were compared with those obtained using routine laboratory procedures. A total of 270 positive blood culture samples were analyzed, with 135 samples processed by each protocol. Both approaches achieved an identification success rate of 93.33%. Rapid AST using the SST method showed error rates of 0.51% minor errors, 0.57% major errors, and 0.23% very major errors, with an overall agreement of 98.69%. The shortened cultivation protocol demonstrated lower error rates (0.46% minor errors and 0.23% major errors) and an overall agreement of 99.31%. These findings confirm that MALDI-TOF MS enables reliable early identification of BSI pathogens and rapid AST, supporting timely optimization of antimicrobial therapy and early detection of multidrug-resistant strains. Full article

Neural progenitor cell (NPC) transplantation is a promising strategy for spinal cord injury repair, as graft-derived neurons can integrate into host circuitry and promote functional recovery. While the brain-regional and dorsoventral identities of NPCs are known to influence graft composition and performance, the importance of axial (rostrocaudal) identity, specifically whether NPCs must be matched to the spinal level of injury, remains poorly understood. To address this, we compared outcomes following transplantation of NPCs isolated from the anterior embryonic spinal cord (A-NPCs) versus the posterior spinal cord (P-NPCs) in a mouse model of C5 cervical dorsal column injury. Following transplantation, NPCs retained their intrinsic molecular axial identities; P-NPC grafts maintained significantly higher expression of the lumbar-associated gene HoxC10 and possessed a higher proportion of Chx10-high V2a neurons compared to A-NPCs. Despite these maintained molecular differences, A-NPC and P-NPC grafts were indistinguishable in neuronal and glial density, axon outgrowth, and their ability to support host axon regeneration, including the corticospinal tract. Long-term behavioral testing and retrograde transsynaptic tracing revealed no significant differences between groups in the recovery of skilled pellet reaching, grip strength, or synaptic integration with host cervical motor circuitry. These findings demonstrate that although transplanted NPCs retain their molecular axial identity in the adult injured environment, this identity is not a primary determinant of anatomical integration or functional outcome. Our findings suggest a degree of plasticity in graft-host interactions and indicate that strict segment-matching is not essential for the efficacy of NPC-based therapies in spinal cord injury. Full article

Background: Extracellular vesicles (EVs) play an important role in tumor progression and intercellular communication, yet the contribution of specific cancer-related genes to EV secretion remains incompletely defined. Methods: To address this, we performed an siRNA-based loss-of-function screen targeting 30 frequently altered (proto-)oncogenes and tumor suppressor genes in the colorectal carcinoma cell line HCT-116 to assess their impact on EV release. EVs were isolated by sequential ultracentrifugation to obtain P14 and P100 fractions pelleting at 14,000× g or 100,000× g, respectively, and were characterized by nanoparticle tracking analysis, EV marker expression, and total protein quantification. Cell viability was assessed to control for potential apoptosis-related effects. Results: With few exceptions, knockdown of the investigated genes led to an increase in EV secretion. Silencing of KRAS and BRAF resulted in significantly elevated P14 EV levels, increased EV marker expression, and higher total protein content, while KRAS knockdown was additionally associated with a shift toward larger particle sizes. Downregulation of CTNNB1 increased P14 and decreased P100 EV secretion, whereas CDH1 knockdown reduced P14 EV levels and slightly increased P100 EVs. No general distinction between tumor suppressor genes and (proto-)oncogenes regarding their effects on EV secretion was observed, and cell viability was not significantly altered under the experimental conditions. Conclusions: These findings suggest that components of the Ras/Raf/MAPK and Wnt signaling pathways may contribute to the regulation of EV secretion in colorectal cancer cells. Full article

