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Search Results (352)

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Keywords = in vitro neutralization assay

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16 pages, 4348 KB  
Article
Varying Corn Flour Inclusion Levels Modulate Fiber Fraction Degradation and Nutritional Value of Rice Straw via Co-Extrusion
by Wenjie Zhang, Siran Wang, Nengxiang Xu, Chenglong Ding and Beiyi Liu
Agriculture 2026, 16(13), 1373; https://doi.org/10.3390/agriculture16131373 (registering DOI) - 24 Jun 2026
Viewed by 60
Abstract
Rice straw, one of the most abundant agricultural residues worldwide, remains significantly underutilized as a ruminant feed source owing to its intrinsic lignocellulosic recalcitrance. This study investigated the effects of co-extruding rice straw with varying proportions of corn flour on nutritional composition and [...] Read more.
Rice straw, one of the most abundant agricultural residues worldwide, remains significantly underutilized as a ruminant feed source owing to its intrinsic lignocellulosic recalcitrance. This study investigated the effects of co-extruding rice straw with varying proportions of corn flour on nutritional composition and in vitro digestibility for ruminant nutrition. Extrusion was conducted using a twin-screw extruder at 180 °C barrel temperature, 5 MPa pressure, and 50% feed moisture content. Five corn levels were formulated on a dry matter basis: pure rice straw (RS100); three blends with increasing corn flour inclusion: RS75:C25 (75% straw + 25% corn flour), RS67:C33 (67% straw + 33% corn flour), and RS60:C40 (60% straw + 40% corn flour); and pure corn flour (C100) as a control. Chemical composition including neutral detergent fiber (NDF), acid detergent fiber (ADF), acid detergent lignin (ADL), cellulose, hemicellulose, water-soluble carbohydrates (WSC), and starch was analyzed. In vitro dry matter digestibility (IVDMD) was determined using a pepsin-cellulase assay. Regression analysis within the practical 0–40% corn flour inclusion range revealed a significant quadratic relationship with IVDMD (R2 = 0.999, p < 0.001). The optimal corn flour proportion was calculated to be approximately 37.5%, which closely matched the RS60:C40 formulation (40% corn flour). Among the tested formulations, RS60:C40 exhibited the greatest extrusion-induced nutritional improvements. Relative to its pre-extrusion values, cellulose decreased by 55.7% (p < 0.05), followed by ADF (16.1%), NDF (12.8%), and hemicellulose (10.2%); IVDMD increased by 34.2% (p < 0.01) and WSC by 56.7% (p < 0.05). Compared with RS100 after extrusion, RS60:C40 raised IVDMD by 49.5% and lowered cellulose by 60.6%. Its IVDMD also surpassed those of RS75:C25 and RS67:C33 (p < 0.05), whereas RS75:C25 showed only marginal improvements. ADL content showed no extrusion-induced change (p > 0.05). Scanning electron microscopy (SEM) of the RS60:C40 formulation revealed that, unlike the intact fibrous structures observed prior to extrusion, post-extrusion samples exhibited extensive disruption of the fibrous matrix. Pearson correlation analysis further supported these findings, showing strong positive correlations between IVDMD and WSC (r = 0.96, p < 0.001) and strong negative correlations between IVDMD and NDF (r = −0.95, p < 0.001). In conclusion, extrusion generally increased IVDMD and WSC while reducing fiber fractions, with the effect depending on corn level. Co-extrusion with 40% corn flour effectively enhanced the nutritional value of rice straw, offering a viable strategy for producing a more digestible ruminant feed. Full article
(This article belongs to the Section Farm Animal Production)
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24 pages, 12189 KB  
Article
Activated Macrophages Promote TNF-α-Associated Tumor Cell Necroptosis in Pituitary Apoplexy Through the PIEZO1–NFATC2/REL Axis
by Xingbo Li, Luowen Zhou, Zhuowei Lei, Sihan Li, Quanji Wang, Haochen Zhao, Linpeng Xu, Juan Chen, Xueyan Wan, Yimin Huang and Ting Lei
Int. J. Mol. Sci. 2026, 27(12), 5635; https://doi.org/10.3390/ijms27125635 (registering DOI) - 22 Jun 2026
Viewed by 96
Abstract
Pituitary apoplexy is an uncommon but clinically urgent complication that often involves intrasellar hemorrhage and tissue necrosis. The mechanisms linking acute tissue injury to the inflammatory tumor microenvironment remain incompletely defined. Here, we characterized the apoplexy-associated microenvironment and examined whether macrophage mechanosensitive signaling [...] Read more.
