Search Results (532)

Resveratrol (RSV), a polyphenol found in a variety of berries and wines, is known for its anti-inflammatory, anticancer, and antioxidant properties. It has been suggested that RSV may play a role in the regulation of metabolic disorders, including diabetes and insulin resistance. However, in recent years, it has been reported to completely inhibit Akt kinase function in liver cells. Akt is a central protein involved in the metabolic function of insulin and is regulated by the phosphatidylinositol-3-kinase (PI3K) pathway. In this study, we examined the effect of RSV on insulin-induced insulin receptor (IR) phosphorylation and proteins involved in the PI3K/Akt pathway in a hepatic cell model, clone 9 (C9), and in hepatoma cells, Hepa 1-6 (H1-6). In both cell lines, RSV inhibited tyrosine phosphorylation of IR and insulin-induced activation of Akt. We also evaluated the effect of RSV on the activation of protein tyrosine phosphatase 1B (PTP1B), which is associated with IR dephosphorylation, and found that RSV increased PTP1B-Tyr¹⁵² phosphorylation in a time- and concentration-dependent manner. Furthermore, we found that the protein kinase C (PKC) inhibitors BIM and Gö6976 prevented the inhibition of Akt phosphorylation by RSV and increased the phosphorylation of Ser/Thr residues in IR, suggesting that PKC is involved in the inhibition of the insulin pathway by RSV. Thus, classical PKC isoforms impair the PI3K/Akt pathway at the IR and GSK3 and GS downstream levels; however, IRS-Tyr⁶³² phosphorylation remains unaffected. These results suggest that RSV can lead to insulin resistance by activating PTP1B and PKC, consequently affecting glucose homeostasis in hepatic cells. Full article

Background: Fowl typhoid (FT), a septicemic infection caused by Salmonella Gallinarum (SG), and H9N2 avian influenza are two economically important diseases that significantly affect the global poultry industry. Methods: We exploited the live attenuated Salmonella Gallinarum (SG) mutant JOL3062 (SG: ∆lon ∆pagL ∆asd) as a delivery system for H9N2 antigens to induce an immunoprotective response against both H9N2 and FT. To enhance immune protection against H9N2, a prokaryotic and eukaryotic dual expression plasmid, pJHL270, was employed. The hemagglutinin (HA) consensus sequence from South Korean avian influenza A virus (AIV) was cloned under the Ptrc promoter for prokaryotic expression, and the B cell epitope of neuraminidase (NA) linked with matrix protein 2 (M2e) was placed for eukaryotic expression. In vitro and in vivo expressions of the H9N2 antigens were validated by qRT-PCR and Western blot, respectively. Results: Oral immunization with JOL3121 induced a significant increase in SG and H9N2-specific serum IgY and cloacal swab IgA antibodies, confirming humoral and mucosal immune responses. Furthermore, FACS analysis showed increased CD4+ and CD8+ T cell populations. On day 28 post-immunization, there was a substantial rise in the hemagglutination inhibition titer in the immunized birds, demonstrating neutralization capabilities of immunization. Both IFN-γ and IL-4 demonstrated a significant increase, indicating a balance of Th1 and Th2 responses. Intranasal challenge with the H9N2 Y280 strain resulted in minimal to no clinical signs with significantly lower lung viral titer in the JOL3121 group. Upon SG wildtype challenge, the immunized birds in the JOL3121 group yielded 20% mortality, while 80% mortality was recorded in the PBS control group. Additionally, bacterial load in the spleen and liver was significantly lower in the immunized birds. Conclusions: The current vaccine model, designed with a host-specific pathogen, SG, delivers a robust immune boost that could enhance dual protection against FT and H9N2 infection, both being significant diseases in poultry, as well as ensure public health. Full article