In this research, Fe-P scaffolds were successfully fabricated by the powder metallurgy method for the first time, using NaCl as the space holder for bone tissue engineering applications, with apparent porosities of approximately 70%. The Fe₃P powder was successfully synthesized by the mechanochemical method under an argon atmosphere using an initial mixture of Fe and P powders. The XRD patterns show that Fe₃P was obtained after sintering the milled powders at 1000 °C. Fe, Fe₃P, and Fe-50 wt% Fe₃P composite scaffolds and bulk pellets were prepared by sintering the milled powder at 1000 °C. Furthermore, the mechanical properties (compression strength) and bioactivity of the Fe-P scaffolds were determined. According to the compression test results, the composite scaffold showed higher compressive strength, lower fracture strain, and higher elastic modulus than the Fe and Fe₃P scaffolds, indicating that adding Fe₃P to Fe improves the mechanical properties. Moreover, among the scaffolds prepared by sintering at 1000 °C, the Fe scaffold exhibited the highest corrosion rate compared to the Fe₃P and composite samples, while the corrosion resistance of the composite sample was 3 times higher than that of the Fe sample. The ICP analysis showed that the amount of Fe released from the bulk pellets during soaking in PBS solution after four weeks was 3220 μg/dL, 4003 μg/dL, and 4774 μg/dL for the composite, Fe₃P, and Fe samples, respectively. The composite sample showed the highest cell viability, while the Fe sample had the lowest. The compressive strength (12.62 MPa) and fracture strain (5.98%) of the porous sintered composite scaffold at 1000 °C were within the range of trabecular bone, while the compressive strength of the composite sample was 17 times higher than that of the Fe sample. Furthermore, the MTS test showed that all the samples had good viability, while the composite sample had the best cell viability. The scaffolds were not cytotoxic. It can be concluded that the mechanical and biological properties of the composite sample were superior to those of the Fe and Fe₃P samples and that it may be a promising candidate for bone tissue engineering applications, especially for trabecular bone replacement. Full article

Proton exchange membranes (PEMs) are essential for fuel cells, yet conventional materials like Nafion suffer from humidity dependence and limited thermal stability. This study introduces sulfonated phenylene-bridged periodic mesoporous organosilicas (PMOs) as promising inorganic–organic hybrid PEMs, synthesized via surfactant-templating with varying alkyl chain lengths for different mesopore sizes. Post-synthetic functionalization involves nitration of phenylene moieties, reduction to amines, and ring-opening of propane or butane sultones to graft sulfonic acid groups via flexible spacers, achieving homogeneous distribution along pore walls. Post-functionalization is confirmed by powder X-ray diffraction (PXRD), revealing preserved 2D hexagonal p6mm ordering and phenylene stacking. N₂ physisorption shows type IV isotherms with reduced pore volumes and pore sizes. ¹H NMR is used to quantify functionalization degrees. Impedance spectroscopy on pressed pellets demonstrates proton conductivities up to 2 × 10⁻³ S cm⁻¹ at 30 °C and 90% RH, depending on the functionalization degree, confirming sulfonic acid-mediated conduction. Full article

A protocol was developed for the isolation and characterization of extracellular vesicles (EVs) from Coffea arabica cell suspension cultures (CSCs). The isolation method involved differential ultracentrifugation of the CSC filtrate, yielding two fractions: the pellet after 100,000×g for 36 min (100k×g) and the pellet obtained from the previous supernatant after 125,000 g for 6 h (125k×g). Both fractions were characterized by size, morphology, and proteomic profiles (ProteomeXchange identifier PXD071909). While no significant differences in average EV size were observed between the two fractions, proteomic analysis revealed distinct quantitative and compositional variations. The 100k×g fraction was enriched in proteins associated with cell periphery, plasma membrane, and extracellular region, whereas the 125k×g fraction predominantly contained proteins from the extracellular region. Proteomic marker analysis confirmed that both fractions contained protein EV markers, such as transmembrane and transport proteins, soluble EV-associated proteins, and proteins targeted to the extracellular environment or cell wall. Conversely, negligible contamination from non-EV-related proteins was detected. Furthermore, transmission electron microscopy (TEM) showed that the average size of the fractions was consistent with that reported for plant EVs. These findings demonstrate that the protocol utilized to isolate EVs from coffee CSC applies to the release of such vesicles without mechanical harsh grinding that leads to tissue/cell rupture and consequent contamination by other cell components. EVs obtained from coffee CSC represent a valuable and scalable platform, paving the way for the development of tools for biotechnological applications. Full article