Pituitary apoplexy is an uncommon but clinically urgent complication that often involves intrasellar hemorrhage and tissue necrosis. The mechanisms linking acute tissue injury to the inflammatory tumor microenvironment remain incompletely defined. Here, we characterized the apoplexy-associated microenvironment and examined whether macrophage mechanosensitive signaling contributes to inflammatory amplification and tissue damage in pituitary neuroendocrine tumors (PitNETs). We combined single-cell RNA sequencing (scRNA-seq), histological validation, clinical stratification, and in vitro functional assays using apoplectic and non-apoplectic human PitNET specimens. Macrophage state transitions, intercellular communication, and transcriptional regulatory programs were analyzed, followed by an experimental assessment of the PIEZO1–Ca2+ axis and macrophage-conditioned medium-induced tumor cell death. Histological validation confirmed macrophage accumulation in apoplectic PitNETs, including a 1.67-fold increase in IBA-1-positive cells (p < 0.001). CellChat-inferred interaction metrics increased descriptively in apoplectic samples. Apoplectic tissues showed higher TNF-α expression (3.00-fold; p < 0.0001) and higher PIEZO1 fluorescence in IBA-1-positive regions (1.39-fold; p = 0.001). Yoda1 increased Calcium 520 fluorescence in macrophages (1.72-fold; p = 0.002), whereas Piezo1 knockdown reduced the Yoda1-associated response (p = 0.003). Conditioned medium from activated macrophages increased total Annexin V/PI-positive death in AtT-20 cells (0.53 ± 0.53% to 32.48 ± 1.14%; p < 0.001) and GH3 cells (0.82 ± 0.50% to 30.92 ± 1.11%; p < 0.001); Piezo1 knockdown or TNF-α neutralization attenuated this effect. Clinically, pathological necrosis was associated with higher symptom frequencies and a greater adjusted likelihood of two or more clinical symptoms. Together, these findings indicate that PIEZO1-related macrophage signaling may participate in TNF-α-associated tumor cell necroptosis in pituitary apoplexy. Pathological necrosis was linked to greater acute symptom burden and perioperative hormonal abnormalities, suggesting that it may identify a clinically severe apoplexy subtype. Full article
(This article belongs to the Section Molecular Immunology)
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13 pages, 7084 KB  
Article
Efficacies of Conventional Antifungals and Complementary and Alternative Medicine as Single or Combination Therapies Against Candida Biofilms in Recurrent Vaginal Candidiasis: An In Vitro Study
by Yihong Pan, Liumei Ye, Lanqian Chen, Lauren Hermann, Panpan Jin, Yingying Cai, Yali Cheng, Weidan Zhang, Cathy J Watson, David McGiffin, Qiong Luo, Xueqiong Zhu and Yue Qu
J. Fungi 2026, 12(6), 415; https://doi.org/10.3390/jof12060415 - 8 Jun 2026
Viewed by 398
Abstract
Objectives: Recurrent vulvovaginal candidiasis (RVVC) is a difficult-to-treat infection, most likely due to the growth of Candida biofilms on the human vaginal epithelium. We assessed in vitro efficacy of conventional antifungals and complementary and alternative medicine (CAM) used in clinical settings, and sought [...] Read more.
Objectives: Recurrent vulvovaginal candidiasis (RVVC) is a difficult-to-treat infection, most likely due to the growth of Candida biofilms on the human vaginal epithelium. We assessed in vitro efficacy of conventional antifungals and complementary and alternative medicine (CAM) used in clinical settings, and sought for Candida biofilm-effective single or combination therapies. Methods: Standard broth microdilution assay and XTT (2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide) assay were used for antifungal and anti-biofilm efficacies of three conventional antifungals, and selected CAM including boric acid, povidone-iodine, and allicin (garlic extract), against Candida clinical isolates grown at neutral and acidic pHs respectively. Fractional inhibitory concentration (FIC) indices were assessed to evaluate interactions between fluconazole and different CAM. Viable count-based cell enumeration and confocal laser scanning microscopy (CLSM) were performed to confirm the efficacy of single or combination therapies against Candida biofilms. Results: All selected conventional antifungals and CAM showed efficacies against planktonic Candida cells. Acidic vaginal microenvironments provided agent-specific protection to Candida cells against conventional antifungals and the CAM. Synergistic or additive interactions were observed between fluconazole at serum achievable concentrations and povidone-iodide at topically achievable concentrations against all tested Candida strains. Most antifungal agents except caspofungin had very limited activities against Candida biofilms. Combining fluconazole at 8 mg/L with povidone-iodine at 2048 mg/L effectively killed Candida biofilms in an acidic vaginal microenvironment to a level that is comparable to that of caspofungin. Conclusions: We provided robust in vitro evidence supporting the combinational use of oral fluconazole and topical CAM povidone-iodine against Candida biofilms in managing RVVC. Full article
(This article belongs to the Special Issue Candida Infections and Antifungal Treatment)
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23 pages, 8706 KB  
Article
Development of Albumin Nanocarriers for Enhanced Curcumin Delivery and In Vitro Anticancer Activity in Colon Cancer Cells
by Aftab Ahmad, Darshana Bagwe, Shagufta Khan, Chetna Dhone, Shilpa Padhare, Anwar A. Alghamdi and Shah Alam Khan
Pharmaceuticals 2026, 19(6), 872; https://doi.org/10.3390/ph19060872 - 30 May 2026
Viewed by 419
Abstract
Objectives: Curcumin possesses well-documented anticancer activity; however, its clinical translation is hindered by poor aqueous solubility and limited bioavailability. The present study aimed to engineer pH-dependent bovine serum albumin (BSA)–based nanocarriers for curcumin delivery and to evaluate their physicochemical characteristics, controlled release behavior [...] Read more.