Monkeypox virus (MPXV) has caused 148,892 confirmed cases and 341 deaths from 137 countries worldwide, as reported by the World Health Organization (WHO), highlighting the urgent need for effective vaccines to prevent the spread of MPXV. Traditional vaccine development is low-throughput, expensive, time consuming, and susceptible to reversion to virulence. Alternatively, a reverse vaccinology approach offers a rapid, efficient, and safer alternative for MPXV vaccine design. Here, MPXV proteins associated with viral infection were analyzed for immunogenic epitopes to design multi-epitope vaccines based on B-cell, CD4+, and CD8+ epitopes. Epitopes were selected based on allergenicity, antigenicity, and toxicity parameters. The prioritized epitopes were then combined via peptide linkers and N-terminally fused to various protein adjuvants, including PADRE, beta-defensin 3, 50S ribosomal protein L7/12, RS-09, and the cholera toxin B subunit (CTB). All vaccine constructs were computationally validated for physicochemical properties, antigenicity, allergenicity, safety, solubility, and structural stability. The three-dimensional structure of the selected construct was also predicted. Moreover, molecular docking and molecular dynamics (MD) simulations between the vaccine and the TLR-4 immune receptor demonstrated a strong and stable interaction. The vaccine construct was codon-optimized for high expression in the E. coli and was finally cloned in silico into the pET21a (+) vector. Collectively, these results could represent innovative tools for vaccine formulation against MPXV and be transformative for other infectious diseases. Full article

The immune landscape of tumor-draining lymph nodes (TDLNs) plays a critical role in shaping antitumor responses and influencing prognosis in oral squamous cell carcinoma (OSCC). Among patients with lymph node (LN) metastasis, clinical outcomes vary widely, yet reliable biomarkers for prognostic stratification remain limited. This study aimed to identify immune features in tumors and LNs that differentiate between favorable and poor outcomes in OSCC patients with nodal metastasis. We analyzed T cell receptor (TCR) CDR3 repertoires and the expression of immune-related genes in primary tumors and paired sentinel LNs from OSCC patients who underwent tumor resection and lymphadenectomy. Patients were divided into three groups: Group A (no nodal metastasis), Group B1 (metastasis without recurrence), and Group B2 (metastasis with recurrence). TCR diversity was assessed using the Shannon index. The expression of immune-related genes (e.g., CD3E, CD4, CD8B, FOXP3, CTLA4, IL2, IL4) was measured by quantitative PCR and normalized to GAPDH. TCR diversity was lower in tumors than in non-metastatic LNs, reflecting clonal expansion. Metastatic LNs exhibited tumor-like diversity, suggesting infiltration by tumor-reactive clones. Tumor gene expression did not differ across groups, but LNs from metastatic cases showed the reduced expression of several immune genes. Notably, CD3E, CD8B, CTLA4, IL2, and IL4 distinguished B1 from B2. The immune profiling of LNs offers superior prognostic value over tumor analysis in OSCC patients with LN metastasis. LN-based evaluation may aid in postoperative risk stratification and personalized postoperative management and could inform decisions regarding adjuvant therapy and follow-up strategies. Full article

Interleukin-1 beta(IL-1β) is the major driving force in neuroinflammation. Here, we report on (i) the role of (IL-1β) in activating a signaling cascade that leads to proliferation and metastasis in glioblastoma cancer pathogenesis as well as (ii) the therapeutic role for IL-1 Receptor Antagonist (IL-1RA) and Tolcapone against untoward aspects of tumor pathogenesis. Here, we report that IL-1β treatment at 50 ng/mL for 48 h increased proliferation and metastasis by 30-fold (p ≤ 0.05), leading to the formation of clones of rapidly dividing cancer cells, leading to the formation of organized glial fibrillary acid protein (GFAP)-immunoreactive, clone-like structures with protruding spikes. Further, IL-1β treatment significantly increased the expression of mRNA levels of the IL-1β-driven pathway TLR-MyD88-NF-κB-TNFα and IL-6 (p ≤ 0.05). IL-1β also increased autophagy via elevation of mRNA and protein levels of cathepsin B, LAMP-2, and LC3B. In contrast, IL-1RA and Tolcapone inhibited this proliferation and the expression of these mRNAs and proteins, inhibiting autophagy by downregulating these autophagy proteins and inducing apoptosis by upregulating the expression of pro-apoptotic proteins like caspase-8 and caspase-3. IL-1β and its receptor can be targeted for successful anticancer therapy, as shown here with the use of IL-1RA and/or Tolcapone. Full article

