Search Results (523)

Thiophene derivatives are particularly attractive for application in drug development for their versatile pharmacological properties. We synthesized a series of four compounds with thiophene carboxamide as a scaffold. The structures were established based on HR-MS and 1D- and 2D-NMR. The purity of the compounds was established to be greater than 92% by thin-layer chromatography and NMR. The cytotoxic effects of the newly synthesized compounds were evaluated against the normal HaCaT cell line and A375, HT-29, and MCF-7 cancer cell lines. The cytotoxic assessment revealed that two compounds exhibit a significant cytotoxic effect on all cancer cell lines. To investigate their potential underlying mechanisms of action, several tests were performed: immunofluorescence imaging, caspase-3/7 assay, mitochondrial membrane potential (JC-1) assay, and 2′,7′–dichlorofluorescein diacetate (DCFDA) assay. MB-D2 proved to be the most cytotoxic and effective in terms of caspase 3/7 activation, mitochondrial depolarization and decrease in ROS production; these effects did not occur in normal HaCaT cells, revealing that MB-D2 has a high selectivity against A375 cancer cells. Full article

Stem cells cultured in cell aggregates exhibit higher cell survival rates and enhanced anti-inflammatory and angiogenic effects compared to single cells, constructing a stable and economical cell aggregate culture system that can accurately adjust the mass transfer distance of nutrients, which contributes to improving the therapeutic effects of stem cell aggregates. In this study, an alginate hydrogel microsphere culture system (Alg-HM) was prepared using electrostatic spraying technology and refined by optimizing the electrostatic spraying technology parameters, such as the sodium alginate concentration, voltage, electrospray injection speed, and nozzle inner diameter. Furthermore, by setting the Tip-dropped culture system (Tip-D culture system, created by dropping the resuspended hMSC aggregate–hydrogel solution with a tip to form the hydrogel microsphere) and Matrigel culture system (created by dropping the resuspended hMSC aggregates–Matrigel solution with a tip to form the Matrigel culture system) as the control group and Alg-HM as the experimental group, the culture effect of hMSC aggregates in the optimized Alg-HM culture system was tested; CCK-8 detection and Ki-67 immunofluorescence staining showed that the Alg-HM culture system significantly enhanced the cell proliferation activity of hMSC aggregates after 7 and 14 days of culture. The Calcein-AM/PI cell staining results showed that the Alg-HM culture system can significantly reduce the central necrosis of hMSC aggregates. The RNA sequencing results showed that the Alg-HM culture system can significantly activate the signaling pathways related to cell proliferation in hMSCs. This culture system is helpful for the culture of cell aggregates in vitro and efficient transplantation in vivo. Full article

The nervous system maintains homeostasis and coordinated behavior through complex neuronal and glial cells. Traditional models, such as primary rodent neurons and human-induced pluripotent stem cell (hIPSC)-derived neurons, have advanced our understanding of neuronal function and neurotoxic damage; however, they are costly and labor-intensive. SH-SY5Y cells, an immortalized human neuroblastoma cell line, provide a more accessible alternative for studying neuronal processes and neurotoxicity. However, their limited capacity to differentiate into specific neuronal phenotypes remains a challenge. To address this limitation, differentiation protocols using neuronal factors and vitamins have been developed, primarily in two-dimensional (2D) cultures, which reduces physiological relevance. Here, we present a novel three-dimensional (3D) SH-SY5Y model incorporating 2D differentiation protocols to generate cholinergic neurons (ChAT+). This model enhances neurotoxicity studies related to pesticides and mycotoxins. Our protocol produces homogeneous spheroids differentiated into cholinergic neurons using serum restriction and specific factors, maintaining viability and circularity for up to 22 days. Differentiation was validated by immunofluorescence and Western blot by Choline acetyltransferase (ChAT) expression. This scalable and reproducible 3D model provides a valuable in vitro tool for neurotoxicological research, improving physiological relevance and enabling the study of cholinergic neuron differentiation and function. Full article