Objectives: Curcumin possesses well-documented anticancer activity; however, its clinical translation is hindered by poor aqueous solubility and limited bioavailability. The present study aimed to engineer pH-dependent bovine serum albumin (BSA)–based nanocarriers for curcumin delivery and to evaluate their physicochemical characteristics, controlled release behavior under gastrointestinal pH conditions, and in vitro anticancer efficacy against the human colon cancer cell line Colo-205. Methods: Curcumin-loaded bovine serum albumin nanoparticles (Cu-BSA-NPs) were fabricated using a desolvation technique followed by chemical crosslinking. Particle size, zeta potential, and polydispersity index (PDI) were assessed by dynamic light scattering. Morphology was examined using scanning electron microscopy (SEM), while structural and thermal properties were evaluated by Fourier-transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC). Drug loading capacity and entrapment efficiency were quantified spectrophotometrically. In vitro drug release was investigated using a gastrointestinal pH-transition model (pH 1.2, 6.8, and 7.4). Cytotoxic activity was assessed using the sulforhodamine B (SRB) assay on Colo-205 cells. Results: The engineered Cu-BSA-NPs exhibited particle sizes ranging from 96.7 ± 10.5 to 126.4 ± 35.8 nm, with PDI values between 0.289 and 0.581 and zeta potentials from −18.2 ± 1.01 to −34 ± 1.0 mV, indicating nanoscale dimensions and moderate colloidal stability. SEM analysis revealed spherical nanoparticles with smooth surfaces and uniform morphology. Entrapment efficiency ranged from 6.59 ± 1.11% to 52.98 ± 0.65%, while drug loading efficiency varied between 1.308 ± 0.206% and 16.744 ± 0.266%. In vitro release studies demonstrated minimal drug release under acidic (pH 1.2) and near-neutral (pH 6.8) conditions, followed by significantly enhanced release at pH 7.4, confirming pH-dependent behavior of the albumin matrix. Cytotoxicity studies showed significant antiproliferative activity against Colo-205 human colon cancer cells. Conclusions: The findings demonstrate successful engineering of albumin-based nanocarriers capable of modulating curcumin release under physiologically relevant pH conditions and enhancing in vitro anticancer activity. Although limited to in vitro evaluation, this study highlights the potential of protein-based nanoplatforms as adaptable delivery systems for colon cancer therapy. Further in vivo investigations are warranted to validate their translational and therapeutic potential. Full article
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12 pages, 1079 KB  
Article
Acid-Tolerant Photosensitizer: Photodynamic Inactivation of Porphyromonas gingivalis by 2′,4′,5′,7′-Tetraiodofluorescein
by Zixiang Wang, Qianwen Deng, Ziyu Huang, Zixing Lin, Haohui Zhu, Janak L. Pathak, Ying Wang and Min Nie
Pathogens 2026, 15(6), 567; https://doi.org/10.3390/pathogens15060567 - 25 May 2026
Viewed by 310
Abstract
Porphyromonas gingivalis (P. gingivalis) is a primary pathogen in periodontitis, yet its elimination is limited by complex anatomical structures. Photodynamic therapy (PDT) is a promising adjunct, but its antimicrobial efficacy is compromised in the acidic microenvironment induced by P. gingivalis. [...] Read more.