Antibody-mediated rejection (ABMR) caused by donor-specific Abs (DSAs) is still the leading cause of late graft loss following clinical organ transplantation, and effective strategies to combat ABMR are still elusive. We previously showed that rIL-2 complexed with anti-IL-2 mAb clone JES6-1A12 (IL-2 cplx) leads to the selective expansion of regulatory T cells (Tregs) and the prolonged survival of MHC-mismatched skin allografts. Although the grafts were eventually rejected, mice failed to develop DSAs. Here, we investigated the impact of IL-2 cplx on the humoral response and germinal center (GC) reaction during allograft rejection. IL-2 cplx treatment prevents Bcl-6 upregulation, leading to suppressed development of GC T and B cells. The IL-2 cplx-induced impairment of GC development limits IgG allo-Ab production but allows for IgM synthesis. By employing a hapten–carrier system to investigate affinity maturation, we found that IL-2 cplx induces a distinct shift in specific Ab production favoring low-affinity IgM while simultaneously decreasing IgG responses. These findings illuminate the potential of IL-2 cplx therapy for inducing humoral tolerance, potentially paving the way for refining strategies aimed at preventing and treating ABMR. Full article

Background and Purpose: Glioblastoma remains a therapeutic challenge with a poor prognosis despite multimodal treatments. Reporter-based magnetic resonance imaging (MRI) offers a promising approach for tumor visualization, but its efficacy depends on sufficient reporter gene expression. This study aimed to develop a chimeric element-regulated ferritin heavy chain 1 (FTH1) reporter system to enhance MRI-based glioma detection while enabling targeted therapy via transferrin receptor (TfR)-mediated drug delivery. Methods: Using gene cloning techniques, we constructed a chimeric FTH1 expression system comprising tumor-specific PEG3 promoter (transcriptional control), bFGF-2 5′UTR (translational enhancement), and WPRE (mRNA stabilization). Lentiviral vectors delivered constructs to U251 glioblastoma cells and xenografts. FTH1/TfR expression was validated by Western blot and immunofluorescence. Iron accumulation was assessed via Prussian blue staining and TEM. MRI evaluated T2 signal changes. Transferrin-modified doxorubicin liposomes (Tf-LPD) were characterized for size and drug loading and tested for cellular uptake and cytotoxicity in vitro. In vivo therapeutic efficacy was assessed in nude mouse models through tumor volume measurement, MR imaging, and histopathology. Results: The chimeric system increased FTH1 expression significantly over PEG3-only controls (p < 0.01), with an increase of nearly 1.5-fold compared to the negative and blank groups and approximately a two-fold increase relative to the single promoter group, with corresponding TfR upregulation. Enhanced iron accumulation reduced T2 relaxation times significantly (p < 0.01), improving MR contrast. Tf-LPD (115 nm, 70% encapsulation) showed TfR-dependent uptake, inducing obvious apoptosis in high-TfR cells compared with that in controls. In vivo, Tf-LPD reduced tumor growth markedly in chimeric-system xenografts versus controls, with concurrent MR signal attenuation. Conclusions: The chimeric regulatory strategy overcomes limitations of single-element systems, demonstrating significant potential for integrated glioma theranostics. Its modular design may be adaptable to other reporter genes and malignancies. Full article

Background: Human cytomegalovirus (CMV) is a major pathogen that poses significant risks to immunocompromised individuals and neonates. The tegument protein pp71, encoded by the UL82 gene, plays a pivotal role in initiating viral lytic replication and evading host immune responses. Despite its clinical relevance, standardized monoclonal antibodies (mAbs) for pp71 remain limited, prompting the need to expand the available repertoire of antibodies targeting this critical protein. Methods: In this study, we constructed a diverse human single-chain variable fragment (scFv) library using RNA derived from the B cells of four healthy donors. The library was expressed in Saccharomyces cerevisiae, and iterative rounds of magnetic-activated cell sorting (MACS) were performed against recombinant pp71. Clonal enrichment was monitored using flow cytometry. Results: Among the isolated clones, one designated ID2 exhibited high sensitivity and specificity for pp71, as demonstrated by flow cytometry, immunofluorescence, an enzyme-linked immunosorbent assay (ELISA), and biolayer interferometry (BLI). Conclusions: Collectively, these findings establish a novel pp71-specific mAb and underscore the utility of yeast surface display combined with MACS for expanding the antibody toolkit available for CMV research and diagnostics. Full article