The aim of this in vitro study is to investigate the adhesion, proliferation, and differentiation of human adipose-derived mesenchymal stem cells (hASC) on Trabecular Titanium scaffolds manufactured with different manufacturing processes (EBM and SLM). The in vitro adhesion and proliferation of hASC on titanium scaffolds with WST assays have been carried out. The comparison of the gene expression profiles of typical bone genes (Alp, Bglap, Col1a1, and Osx) through real-time PCR assays and the evaluation of extracellular matrix composition with immunofluorescence and SEM analysis have been performed. In addition, the possible osteoinductive properties of the two scaffolds have been investigated through real-time PCR and ALP assays. Data showed that Trabecular Titanium supports human adipose-derived mesenchymal stem cell colonization and induces differentiation in bone with the deposition of the abundant extracellular mineralized matrix regardless of the manufacturing process, proving that the micro- and macro-design features are the key factors responsible for the osteoinduction behavior. These features can only be achieved through tailored 3D printing process parameters. Full article

Background/Objectives: Non-melanoma skin cancer (NMSC) is among the most common cancers in the United States, with solar ultraviolet (UV) radiation being a primary etiological factor. T-LAK cell-originated protein kinase (TOPK), a serine/threonine kinase activated by solar UV, has been implicated in skin carcinogenesis. This study aimed to investigate the mechanistic role of TOPK in solar UV-induced skin damage and tumor development. Methods: RNA sequencing (RNA-seq) was performed on skin tissues from wild-type (WT) and TOPK knockout (KO) mice, with or without solar UV exposure, to identify TOPK-regulated genes and pathways. Follow-up experiments using Western blotting, immunofluorescence, and luciferase assays were conducted in vitro and in vivo. Functional assays included 3D spheroid and Transwell co-culture systems involving cutaneous squamous cell carcinoma (cSCC) and fibroblast cells. Results: TOPK deletion altered gene expression profiles and inhibited solar UV-induced activation of multiple signaling pathways, including cytokine–cytokine receptor interaction, PI3K/AKT, MAPKs, PKG, cAMP, and calcium signaling. RNA-seq and protein analyses identified interleukin-19 (IL19) as a key downstream effector suppressed by TOPK deletion. In cSCC and fibroblast cells, TOPK knockdown reduced IL19 expression and secretion. IL19 promoted cSCC growth and activated PI3K/AKT, ERK, and TOPK pathways. Additionally, chronic TGFβ exposure increased IL19 expression and activated fibroblasts, as indicated by elevated αSMA and FAPα levels. Conclusions: These findings establish TOPK as a central regulator of solar UV-induced skin carcinogenesis, partially via modulation of IL19 signaling and fibroblast activation. Targeting TOPK may offer a novel strategy for the prevention and treatment of NMSC. Full article

This study aimed to develop monoclonal antibodies (mAbs) against the gD protein of FHV-1 for rapid and specific virus detection. The gD protein, a highly conserved part of the FHV-1 envelope, is crucial for viral entry into host cells, making it an ideal detection target. We immunized BALB/c mice with an mRNA vaccine encoding the gD gene, achieving a serum antibody titer of 1:140,000 after three immunizations. The mice were then boosted with recombinant gD protein. Through cell fusion and multiple subcloning rounds, we obtained five hybridoma cell lines (D7, E4, E9, E10, and E19) that stably secrete anti-gD protein mAbs. Characterization by indirect immunofluorescence and Western blot showed that mAbs D7 and E4 have high specificity and strong binding activity against FHV-1, detectable at 2 μg/mL. These mAbs provide specific tools for FHV-1 detection and a basis for developing rapid diagnostic methods using ELISA, colloidal gold, and other technologies. Full article