Porphyromonas gingivalis (P. gingivalis) is a primary pathogen in periodontitis, yet its elimination is limited by complex anatomical structures. Photodynamic therapy (PDT) is a promising adjunct, but its antimicrobial efficacy is compromised in the acidic microenvironment induced by P. gingivalis. Given that 2′,4′,5′,7′-tetraiodofluorescein (TIF) exhibits robust and stable photodynamic activity under acidic conditions, this study investigated the antibacterial effect of TIF-mediated PDT (TIF-PDT) against P. gingivalis. P. gingivalis ATCC 33277 was treated with TIF (5, 10, 20, and 40 μM), light (525 nm), or both. Reactive oxygen species (ROS) generation was assessed at pH 4.5 or 7.4. Bacterial viability and membrane integrity were evaluated by colony-forming unit (CFU) assay and LIVE/DEAD staining. CFU assays demonstrated that TIF-PDT groups achieved an approximately 4-log reduction in bacterial viability compared to the DEMI, Light, and TIF groups, with no dark cytotoxicity and light-alone effects. LIVE/DEAD staining revealed bright yellow fluorescence in the TIF-PDT (40 μM), indicating membrane damage and significantly lower survival rates than controls. TIF-PDT at 10, 20, and 40 μM produced ROS under both neutral and acidic conditions, exhibited low dark cytotoxicity, and demonstrated potent antibacterial activity against P. gingivalis in vitro, suggesting its potential as an acid-tolerant photosensitizer for periodontitis adjunctive therapy. Full article
(This article belongs to the Section Bacterial Pathogens)
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16 pages, 1461 KB  
Article
Evaluation of the Anti-Inflammatory Activity of Selected Plant Extracts in an In Vitro Model of Inflammation Using LPS-Stimulated Macrophages
by Karolina Merecz, Kinga Suska, Olga Biniszewska, Mikołaj Hirsa, Aneta Wojdyło, Aleksandra Tarasiuk-Zawadzka and Jakub Fichna
Biomedicines 2026, 14(5), 1174; https://doi.org/10.3390/biomedicines14051174 - 21 May 2026
Viewed by 491
Abstract
Background: Inflammatory bowel disease (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC), is a group of chronic gastrointestinal (GI) diseases with complex and multifactorial pathophysiology. The global prevalence of IBD is increasing, highlighting the need to develop new therapeutic approaches. Plant-derived extracts [...] Read more.
Background: Inflammatory bowel disease (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC), is a group of chronic gastrointestinal (GI) diseases with complex and multifactorial pathophysiology. The global prevalence of IBD is increasing, highlighting the need to develop new therapeutic approaches. Plant-derived extracts have recently gained prominence due to their anti-inflammatory properties. Methods: This study investigated: apricot leaves (ALE), peach leaves (PLE), black chokeberry fruit (BCHE), rosehip seeds (RSE), passion fruit seeds (PSE), and linden blossom (LBE) (all at the concentration 10–200 µg/mL) in RAW 264.7 mouse macrophages. Cytotoxicity was assessed using the neutral red uptake (NRU) assay, and anti-inflammatory activity was assessed using Griess assay in the lipopolysaccharide (LPS)-induced inflammation. Additionally, the mRNA expression levels of key inflammatory genes (interferon-γ (Ifn-γ), interleukin-6 (Il-6), nitric oxide synthase (Nos2), and tumor necrosis factor-α (Tnf-α)) were analyzed. Results: ALE and PLE exhibited minimal cytotoxicity and strong anti-inflammatory activity, reducing the expression of all analyzed genes. PSE demonstrated anti-inflammatory activity in the Griess assay, but did not alter mRNA expression. Conclusions: ALE and PLE exhibit promising anti-inflammatory properties and warrant further preclinical investigation. Comprehensive in vitro and in vivo studies are necessary to confirm these results. Full article
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19 pages, 5415 KB  
Article
An mRNA Vaccine with Tandem Mutated HA-NA Confers Protection Against Multiple Strains of H1N1 Influenza
by Xuena Du, Yuxia Yuan, Cong Tang, Yanwen Li, Zhaolan Guo, Yun Yang, Hao Yang, Yanan Zhou, Qing Huang, Hongyu Chen, Wenqi Quan, Junbin Wang and Shuaiyao Lu
Vaccines 2026, 14(5), 454; https://doi.org/10.3390/vaccines14050454 - 19 May 2026
Viewed by 488
Abstract
Background/Objectives: Recurrent influenza epidemics impose a severe global burden, with conventional vaccines constrained by production time lags and rapid viral mutation. This study aims to explore a novel influenza mRNA vaccine design that balances conserved and mutable antigen regions. By combining hemagglutinin (HA) [...] Read more.
Background/Objectives: Recurrent influenza epidemics impose a severe global burden, with conventional vaccines constrained by production time lags and rapid viral mutation. This study aims to explore a novel influenza mRNA vaccine design that balances conserved and mutable antigen regions. By combining hemagglutinin (HA) and neuraminidase (NA) into a dual-target approach, the objective is to simultaneously block viral entry and inhibit progeny release, potentially establishing a proposed “front-blockade, rear-containment” dual protective barrier against multiple H1N1 strains. Methods: We engineered a dual-target tandem mRNA vaccine linking mutated HA with conserved NA, with strategic amino acid mutations introduced into key antigenic sites within the HA head domain. Vaccine efficacy was evaluated in a mouse model. Humoral immunity was assessed by measuring antigen-specific antibody titers, and cellular immunity was evaluated via ELISpot assay. Protective capacity was determined through lethal challenge experiments using diverse H1N1 viral strains. Results: The vaccine successfully expressed the HA-NA tandem antigen at 130 kDa, and the in vitro-expressed antigen exhibited normal neuraminidase activity. Preliminary evidence supported the dual-target concept in model mice: hemagglutination-inhibiting and micro-neutralizing antibodies targeting HA were detected, and serum neuraminidase-inhibiting activity was also observed. In addition to triggering potent cellular immune responses, the vaccine offered total protection against lethal doses of various H1N1 variants. Conclusions: This study suggests a promising dual-target strategy that harmonizes antigen conservation and mutation while potentially establishing a synergistic front-blockade and rear-containment defense. The approach offers a viable pathway for developing improved H1N1 influenza vaccines. Full article
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28 pages, 3157 KB  
Article
Integrated Evaluation of Urtica dioica Extract Assessing Physiochemical Analysis with Antioxidant, Antiviral, and Immunomodulatory Effects Against SARS-CoV-2
by Gulsah Akbas, Seyma Aydinlik, Jenya Dursun, Frederick Lia, Mustafa Emrem, Banu Mansuroğlu and Yuksel Cetin
Pharmaceuticals 2026, 19(5), 693; https://doi.org/10.3390/ph19050693 - 28 Apr 2026
Viewed by 679
Abstract
Background: A major challenge in antiviral development is the identification of novel virus–host interactions while ensuring therapeutic efficacy and safety. These challenges have renewed interest in phytochemicals derived from medicinal plants as alternative antiviral agents. Objectives: In this study, we investigated the antioxidant, [...] Read more.