Background/Objectives: The oncoprotein EWS::FLI1 is a chimeric transcription factor that aberrantly brings transcriptional deregulation relevant to Ewing sarcoma. It is also regarded as a therapeutic target for suppressing oncogenic progression, but the inhibition and clearance of the EWS::FLI1 oncoprotein remain a challenge. Methods: We apply a polyglutamine (polyQ) fusion strategy to directly target EWS::FLI1 in suppression of its transcriptional malfunction in A673 cells derived from Ewing sarcoma. Based on the template of the N-terminal fragment of polyQ-expanded ataxin-7 (Atx7_93Q-N172) and the homologous peptides of EWS::FLI1, we have designed and constructed three polyQ fusion proteins, namely Atx7_93Q-N172-SYGQ1, Atx7_93Q-N172-SYGQ2, and Atx7_93Q-N172-LCD. Results: Supernatant/pellet fractionation and immunofluorescence imaging reveal that the polyQ fusion proteins co-precipitate and co-localize with EWS::FLI1 in A673 cells, indicating that the polyQ fusions we have designed can sequester endogenous EWS::FLI1 into insoluble aggregates and reduce its cellular availability. Moreover, these polyQ fusions, especially Atx7_93Q-N172-LCD, alter the expression of EWS::FLI1 downstream genes, with an increase in P21 (CDKN1A) and a decrease in c-Myc. Conclusions: These results demonstrate that the engineered polyQ fusions entrap endogenous EWS::FLI1 protein into aggregates and reduce its soluble fraction in Ewing sarcoma cells. This study provides an alternative potential for treating Ewing sarcoma and other tumors by directly targeting the oncogenic proteins in the future. Full article

Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) possess the potential for chondrogenic differentiation, offering a promising alternative source for cartilage regeneration. To address the limited availability and expansion capacity of autologous chondrocytes, we investigated the effect of co-overexpression of Sox9, TGFβ1, and type II collagen (Col II) on the chondrogenic differentiation of hUC-MSCs using both 2D and 3D pellet culture systems. Following transfection, the cells exhibited a chondrocyte-like morphology and a marked downregulation of the stemness marker Stro-1. After 21 days in a 3D pellet culture system, the cells formed cartilage-like tissue characterized by the strong expression of chondrocyte-specific genes (Sox9, TGFβ1, Col II, Aggrecan) along with the significant secretion of sulfated glycosaminoglycans (sGaGs). These effects were attributed to enhanced cell–cell contact and extracellular matrix interactions promoted by the 3D environment. Our findings suggest that genetically modified hUC-MSCs cultured in a 3D pellet system represent a robust in vitro model for cartilage regeneration, with potential applications in transplantation and drug toxicity screening. Full article

In this present study, sodium- and strontium-modified bismuth titanate—Bi_0.5Na_0.5TiO₃ (BNT) and Bi_0.5Sr_0.5TiO₃ (BST)—were synthesized using the auto-combustion technique with citric acid (C₆H₈O₇) and glycine (C₂H₅NO₂) as fuels in an optimized ratio of 1.5:1. The resulting powders were characterized using X-ray diffraction (XRD), energy-dispersive X-ray (EDX) spectroscopy, UV–Visible diffuse reflectance spectroscopy (DRS), and Fourier-transform infrared (FT-IR) spectroscopy. The electrical behavior of the samples was studied using an LCR meter. XRD analysis confirmed the formation of a single-phase perovskite structure with average crystallite sizes of 18.60 nm for BNT and 22.03 nm for BST, attributed to the difference in ionic radii between Na⁺ and Sr²⁺. An increase in crystallite size was accompanied by a corresponding increase in lattice parameters and unit-cell volume. The Williamson–Hall analysis further validated the strain-size contributions. EDX (Energy-Dispersive X-ray analysis) results confirmed successful incorporation of Na⁺ and Sr²⁺ without detectable impurity phases. Optical studies revealed distinct absorption peaks at 341 nm for BNT and 374 nm for BST, and the optical bandgap (E_g), calculated using Tauc’s relation, was found to be 2.6 eV and 2.0 eV, respectively. FT-IR spectra exhibited characteristic Ti-O vibrational bands in the range of 420–720 cm⁻¹, consistent with the perovskite structure. For electrical characterization, the powders were pelletized under 3-ton pressure and sintered at 1000 °C for 3 h. The dielectric constant (ε_r), dielectric loss (tan δ), and ac conductivity (σ) of both samples increased with frequency. The combined structural, optical, and electrical results indicate that the optimized compositions of BNT and BST possess properties suitable for use in capacitors and other energy-storage applications. Full article