The rabies virus (genus Lyssavirus), is a deadly zoonotic agent affecting humans and animals. Although Mexico has been declared free of canine rabies (V1), sylvatic rabies persists. This study aimed to determine the prevalence of the virus in Desmodus rotundus and other non-hematophagous bat species in Oaxaca. The methodology comprised four stages: a literature review, data requests to the Servicio Nacional de Sanidad, Inocuidad y Calidad Agroalimentaria (SENASICA), fieldwork using mist nets across 15 municipalities in Oaxaca, and diagnosis via direct immunofluorescence at the Centro Nacional de Servicios de Diagnóstico en Salud Animal (CENASA). SENASICA reported 89 positive rabies cases (2014–2023) across six laboratories, with the majority (67.02%) attributed to the Oaxaca State Public Health Laboratory. Among the 194 bats analyzed (129 D. rotundus), only three tested positive for the virus, yielding a prevalence of 1.54%. Positive cases were exclusively identified in D. rotundus from San Lucas Ojitlán and The Heroic City of Tlaxiaco. This prevalence aligns with that of national studies, which ranges from 0.05% to 3%. These findings underscore the need to maintain epidemiological surveillance in wild and domestic fauna, alongside public awareness campaigns highlighting bats’ ecological importance for ecosystem conservation and the risks associated with their decline. Full article

Chemotherapy resistance is a major obstacle in the treatment of ovarian cancer, often resulting in disease recurrence and poor prognosis for patients. A key contributor to this resistance is the overexpression of ATP-binding cassette (ABC) transporters, including breast cancer resistance protein (BCRP/ABCG2), which actively effluxes chemotherapeutic agents such as topotecan (TOP) or mitoxantrone (MIT), limiting their intracellular accumulation and efficacy. This study investigated the potential of elacridar (GG918), a potent dual P-gp and BCRP inhibitor, to overcome drug resistance in ovarian cancer cell lines. Both TOP-sensitive and TOP-resistant ovarian cancer cells were grown in two-dimensional (2D) monolayers and three-dimensional (3D) spheroid models to better mimic the tumor microenvironment. The expression of the ABCG2 gene was quantified via qPCR and BCRP protein levels were assessed by western blotting and immunofluorescence. Drug response was evaluated using MTT viability assays, while BCRP transporter activity was examined using flow cytometry and microscopic assessment of the intracellular retention of BCRP fluorescent substrates (Hoechst 33342 and MIT). In both 2D and 3D cultures, elacridar effectively inhibited BCRP function and significantly enhanced sensitivity to TOP. These findings suggest that elacridar can inhibit BCRP-mediated drug resistance in ovarian cancer cell models. Full article

Sjogren’s disease (SjD) is a systemic autoimmune disease primarily affecting the exocrine glands. Systemic manifestations, including hepatic involvement, are increasingly recognized. This study aimed to delineate the clinical features and associated factors of autoimmune hepatic involvement in SjD. A retrospective analysis was conducted on patients diagnosed with SjD at Seoul St. Mary’s Hospital over the past 10 years. Autoimmune hepatic involvement was defined by fulfilling diagnostic criteria for autoimmune hepatitis (AIH) or primary biliary cholangitis (PBC). Clinical, serological, and demographic data were obtained from medical records. Among 1119 patients with SjD, 51 (4.6%) had autoimmune hepatic involvement. AIH (64.7%) was the most common type, followed by PBC (27.5%) and overlapping disease (7.8%). Compared to those without hepatic involvement, affected patients were older at diagnosis (p = 0.003) and showed higher frequencies of thrombocytopenia, splenomegaly, anti-centromere antibody (ACA), and elevated antinuclear antibody titers as measured by indirect immunofluorescence (IFI-HEp-2) (all p < 0.001). Multivariable analysis identified splenomegaly, elevated IFI-HEp-2 titer, and ACA positivity as independent factors associated with hepatic involvement. Most patients responded well to immunosuppressive therapy, with only a small proportion (15.7%) progressing to liver fibrosis. Autoimmune hepatic involvement is relatively uncommon but clinically meaningful in patients with SjD. Full article