Background: A major challenge in antiviral development is the identification of novel virus–host interactions while ensuring therapeutic efficacy and safety. These challenges have renewed interest in phytochemicals derived from medicinal plants as alternative antiviral agents. Objectives: In this study, we investigated the antioxidant, antiviral, and immunomodulatory properties of a Mediterranean Urtica dioica extract (UdE) against SARS-CoV-2 using chemical, biochemical, and in vitro approaches. Methods: The physicochemical properties of UdE were characterized using microtiter assays and HPLC analysis. Cytocompatibility was evaluated in HEK293T, Vero E6, Caco-2, and Calu-3 cell lines while antioxidant activity was assessed using both chemical and cell-based assays. Antiviral activity was evaluated by assessing inhibition of SARS-CoV-2 receptor binding domain (RBD)–ACE2 interaction using ELISA, inhibition of SARS-CoV-2 main protease (Mpro) activity via FRET assay and inhibition of viral entry using SARS-CoV-2 S1 pseudovirus neutralization assay. Results: UdE (100 µg/mL) inhibited RBD–ACE2 binding by 94% and suppressed Mpro activity by 74%, while reducing moderate but significant inhibition of pseudovirus entry (33.6%) at 300 µg/mL dose level in ACE2 expressing HEK293T cells. Immunomodulatory analysis revealed significant suppression of IL-1β and IL-6 production, accompanied by increased TNF-α and IL-8 levels. Conclusions: Collectively, these findings highlight that UdE exhibits multi-target in vitro antioxidant, antiviral, and immunomodulatory activity against SARS-CoV-2; therefore, UdE represents a promising bioactive extract for the management of SARS-CoV-2 infection. Full article
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19 pages, 787 KB  
Article
Physicochemical Characteristics, In Vitro Ruminal Digestibility, Bioactive Compounds, and Estimated Methane Production of Wild Floral Species in Goats from the Republic of Malta: A Descriptive Study
by Jamie Buttigieg, Emmanuel Sinagra and Everaldo Attard
Vet. Sci. 2026, 13(5), 427; https://doi.org/10.3390/vetsci13050427 - 28 Apr 2026
Viewed by 938
Abstract
Pasture plants can contribute to ruminant nutrition and may, depending on composition, influence rumen fermentation and methane production. This study evaluated the nutritional composition, bioactive compounds, and methane production potential of 32 terrestrial plant species commonly foraged by goats in Malta. Dried plant [...] Read more.
Pasture plants can contribute to ruminant nutrition and may, depending on composition, influence rumen fermentation and methane production. This study evaluated the nutritional composition, bioactive compounds, and methane production potential of 32 terrestrial plant species commonly foraged by goats in Malta. Dried plant samples were analysed for proximate composition using near-infrared spectroscopy, total polyphenols using the Folin–Ciocalteu assay, antioxidant activity using the DPPH assay, and methane production using an in vitro rumen fermentation system incubated for 48 h, with rumen fluid pooled from three goats (analyses performed in triplicate). Crude protein ranged from 1.16 to 31.97% DM, neutral detergent fibre from 12.29 to 48.89%, and ash from 9.69 to 17.20% across species. Total polyphenolic content varied from 0.07 to 1.30% (w/w), while antioxidant activity (IC50) ranged from 0.37 to 55.9 mg/mL. Methane production after 48 h ranged from 30.39 to 198.26 L CH4 kg−1, indicating variation in fermentation characteristics among species. These results indicate that Rumex bucephalophorus and Urtica pilulifera demonstrated relatively high protein or bioactive values and comparatively lower in vitro methane-related parameters under the conditions tested. Full article
(This article belongs to the Section Nutritional and Metabolic Diseases in Veterinary Medicine)
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20 pages, 3245 KB  
Article
Dual Specificity Phosphatase 4 Enhances Immunotherapy Response by Inhibiting TGF-β1 Secretion in Hepatocellular Carcinoma
by Lian-Pan Su, Wei-Yi Wang, Xiao-Dan Ma and Shi-Hui Hao
Cancers 2026, 18(8), 1289; https://doi.org/10.3390/cancers18081289 - 19 Apr 2026
Viewed by 669
Abstract
Background: Tumor immune microenvironment (TIME) heterogeneity limits immunotherapy efficacy in hepatocellular carcinoma (HCC), underscoring the need for predictive biomarkers and therapeutic targets. We previously identified dual specificity phosphatase 4 (DUSP4) as a mediator of sorafenib resistance, but its immunomodulatory role remains unknown. [...] Read more.