This study looked at a fungal–cyanobacterial co-pellet system for cleaning up coffee waste and producing high-value polymers. Optimization focused on the pelletization process, waste removal efficiency, and biomass yield. Optimal conditions, including pH (6.5), glucose concentration (6 g/L), and shaking speed (130 rpm), achieved a maximum cyanobacterial immobilization efficiency of up to 97% on the fungal mycelium. Scanning electron microscopy (SEM) confirmed the formation of an integrated co-pellet structure, with fungal hyphae acting as a physical scaffold and extracellular polymeric substances (EPSs) enhancing cell–cell adhesion. The co-culture system exhibited superior performance compared to fungal (20.56 g/L) and algal (1.09 g/L) monocultures. It effectively removed major coffee effluent pollutants, achieving a significant reduction in total phenolic compounds (74.5%). Furthermore, the co-pellets displayed a remarkable final biomass yield (24.33 g/L) and high production of extracellular polymeric substances (EPSs) (5.28 g/L) and intracellular polymeric substances (IPSs) (3.84 g/L). The synergistic relationship was further confirmed by high nitrogen contents in the co-pellets (15.24%), which significantly surpassed that of the individual fungal biomass, suggesting interspecies nutrient transfer. Valuable glycerol-lipids were detected and identified in the fermentative broth of the co-culture confirming a highly efficient bioconversion process. Analyses revealed a targeted metabolic flow toward the accumulation of monoglycerides, notably monooleoylglycerol and monopalmitin, highlighting a powerful cooperative compatibility for producing high-value emulsifiers. Overall, these findings firmly establish the cyano-fungal co-pellet system as a robust and sustainable biorefinery approach for treating complex industrial wastewater while producing a high-quality, value-added biomass suitable for utilization as a biofertilizer or animal feed. Full article

This study characterized leaf extracts of Cymbopogon flexuosus (Ryukyu Lemongrass Corporation, Okinawa, Japan) and evaluated the bioaccessibility and bioactivities of phenolic compounds following a simulated in vitro gastrointestinal model of digestion (in vitro GID) of plant material. Undigested (controls, AqC, EtC) and digested aqueous (AqD) and ethanolic (EtD) extracts were analyzed. Control extracts contained higher total phenolics and flavonoids than digested ones, with EtC showing the highest values. UHPLC-QToF-MS (ultra-high-performance liquid chromatography system coupled to a quadrupole time-of-flight mass spectrometer) identified 32 compounds, including phenolic acids, flavone aglycones, C-glycosides, and derivatives. Hydroxybenzoic acids, coumaric acid, caffeic esters, flavones, tricin derivatives, vitexin, and isoorientin exhibited reduced recovery, while coumaric acid hexoside, ferulic acid hexoside, and isoschaftoside/schaftoside exceeded 100% recovery, suggesting release from the matrix. Some compounds were absent from AqD, and many were found in the pellet, indicating potential colonic metabolism. Antioxidant activity (DPPH, reducing power, β-carotene/linoleic acid) was stronger in controls but always weaker than BHT/ascorbic acid. Extracts mildly inhibited α-amylase but more strongly inhibited α-glucosidase as shown with applied enzyme inhibition assays, especially EtD (76.93% at a concentration of 10 mg/mL), which showed stronger activity than controls but remained below acarbose (87.74% at 1 mg/mL). All extracts promoted HaCaT keratinocyte growth and reduced HCT-116 colon cancer cell viability at 250 µg/mL, with the strongest effects in AqC and AqD. Overall, GID decreased antioxidant activity but enhanced antidiabetic potential, confirming the safety and selective anticancer effects of C. flexuosus extracts. Full article