Background: The principal function of mammary glands is to produce milk to nourish the newborn. Optimal lactation is controlled by various hormones, growth factors, and cytokines. Objectives: Using 3D cultures of primary mouse mammary epithelial cells, we explored the effects of T helper (Th)1 cytokines, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α on the structure and function of mammary cells as well as the underlying mechanism. Methods: Three-dimensional cultures of mammary cells were treated with IFN-γ/TNF-α, and milk protein expression and acinar structures were analyzed by immunoblotting and immunofluorescence microscopy. Results: Our results revealed that combined treatment with IFN-γ and TNF-α inhibits prolactin-induced STAT5 tyrosine phosphorylation and β-casein expression. These cytokines also disrupted the structure of mammary acini, resulting in smaller or no lumens, disordered cell arrangements, and multilayered cells in certain regions. Additionally, some cells became elongated rather than maintaining their usual cube-like shape. Since cell proliferation and death can modulate the structural organization of acini, we examined the influences of IFN-γ and TNF-α on these events. Combined cytokine treatment moderately increased cell proliferation and cell death. Notably, stimulation with IFN-γ and TNF-α induced the expression of inducible nitric oxide synthase (iNOS), and the inhibition of iNOS partially restored acinar morphology and β-casein expression, revealing a novel mechanism for cytokine-induced acinar disruption. Conclusions: When a Th1 cytokine milieu is dominant, such as during inflammation and infection, IFN-γ and TNF-α might cause mammary gland ductal occlusion and lactation insufficiency. Full article

Rodents are recognized as reservoirs of a wide range of pathogens, including microsporidia. The presence of microsporidia in the environment of mainland Spain and its islands has become increasingly known, as the number of studies has multiplied over time. The present study was conducted to determine the occurrence and diversity of microsporidia in three rodent species (Mus musculus, Rattus norvegicus, and Rattus rattus) in the Canary Islands, Spain. Ninety-three fecal samples were obtained from wild rodents on La Gomera and Gran Canaria Islands. Each sample was tested using Weber’s modified trichrome staining and immunofluorescence antibody tests (IFATs) against the Encephalitozoon genus and Enterocytozoon bieneusi. The microscopy-positive samples were subsequently analyzed using a nested polymerase chain reaction (PCR) followed by Sanger sequencing. The staining technique showed 38.7% (36/93) positivity, whereas the IFATs for Encephalitozoon spp. and Ent. bieneusi revealed 3.2% (3/93) and 6.5% (6/93) positivity, respectively. Finally, the nested PCR and nucleotide sequence analysis confirmed a 9.7% (9/93) occurrence of Ent. bieneusi and 17.2% occurrence (16/93) of different undetermined microsporidia species, whereas no Encephalitozoon spp. were detected. Seven different Ent. bieneusi genotypes were detected as follows: three known (AAE1, D, and SBM1) and four novel (GRE1, GRE2, LGE1, and LGE2), all of which belonged to Group 1. The results demonstrate, for the first time, that microsporidia are present in the rodent populations of the Canary Islands. Further studies are needed to determine the impact of the presence of microsporidia in rodents on the zoonotic transmission of these parasites. Full article

In fibrotic skeletal muscles, excessive extracellular matrix (ECM) deposition is a result of increased activation and decreased apoptosis of myofibroblasts. The aim of this study is to investigate whether treatment with quercetin, kaempferol or capsaicin can reduce the transforming growth factor-beta 1 (TGF-β1)-induced myofibroblast differentiation and fibrotic ECM expression in differentiated C2C12 cells. Two-day-differentiated C2C12 cells were treated with TGF-β1 for 48 h to induce myofibroblast differentiation. Twenty-four hours before (pre-treatment) and for forty-eight hours with (co-treatment) TGF-β1 treatment, cells were exposed to quercetin (25, 50 µM), kaempferol (10, 25, 50 µM) or capsaicin (25, 50 µM). The immunofluorescence intensity of alpha smooth muscle actin (αSMA) and collagen type I/III gene expression were assessed as myofibroblast markers. MyoD immunofluorescence intensity was measured as a myogenic marker. Co-treatment of TGF-β1 with the phytochemicals was most effective, resulting in a decreased number of αSMA-positive cells (all three compounds), decreased collagen type I (kaempferol, capsaicin) and type III (kaempferol) gene expression, and increased MyoD (kaempferol, capsaicin) protein expression compared to TGF-β1 treatment. This study demonstrates that treatment with quercetin, kaempferol or capsaicin can reduce myofibroblast markers. This suggests a possible anti-fibrotic effect of the phytochemicals in skeletal muscle. Full article