Background: Tumor immune microenvironment (TIME) heterogeneity limits immunotherapy efficacy in hepatocellular carcinoma (HCC), underscoring the need for predictive biomarkers and therapeutic targets. We previously identified dual specificity phosphatase 4 (DUSP4) as a mediator of sorafenib resistance, but its immunomodulatory role remains unknown. Methods: Glypican-3 (GPC3)-specific chimeric antigen receptor (CAR) T-cell cytotoxicity assays were performed to assess the impact of DUSP4 on HCC immune susceptibility. A subcutaneous tumor model using Dusp4-overexpressing cells in female C57BL/6J mice was established to evaluate DUSP4-mediated microenvironment remodeling and anti-PD-L1 therapy efficacy. Bulk RNA sequencing of DUSP4-overexpressing HCC cells identified downstream pathways. Public datasets were interrogated to correlate DUSP4 expression with immune checkpoint blockade (ICB) response and immune infiltration in HCC. Results: DUSP4 overexpression significantly enhanced HCC cell susceptibility to CAR-T cell killing in vitro and potentiated anti-PD-L1 efficacy in vivo, accompanied by TIME remodeling. Mechanistically, RNA sequencing revealed DUSP4-mediated downregulation of the TGF-β signaling pathway, functionally confirmed using a neutralizing antibody that abrogated the enhanced CAR-T killing. Public datasets confirmed associations between DUSP4 expression and enhanced immune cytolytic activity with favorable prognostic outcomes in HCC. Conclusions: DUSP4 serves as a critical molecular nexus linking targeted therapy resistance to enhanced immunotherapy sensitivity. By attenuating the TGF-β signaling pathway, DUSP4 reprograms TIME toward an immunologically active state, thereby augmenting the efficacy of immunotherapy. These findings establish DUSP4 as a promising dynamic biomarker for guiding sequential therapy in HCC and highlight its potential as a novel therapeutic target to improve outcomes in solid tumor immunotherapy. Full article
(This article belongs to the Section Cancer Immunology and Immunotherapy)
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11 pages, 220 KB  
Article
Effects of Different Proportions of Corn Silage and Ramie Silage on In Vitro Rumen Fermentation Characteristics and Methane Production
by Honghui Qi, Cheng Gao, Zhicai Li and Duanqin Wu
Animals 2026, 16(8), 1250; https://doi.org/10.3390/ani16081250 - 18 Apr 2026
Viewed by 382
Abstract
This study investigated the interactive effects of corn silage and ramie silage on in vitro rumen fermentation characteristics, aiming to provide a scientific basis and empirical evidence for the rational incorporation of ramie into ruminant diets. Four binary substrate mixtures were formulated based [...] Read more.
This study investigated the interactive effects of corn silage and ramie silage on in vitro rumen fermentation characteristics, aiming to provide a scientific basis and empirical evidence for the rational incorporation of ramie into ruminant diets. Four binary substrate mixtures were formulated based on dry matter (DM) mass ratios of corn silage to ramie silage: 100:0 (CON), 60:40 (R40), 20:80 (R80), and 0:100 (R100). Rumen fluid was collected from three adult Liuyang black goats surgically fitted with permanent rumen cannulas, and a standardized 48 h in vitro batch culture assay was conducted. Results demonstrated that increasing the proportion of ramie silage significantly decreased (p < 0.05) the DM degradation rate, neutral detergent fiber (NDF) degradation rate, acid detergent fiber (ADF) degradation rate, and total gas production per gram of substrate DM. Specifically, CON and R40 exhibited significantly higher values for all four parameters than R80 and R100 (p < 0.05). Methane production was significantly reduced in all ramie-containing treatments relative to CON (p < 0.05), whereas hydrogen production increased progressively with ramie inclusion level, with CON yielding significantly less H2 than both R80 and R100 (p < 0.05). Regarding fermentation parameters, increasing ramie proportion elevated (p < 0.05) both fermentation fluid pH and the acetate-to-propionate ratio, while total volatile fatty acid (TVFA) concentration declined linearly (p < 0.05). TVFA concentrations did not differ significantly between CON and R40, yet both were significantly greater than those in R80 and R100 (p < 0.05). Collectively, these findings indicate that ramie silage is a nutritionally valuable forage with potential as a high-quality partial replacement for conventional silages in ruminant feeding systems; however, its inclusion in corn–ramie mixed silages should not exceed 40% (on a DM basis) to maintain optimal fermentative efficiency and nutrient degradability. Full article
21 pages, 3234 KB  
Article
The Effects of Past COVID-19 and Vaccination on Antibody Levels, Cellular Immunity, and Cytokine Production by Peripheral Blood Mononuclear Cells
by Yulia A. Desheva, Tatiana V. Gupalova, Polina A. Kudar, Galina F. Leontieva, Igor V. Kudryavtsev, Andrey S. Trulioff, Danila S. Guzenkov, Victoria A. Matyushenko, Elena A. Bormotova, Daniil D. Sokolovsky, Georgy A. Matveev, Boris P. Nikolaev and Alexander N. Suvorov
Biomedicines 2026, 14(4), 923; https://doi.org/10.3390/biomedicines14040923 - 17 Apr 2026
Viewed by 727
Abstract
Background/Objective: This study is a cross-sectional investigation of long-term immune responses measured at different time intervals after COVID-19 infections, vaccinations, or combined exposure. The focus is on immune reactivity against recombinant spike (S) and nucleocapsid (N) protein antigens. Materials and Methods: Serum antibody [...] Read more.