Influenza virus is one of the most frequent causes of respiratory infection in humans. Recent studies suggest that extracellular vesicles (EVs)—small particles released by cells during influenza virus infection—can influence the immune response and viral pathogenesis. However, during viral replication, infected cells can also release EVs, which may include different subtypes. This study aimed to purify and characterize viral preparations and EVs using sequential ultracentrifugation methods. Influenza A/H1N1, A/H3N2, and B virus strains were produced in human Calu-3 and A549 cell lines. Viral supernatants then underwent a series of differential ultracentrifugation steps at 3000× g, 17,000× g, and 100,000× g. Dynamic light scattering analysis (DLS) validated size heterogeneity for all three types of EVs. Measurement of infectious virus particles for all three pellets showed virus enrichment at 17,000× g and 100,000× g. Dot blot analysis confirmed the enrichment of virus particles in these fractions and the presence of EV protein. This study demonstrates the presence of EVs in virus preparations and highlights the need for improved separation methods to characterize them better and explore their role in viral infection pathogenesis. Full article

Background/Objectives: Tubo-ovarian high-grade serous carcinoma (HGSC) is a highly lethal malignancy, usually diagnosed at an advanced stage due to the lack of early symptoms and biomarkers. Contraceptive hormone use is associated with a reduced risk of HGSC, but the relative contributions of natural versus synthetic progestins, and their interaction with estrogens, are poorly understood. Methods: We evaluated the chemo-preventive efficacy of a synthetic progestin medroxyprogesterone acetate (MPA), progesterone (P4), and combined 17β-estradiol-progesterone (E2 + P4) in a well-characterized genetically engineered mouse model (GEMM) of oviductal HGSC based on the conditional inactivation of one or both alleles of the Brca1, Trp53, Rb1, and Nf1 tumor suppressor genes (BPRN-het and BPRN-homo mice, respectively). Mice received hormones or placebo via slow-release pellets implanted subcutaneously. After induction of tumor formation, the mice were monitored for tumor development, progression, and survival. Tumor incidence was assessed histologically, and hormone effects were further explored via RNA-seq analysis of oviductal tissues. Results: MPA significantly reduced HGSC incidence and delayed tumor progression compared to the placebo, P4, and P4 + E2 in both BPRN-homo and BPRN-het mice, with up to 78% tumor-free survival in the MPA-treated BPRN-het cohort. P4 monotherapy did not provide significant protection vs. the placebo, but the effects of P4 could have been impacted by a failure to achieve sustained release of the hormone beyond 4–8 weeks. The E2 + P4 combination accelerated tumorigenesis and reduced survival (p < 0.0001 in BPRN-homo and p = 0.0004 in BPRN-het mice). MPA did not affect tumorigenesis in a colon cancer GEMM, or the growth of mouse HGSC-derived cells in vivo, suggesting the role of MPA in the early stages of HGSC development. Gene expression analyses showed that P4 and MPA downregulated cholesterol homeostasis, early and late estrogen response, and epithelial–mesenchymal transition pathways, though only MPA afforded tumor protection. Conclusions: These findings demonstrate that a synthetic progestin, specifically MPA, confers robust protection against HGSC development, while a combination including E2 (E2 + P4) increases risk. This work also illustrates how HGSC GEMMs can be used to compare the chemo-preventive effects of various synthetic progestins on HGSC development in order to prioritize the most effective ones for use in preventing HGSC in both general and high-risk populations. Full article