Background: Induction of apoptosis is an important strategy for the treatment of prostate cancer. DR5 is a member of the death receptor superfamily and targeting DR5 is an effective way to induce apoptosis. Pyripyropene O is a natural compound isolated from the marine fungus Aspergillus fumigatus SCSIO 41220. We found it has anti-prostate cancer potential by inducing apoptosis; Methods: The effects of pyripyropene O on the viability, proliferation, cell cycle, apoptosis and migration of prostate cancer cells were investigated by MTT assay, plate clone formation assay, 3D cell sphere assay, flow cytometry and real-time cell analysis. Transmission electron microscopy was used to observe the changes in the internal structure of prostate cancer cells after treatment with pyripyropene O. After determining the mode of cell death, the mechanism of action of pyripyropene O on prostate cancer was further investigated using apoptotic protein microarray, western blot, qPCR, molecular docking, cellular immunofluorescence staining and cellular thermal shift assay. After explaining the mechanism of action of pyriproxyfen O, the in vivo absorption, distribution, metabolism, excretion and potential toxicity of pyriproxyfen O were investigated using ADMETLab 2.0 software. Finally, a zebrafish xenograft tumour model was developed to evaluate the anti-prostate cancer effects of pyriproxyfen O in vivo; Results: The experimental results at the cellular level showed that pyripyropene O inhibited the survival, proliferation and migration of prostate cancer cells, and also showed that pyripyropene O blocked the prostate cancer cell cycle at the G2/M phase and induced apoptosis. At the molecular level, pyripyropene O binds to the transcription factor YY1, promotes YY1 nuclear translocation, regulates the transcription level of DR5, a target gene of YY1, and upregulates the expression of DR5 mRNA and protein. The in vivo results showed that pyripyropene O effectively inhibited the development of prostate cancer in zebrafish; Conclusions: Pyripyropene O has a clear anti-prostate cancer effect at both cellular and animal levels, inhibiting the survival and proliferation of prostate cancer cells by binding to the transcription factor YY1 to activate the expression of DR5 to promote apoptosis, thus exerting an inhibitory effect on prostate cancer. Full article

30 percent of infertile men are diagnosed with idiopathic infertility. This study aimed to assess oxidative stress in the semen of 77 patients with idiopathic infertility by measuring F₂-isoprostane (F₂-IsoP), resolvin D1 (RvD1) levels, and semen parameters. The presence and localization of 8-IsoProstaglandin F_2α were determined using immunofluorescence. No significant correlations were observed for F₂-IsoP and RvD1 levels with the semen variables. Based on F₂-IsoP levels, individuals were classified into two groups: Group 1 (F₂-IsoPs ≤ 29.96 ng/mL, 51%) and Group 2 (F₂-IsoPs > 29.96 ng/mL, 49%). In comparison to Group 1, Group 2 showed significantly higher F₂-IsoP levels (13.33 ng/mL vs. 44.80 ng/mL; p < 0.05), a lower progressive motility percentage (30% vs. 25%; p < 0.05), and increased RvD1 levels (36.09% vs. 44.94%). Immunofluorescence analysis revealed a different localization of 8-IsoProstaglandin F_2α in the ejaculated sperm of Group 1 compared to that observed in Group 2. A weak signal was detected in the sperm tail (Group 1, 79.1% vs. Group 2, 36.9; p < 0.01). In spermatozoa of Group 2 patients, a strong signal in the acrosome, midpiece, and tail was highlighted. These findings suggest the need to test oxidative stress during routine semen analysis in patients with idiopathic infertility to improve diagnosis and treatment. Full article