Background/Objective: This study is a cross-sectional investigation of long-term immune responses measured at different time intervals after COVID-19 infections, vaccinations, or combined exposure. The focus is on immune reactivity against recombinant spike (S) and nucleocapsid (N) protein antigens. Materials and Methods: Serum antibody levels were assessed up to four to four and a half years after infection or immunization, including virus-specific immunoglobulin G (IgG), IgA and IgM antibodies, as well as neutralizing antibodies against the S-protein. Cellular immunity was assessed by analyzing peripheral blood mononuclear cells (PBMC; n = 43 in first cohort, n = 32 in second cohort), including T-helper memory and cytotoxic subsets, and cytokine production after in vitro stimulation with recombinant SARS-CoV-2 proteins. A multiplex cytokine assay was used to analyze effector and regulatory immune responses. Results: Virus-specific IgG antibodies persisted for years after exposure to SARS-CoV-2, with IgG against the receptor-binding domain (RBD) correlating most strongly with neutralizing activity. Vaccinated individuals demonstrated higher IgA responses, whereas antibodies to the N-protein were associated with previous infection. No IgM antibodies were detected in any subjects, suggesting an immune response based on memory rather than ongoing infection. PBMCs from individuals with a history of both COVID-19 exposure and vaccination exhibited enhanced responsiveness, characterized by increased frequencies of memory T cells compared to vaccination alone. Stimulating with the S-protein induces higher cytokine production, including IFN-gamma, TNF-alfa, and IL-12(p70), compared with stimulation by the N-protein. Cytokines such as IL-10 and TGF-beta are also elevated, suggesting immune regulation rather than persistent inflammation. Conclusions: SARS-CoV-2 infection and vaccination are associated with persistent humoral and cellular immune responses detectable several years after exposure. Individuals with hybrid immunity exhibit broader and functionally enhanced immune reactivity, indicating more robust long-term immune memory. Future studies should focus on the long-term consequences of hybrid immunity and optimize other vaccine strategies, including recombinant antigen vaccines. Full article
(This article belongs to the Section Molecular and Translational Medicine)
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12 pages, 1794 KB  
Article
Evaluating Neutralizing Antibody Titers by Recombinant Feline Calicivirus with Heterologous Capsid Protein VP1
by Yang Wang, Wei Lin, Yue Zhang, Hongling He, Yueming Wang, Saisai Li, Qiuyuan Zhang, Shile Huang, Jun Luo and Xiaofeng Guo
Animals 2026, 16(8), 1237; https://doi.org/10.3390/ani16081237 - 17 Apr 2026
Viewed by 405
Abstract
Feline calicivirus (FCV) is a major pathogen that threatens feline health worldwide. Its global prevalence, extensive genetic variability, and limited cross-protection among strains present significant challenges for vaccine development. In this study, an infectious clone of the FCV-GDJM202201 strain was constructed using the [...] Read more.