CK2α is a kinase important for essential cellular and biological processes. CK2α is ubiquitously expressed, albeit at different tissue levels, and its transcript levels are dysregulated in disease. However, there is limited knowledge on the regulation of CK2α gene expression. The best one studied, the human CSNK2A1 (CK2α) gene promoter, contains uncharacterized binding motifs for NF-κB. Our goal was to investigate the role of NF-κB in Csnk2a1 promoter regulation. We cloned the mouse Csnk2a1 promoter which had significant sequence homology with the human CSNK2A1 promoter. Using promoter deletions, we identified a minimal promoter region containing transcription factor motifs (NF-κB, Ets-1, Sp1) consistent with those published for the CSNK2A1 promoter. Electrophoretic mobility shift assays demonstrated specific NF-κB subunit binding to the minimal promoter. NF-κB subunit transfection and extracellular NF-κB stimulation in non-tumor cell lines led to increased transactivation of the mouse minimal promoter. These data, together with data on the regulation of NF-κB by CK2 kinase activity, suggest a positive-feedback loop between CK2α and NF-κB. Non-tumor cell line re-plating and increased percent confluence upregulated Csnk2a1 transcript levels which differed from tumor cell line published data. In summary, Csnk2a1 promoter is regulated by NF-κB signaling and during cellular proliferation. Full article

Peptide vaccines possess several advantages over mRNA vaccines but are generally less effective at inducing antitumor immunity. The bottlenecks limiting peptide vaccine efficacy could be elucidated by tracking and comparing vaccine-induced T-lymphocytes in successful and unsuccessful cases. Here we have applied our recent database of neoantigen-specific T cell receptors (TCRs) to profile tumor-specific T cells following vaccination with a neoantigen peptide vaccine and to correlate this with the response. Mice were vaccinated prophylactically with p30 peptide encoding B16 melanoma neoantigen (K739N mutation in Kif18b gene). The B16F0 melanoma in the vaccinated mice was additionally treated by a CTLA-4 checkpoint blockade. T cells from the tumors, tumor-draining lymph nodes (tdLNs) and vaccine depots were isolated, phenotyped, sorted by subsets and sequenced for TCR repertoires. The vaccine induced the accumulation of tumor-specific CD4+ Th cells in the tdLNs, while in the tumors these cells were present and their frequencies were not changed by the vaccine. These cells also accumulated at the vaccine depots, where they were phenotypically skewed by the vaccine components; however, these effects were minor due to approximately 50-fold lower cell quantities compared to the tdLNs. Only some of the p30-specific Th cells showed tumoricidal activity, as revealed by the reverse correlation of their frequencies in the tdLNs with the tumor size. The CTLA-4 blockade did not affect the tumor growth or the frequencies of tumor-specific cells but did stimulate Th cell motility. Thus, we have shown that tumor-specific Th clones accumulate and/or expand in the tdLNs, which correlates with tumor suppression but only for some of these clones. Tumor infiltration by these clones is not correlated with the growth rate. Full article

In B-cell chronic lymphocytic leukemia (B-CLL), hypodiploidy is a rare but aggressive subtype of the disease with a very bad prognosis. Hypodiploidy, in contrast to normal B-CLL chromosomal aberrations, is marked by widespread genomic instability, which promotes treatment resistance and quick illness development. Its persistence after treatment implies that chromosomal loss gives cancerous clones a selection edge, which is made worse by telomere malfunction and epigenetic changes. Since thorough genetic profiling has a major impact on patient outcomes, advanced diagnostic methods are crucial for early detection. Treatment approaches must advance beyond accepted practices because of its resistance to traditional medicines. Hematopoietic stem cell transplantation (HSCT) and chimeric antigen receptor (CAR) T-cell therapy are two potential new therapeutic modalities. Relapse and treatment-related morbidity continue to be limiting concerns, despite the noteworthy improvements in outcomes in high-risk CLL patients receiving HSCT. Although more research is required, CAR T-cell treatment is effective in treating recurrent B-ALL and may also be used to treat B-CLL with hypodiploidy. Novel approaches are essential for enhancing patient outcomes and redefining therapeutic success when hypodiploidy challenges established treatment paradigms. Hypodiploidy is an uncommon yet aggressive form of B-CLL that has a very bad prognosis. Hypodiploidy represents significant chromosomal loss and structural imbalance, which contributes to a disordered genomic environment, in contrast to more prevalent cytogenetic changes. This instability promotes resistance to certain new drugs as well as chemoimmunotherapy and speeds up clonal evolution. Its persistence after treatment implies that hypodiploid clones have benefits in survival, which are probably strengthened by chromosomal segregation issues and damaged DNA repair pathways. Malignant progression and treatment failure are further exacerbated by telomere erosion and epigenetic dysregulation. The need for more sensitive molecular diagnostics is highlighted by the fact that standard karyotyping frequently overlooks hypodiploid clones, particularly those concealed by endoreduplication, despite the fact that these complications make early and correct diagnosis crucial. Hypodiploidy requires a move toward individualized treatment because of their link to high-risk genetic traits and resistance to conventional regimens. Although treatments like hematopoietic stem cell transplantation and CAR T-cells show promise, long-term management is still elusive. To improve long-term results and avoid early relapse, addressing this cytogenetic population necessitates combining high-resolution genomic technologies with changing therapy approaches. Full article