Purpose: The aim of this study was to investigate the protective effects of systemically administered tauroursodeoxycholic acid (TUDCA) in an optic nerve crush (ONC) mouse model of retinal ganglion cell (RGC) death. Methods: C57BL/6J mice were injected intraperitoneally (i.p.) three times per week with TUDCA (500 mg/kg) for two weeks, after which unilateral ONC was performed. A control cohort was identically treated with a drug vehicle (phosphate buffered saline; PBS). A separate cohort did not undergo any injections or surgeries (this was termed the “Naïve” group). Pattern electroretinography (PERG) was recorded 3 days after ONC. Retinas were harvested for whole-mount immunofluorescence staining with an antibody against RGC marker Brn3a and imaged by fluorescent confocal microscopy. Apoptotic cells in the ganglion cell layer (GCL) were detected by Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick End Labeling (TUNEL) performed on fixed retina sections. Glial fibrillary acidic protein (GFAP) immunostaining on fixed retina sections was conducted to detect the activation of Müller cells. Total RNA was extracted from retinas and expression of interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and IL-10 was determined by digital droplet PCR (ddPCR). Results: TUDCA treatment preserved visual function as assessed by PERG. P1 and N2 amplitudes from the PBS-treated ONC group were significantly diminished compared to those of the Naïve group (p < 0.001). TUDCA treatment prevented this diminution. The amplitudes of P1 and N2 in the TUDCA-treated ONC group were statistically indistinguishable from those of the Naïve group and were higher than the PBS-treated ONC group (TUDCA+ONC vs. PBS+ONC, P1: 6.99 ± 0.89 µV vs. 3.60 ± 0.69 µV, p < 0.01; N2: −9.30 (IQR: −13.43–−6.44) µV vs. −4.47 (IQR: −10.26–−2.17) µV). TUDCA treatment preserved RGCs. The ONC-vehicle-only group had 25% fewer RGCs (Brn3a-positive cells) than Naïve eyes (p < 0.0001). TUDCA treatment nearly completely prevented this loss, preserving all but 7.7% of the RGCs, and the number of RGCs in the TUDCA-treated ONC group was significantly higher than in the PBS-treated ONC group (TUDCA+ONC vs. PBS+ONC, 1738.00 ± 14.43 cells per field vs. 1454.00 ± 6.55 cells per field, p < 0.0001). The number of TUNEL-positive cells in the GCL (Naïve vs. PBS+ONC group: 1.00 (IQR: 0.00–2.00) % vs. 37.00 (IQR: 8.50–48.50) %, p < 0.05) and GFAP-positive fibers transversing retina sections (Naïve vs. PBS+ONC group: 33.00 ± 1.15 vs. 185.70 ± 42.37 fibers/retina, p < 0.05), and the expression of IL-6, TNF-α were significantly greater in the PBS-treated ONC group compared to that of the Naïve group (Naïve vs. PBS+ONC group, IL-6: 0.07 (IQR: 0.06–0.31) vs. 0.99 (IQR: 0.56–1.47), p < 0.05, TNF-α: 0.19 ± 0.069 vs. 1.39 ± 0.23; p < 0.01), an increase not observed with TUDCA treatment. Conclusions: Systemic TUDCA treatment significantly preserved RGC function and survival in the mouse ONC model of RGC damage. TUDCA treatment prevented RGC apoptosis, Müller glial cell activation, and retinal expression of several inflammatory cytokines. These data suggest that TUDCA is a promising therapeutic candidate for preserving RGC numbers and function. Full article