Feline calicivirus (FCV) is a major pathogen that threatens feline health worldwide. Its global prevalence, extensive genetic variability, and limited cross-protection among strains present significant challenges for vaccine development. In this study, an infectious clone of the FCV-GDJM202201 strain was constructed using the eukaryotic expression plasmid pcDNA3.1 under the control of the cytomegalovirus (CMV) promoter. The rescued virus, rGDJM-A4822T, exhibited growth kinetics comparable to those of the parental strain in vitro. Subsequently, two recombinant viruses, rGDJM-VP1JL and rGDJM-VP1SH, were generated by replacing the VP1 gene in the GDJM202201 backbone with those from heterologous FCV strains. Notably, these recombinant viruses exhibited reduced viral titers compared to rGDJM-A4822T. Finally, neutralization assays revealed differential neutralizing antibody titers among the recombinant FCVs, with rGDJM-A4822T inducing higher neutralizing antibody titers and cross-neutralizing activity. Collectively, this study establishes an FCV infectious clone that can be used to rescue recombinant viruses carrying heterologous VP1 proteins and to evaluate neutralizing antibody responses. Full article
(This article belongs to the Section Companion Animals)
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22 pages, 5581 KB  
Article
Enhanced Th1 Cellular Immunity Induced by an RSV-F mRNA Vaccine Rationally Designed Using NLP Algorithms
by Zhi-Wu Xia, Qi Tang, Jun-Jie Pan, Jing Liu, Lan-Xin Jia, Guo-Mei Zhang, Man-Ni Xie, Jia-Hao Zheng, Chuan-Shuo Lv, Lei Zhang, Yan-Hong Shi, Liang He, Min Luo and Jun-Long Zhao
Vaccines 2026, 14(4), 356; https://doi.org/10.3390/vaccines14040356 - 16 Apr 2026
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Abstract
Background: Respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract infections in infants, seniors, and immunocompromised individuals, contributing substantially to the global disease burden. Given the limited preventive options available, developing an effective and safe vaccine remains a public [...] Read more.
Background: Respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract infections in infants, seniors, and immunocompromised individuals, contributing substantially to the global disease burden. Given the limited preventive options available, developing an effective and safe vaccine remains a public health priority. Methods: An mRNA vaccine encoding the RSV PreF protein was designed and prepared. Antigen properties were evaluated in silico, and the coding sequence was optimized using NLP algorithms. The stability and translational efficiency of the mRNA constructs were verified through in vitro and in vivo assays, followed by immunogenicity evaluation of the formulated mRNA vaccines in a BALB/c mouse model. Results: The optimized mRNA showed predicted improvements in structural stability and a lower free energy state, which were associated with increased translational efficacy in vitro. Correct antigen conformation and retention of key epitopes were confirmed by intracellular staining followed by flow cytometry. A balanced Th1-biased immune response was induced in mice, characterized by high levels of neutralizing antibodies and antigen-specific T-cell immunity, along with enhanced memory T-cell proliferation and differentiation, indicating long-term immunological memory. Conclusions: A novel RSV PreF mRNA vaccine was successfully developed via optimization of protein structure and mRNA sequence. Superior immunogenicity was demonstrated in the BALB/c mouse model, together with promising potential in terms of vaccine safety and immunological persistence. These findings represent a promising step forward in the pursuit of an effective RSV vaccine and suggest the potential of the developed mRNA vaccine to induce substantial immune responses that may correlate with protection in future challenge studies. Full article
(This article belongs to the Section Vaccine Design, Development, and Delivery)
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18 pages, 2840 KB  
Article
AUBFM01 Phage as a Therapeutic Candidate Against MDR Acinetobacter baumannii: Characterization, and Immune-Aware Profiling
by Dina Kabbara, Layane Nakib, Zahraa Shokor, Tasnime A. Abdo Ahmad, May F. Mrad, Ghassan G. Matar and Esber S. Saba
Microorganisms 2026, 14(4), 903; https://doi.org/10.3390/microorganisms14040903 - 16 Apr 2026
Viewed by 593
Abstract
Multidrug-resistant Acinetobacter baumannii is a major nosocomial pathogen for which bacteriophages are being explored as alternative antibacterial agents. In this study, we isolated and characterized AUBFM01, a lytic phage active against MDR A. baumannii, and performed an initial assessment of its interaction [...] Read more.
Multidrug-resistant Acinetobacter baumannii is a major nosocomial pathogen for which bacteriophages are being explored as alternative antibacterial agents. In this study, we isolated and characterized AUBFM01, a lytic phage active against MDR A. baumannii, and performed an initial assessment of its interaction with PMA-differentiated THP-1 macrophages. AUBFM01 was evaluated by host range testing, adsorption and one-step growth assays, lytic activity, stability testing, biofilm disruption, whole-genome sequencing, and flow cytometry-based macrophage profiling. The phage showed rapid adsorption, a short latent period of approximately 30 min, and a burst size of about 165 phage particles per infected cell. It remained stable under moderate temperature and near-neutral pH conditions and significantly reduced preformed A. baumannii biofilm biomass in vitro. Genomic analysis identified a 41,354-bp double-stranded DNA genome lacking detectable lysogeny-associated genes, antibiotic resistance determinants, and known bacterial virulence factors. In THP-1 macrophages, AUBFM01 exposure was associated with reduced cell viability and with enrichment of a resting/intermediate-like CD86-defined phenotype among the remaining cells, including after endotoxin reduction. These findings identify AUBFM01 as a lytic anti-Acinetobacter phage with antibiofilm activity and notable macrophage-associated effects that warrant further mechanistic and safety investigation. Full article
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