Fibronectin (FN), a primary component of the extracellular matrix (ECM), features multiple structural domains closely linked to various cellular behaviors, including migration, spreading, adhesion, and proliferation. The FN3 domain, which contains the RGD sequence, is critical in tissue repair because it enables interaction with integrin receptors on the cell surface. However, the large molecular weight of wild-type FN presents challenges for its large-scale production through heterologous expression. Therefore, this study focused on cloning the FN3 functional domain of full-length FN for expression and validation. This study selected Bacillus subtilis as the expression host due to its prominent advantages, including efficient protein secretion, absence of endotoxins, and minimal codon bias. The recombinant vector pHT43-FN3 was successfully constructed through homologous recombination technology and transformed into Bacillus subtilis WB800N. The FN3 protein was successfully expressed after induction with IPTG. Following purification of the recombinant FN protein using a His-tag nickel column, SDS-PAGE analysis showed that the molecular weight of FN3 was approximately 27.3 kDa. Western blot analysis confirmed the correct expression of FN3, and the BCA protein assay kit determined a protein yield of 5.4 mg/L. CCK8 testing demonstrated the good biocompatibility of FN3. In vitro cell experiments showed that FN3 significantly promoted cell migration at a 20 μg/mL concentration and enhanced cell adhesion at 10 μg/mL. In summary, this study successfully utilized Bacillus subtilis to express the FN3 functional domain peptide from FN protein and has validated its ability to promote cell migration and adhesion. These findings not only provide a strategy for the expression of FN protein in B. subtilis, but also establish an experimental foundation for the potential application of FN3 protein in tissue repair fields such as cutaneous wound healing and cartilage regeneration. Full article

Viral evasion from effective human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T-cell responses and from antiretroviral therapy through viral sequence variation is frequently accompanied by a loss in viral fitness. The impact of sequence variations on replication capacity in vitro was mostly studied by introducing single mutations into a specific clonal strain such as NL4-3. How the specific viral backbone itself impacts replicative fitness remains elusive. To test for a potential effect of the viral backbone, we constructed HIV-1 clade B clones with consensus sequences for gag and/or pol and evaluated the infectivity of viral variants harboring well-defined cytotoxic T-lymphocyte (CTL) escape mutations or drug resistance mutations within this backbone or the clonal NL4-3 strain. Viral variants with consensus sequences were replication-competent in vitro, although at lower rates than the NL4-3 virus. Introduction of the dominant CTL escape mutation R₂₆₄K into the newly constructed viruses or into NL4-3 led to a dramatic reduction in infection rates. In contrast to the NL4-3 backbone, the combination of R₂₆₄K with its compensatory mutation S₁₇₃A on the consensus backbone led to higher infection rates as compared to the same virus in the absence of R₂₆₄K and S₁₇₃A. Furthermore, 2 out of 10 drug resistance mutations in pol led to opposing effects, with an increase in infection rates on the consensus gag/pol backbone and a reduction on NL4-3. Therefore, the effect of the respective viral backbone on infectivity observed in vitro might constitute an additional factor to explain differential kinetics of mutational evasion from immune and pharmaceutical pressure. Full article

Background: hMPXV poses a major public health risk due to its human-to-human transmissibility, severe complications, especially in immunocompromised individuals, and global spread, necessitating effective surveillance and stringent prophylactic measures to mitigate its colossal impact. Objective: The study aimed to annotate hMPXV(IMV) proteins to propose a potential reverse vaccinology-based vaccine against hMPXV. Methods: The target MPXV(IMV) protein’s sequences, formatted in FASTA, were sourced from genome/proteome databases (BV-BRC and UniProt) (accessed on 6 November 2024), followed by CD-Hit-based redundancy removal. Epitope prediction for B-cells (lymphocytes), cytotoxic T-cells or cytotoxic T-lymphocytes (CTLs), and helper T-cells (HTLs) was executed using ABCpred, IEDB’s ANNs 4.0, and an artificial neural network-based alignment tool (NN-align 2.3)/ML-based tool (NetMHCII 2.3). Various immunoinformatics filters (antigenicity, toxicity, and allergenicity) were applied to substantiate the potency and safety of the formulated vaccine candidate. The constructed vaccine’s physiochemical and structural features (secondary and tertiary), with structural stability (confirmed by molecular docking followed by dynamic simulation with TLRs (TLR4 & TLR2) and MHCs), were determined. Additionally, cloning (using pET-28a(+) vector) was conducted to verify the vaccine’s expression potential and translation efficiency. The construct’s population coverage was also ascertained. Results: The MPXV-2-Beta vaccine constructs, of the six initially designed constructs, was identified as the most promising candidate, signifying nonallergenic profile and nontoxic features, with a predicted antigenicity score (PAS) = 0.7202, 407 residues, a molecular weight of 43,102.1 Da, pI of 9.2, and favorable stability parameters (AI: 65.65, GRAVY: −0.597, I-i: 25.92). It showed high solubility (score: 0.942). The ProSA Z-score of −9.38 confirmed the structural stability, reliability, and precision of the MPXV-2-Beta 3D model, which is comparable to experimental structures. Furthermore, 98.8% of all the residues nested within favored or allowed regions in a critical Ramachandran plot signified the model’s exceptional structural integrity and quality. Docking and dynamic simulation of MPXV-2-Beta with TLRs (TLR4 & TLR2) and MHCs demonstrated stiffer docking stability (strong polar and nonpolar interaction) and negative eigenvalue value (during dynamic simulation), suggesting its ability to enhance immune receptor activation under physiological conditions. MPXV-2-Beta was predicted to trigger a robust immune response (IR) with comprehensive world population coverage (98.55%, SD = 10.41). Conclusions: Based on the evaluated parameters, the MPXV-2-Beta designed in this study exhibited significant potential as an effective candidate against hMPXV. This study establishes a foundation for developing an efficient vaccine against hMPXV, requiring further experimental and clinical validation to confirm computational findings. Full article

The phytochemical screening of extracts of Tacca leontopetaloides tubers has afforded the isolation of two novel chalcones, tarkalynins A and B, along with taccalonolide A and its 12-propanoate. The screening of Zanthoxylum zanthoxyloides stem bark yielded taraxerol acetate, dihydrochelerythrin and fagaramide. These compounds were obtained through column and thin-layer chromatography and identified using NMR and LC-HRMS. The compounds were tested against Trypanosoma brucei brucei s427 and its multi-drug-resistant clone B48, against Trypanosoma evansi, Trypanosoma equiperdum and Trypanosoma congolense, and against Leishmania mexicana. Cytotoxicity was tested against the human HEK293 cell line. The highest activities were observed with dihydrochelerythrin and fagaramide against T. b. brucei s427 and B48, T. evansi, and L. mexicana, with EC₅₀ values of 1.37, 2.559, 1.09, and 5.44 µM and 17.8, 10.9, 10.9, and 13.3 µM, respectively. In addition, tarkalynin A and taraxerol acetate displayed promising activity against T. equiperdum (EC₅₀ = 21.4 and 21.3 µM, respectively). None of these compounds showed significant cross-resistance with existing trypanocides (RF ≈ 1; p > 0.05). The compounds displayed low toxicity to human cells, with most exhibiting no growth inhibition at concentrations of 100, or even 300 µM. This report provides further evidence of the potential use of natural products for combating parasitic diseases. Full article