Search Results (1,432)

Skeletal muscle atrophy underlies sarcopenia, frailty, and muscular dystrophies, but the molecular mechanisms linking oxidative stress to muscle degeneration remain incompletely understood. We previously identified protein complex formation between transient receptor potential canonical 3 (TRPC3) and NADPH oxidase 2 (Nox2) as a key driver of anthracycline-induced myocardial atrophy. Here, we investigated whether this complex also contributes to skeletal muscle wasting. In skeletal muscle from sciatic nerve transection model mice and Duchenne muscular dystrophy (mdx) mice, TRPC3-Nox2 complex formation was enhanced. TRPC3 deletion significantly attenuated denervation-induced soleus atrophy and reduced reactive oxygen species (ROS) production. TRPC3-Nox2 complex formation was upregulated in the soleus muscle (SM) of mdx mice. Pharmacological disruption of the TRPC3-Nox2 interaction improved muscle size and strength and reduced plasma creatine kinase in mdx mice. A recombinant adeno-associated virus (AAV) encoding a TRPC3 C-terminal peptide was used to suppress TRPC3-Nox2 complex formation in vivo. AAV-mediated expression of TRPC3 C-terminal peptide mitigated muscle wasting (CSA) in mdx mice, while muscle strength and plasma CK were not significantly improved. Thus, TRPC3-Nox2 complex formation may be a pivotal driver of oxidative stress-mediated skeletal muscle atrophy. Targeting this protein–protein interaction represents a promising therapeutic strategy for Duchenne muscular dystrophy (DMD) and other intractable muscle-wasting disorders. Full article

Background/Objectives: Notwithstanding the declaration by the World Health Organization in May 2023 regarding the conclusion of the COVID-19 pandemic, new cases of this potentially lethal infection continue to be documented globally, exerting a sustained influence on the worldwide economy and social structures. Contemporary SARS-CoV-2 variants, while associated with a reduced propensity for severe acute pathology, retain the capacity to induce long-term post-COVID syndrome, including in ambulatory patient populations. This clinical phenomenon may be attributable to potential autoimmune reactions hypothetically triggered by antiviral antibodies, thereby underscoring the need for developing novel, universal vaccines against COVID-19. The nucleocapsid protein (N), being one of its most conserved and highly immunogenic components of SARS-CoV-2, presents a promising target for such investigative efforts. However, the protective role of anti-N antibodies, generated during natural infection or through immunization with N-based vaccines, alongside the potential adverse effects associated with their production, remains to be fully elucidated. In the present study, we aim to identify potential sites of homology in structures or sequences between the SARS-CoV-2 N protein and human antigens detected using hyperimmune sera against N protein obtained from mice, rabbits, and hamsters. Methods: We employed Western blot analysis of lysates from human cell lines (MCF7, HEK293T, THP-1, CaCo2, Hep2, T98G, A549) coupled with mass spectrometric identification to assess the cross-reactivity of polyclonal and monoclonal antibodies generated against recombinant SARS-CoV-2 N protein with human self-antigens. Results: We showed that anti-N antibodies developed in mice and rabbits exhibit pronounced immunoreactivity towards specific components of the human proteome. In contrast, anti-N immunoglobulins from hamsters showed no non-specific cross-reactivity with either hamster or human proteomic extracts because of the lack of autoreactivity or immunogenicity differences. Subsequent mass spectrometric analysis of the immunoreactive bands identified principal autoantigenic targets, which were predominantly heat shock proteins (including HSP90-beta, HSP70, mitochondrial HSP60, and HSPA8), histones (H2B, H3.1–3), and key metabolic enzymes (G6PD, GP3, PKM, members of the 1st family of aldo-keto reductases). Conclusions: The results obtained herein highlight the differences in the development of anti-N humoral responses in humans and in the Syrian hamster model. These data provide a foundational basis for formulating clinical recommendations to predict possible autoimmune consequences in COVID-19 convalescents and are of critical importance for the rational design of future N protein-based, cross-protective vaccine candidates against novel coronavirus infections. Full article

Epizootic hemorrhagic disease (EHD) is an important livestock disease caused by Epizootic hemorrhagic disease virus (EHDV). The recent incursion and wide distribution of EHDV in Europe have increased the need for effective vaccine candidates. Background/Objectives: The VP2 protein of EHDV forms the outer capsid layer of the virion and is essential for viral assembly and host cell entry. Owing to its antigenic properties, VP2 represents a major target for vaccine development. However, the recombinant production of VP2 is limited by low stability and poor yields, representing a significant barrier for the generation of safe and effective subunit vaccines. Methods: To overcome these limitations, the VP2 protein from EHDV serotype 8 (EHDV-8) was rationally engineered with targeted modifications at both the amino and carboxyl termini of its coding sequence. Recombinant expression was performed using a baculovirus vector-mediated system in Trichoplusia ni pupae (CrisBio^® technology), employed as living biofactories. Results: The engineering of VP2 resulted in up to a tenfold increase in protein yields compared with the wild-type sequence, while maintaining the trimeric structural integrity of the recombinant protein. Both wild-type and engineered VP2 protein variants were formulated and used to immunize IFNAR(−/−) mice, a model susceptible to EHDV infection. Both engineered and wild-type VP2 formulations elicited comparable neutralizing antibody responses in vaccinated animals. Furthermore, immunization with either formulation conferred full protection against lethal EHDV-8 challenge. Conclusions: In this work, we demonstrated that the rational engineering of the VP2 protein significantly improved recombinant expression yields in a baculovirus-based system without compromising structural integrity or immunogenicity. These findings additionally demonstrate the feasibility of producing high-quality VP2 antigens in T. ni pupae using CrisBio^® technology and support their potential application in the development of subunit vaccines against EHDV. Full article

The therapeutic clotting factor VIII (FVIII) is known for its particular immunogenicity, with nearly 30% of hemophilic patients developing neutralizing antibodies against the infused protein. The root cause of this immunogenicity is still not well understood, but intrinsic factors, such as FVIII byproducts, have been linked to the immunological response elicited. Bioengineering of the FVIII molecule has been improving its recombinant (rhFVIII) production in many aspects, mainly enhancing its expression and stability. Assessment of immunogenicity for novel recombinant isoforms is crucial for further development and scaling-up processes, particularly due to the unpredictable antigenic properties and their impact on neutralizing antibody formation. In the present study, we describe a bioengineered human recombinant FVIII (rhFVIII-H6A), which induces lower immunogenicity in a murine model of hemophilia A. The rhFVIII-H6A product is characterized by a B-domain-deleted heavy chain (HCh), with the C-terminal of the B-domain fused to the light chain (BΔ-LCh). Compared to plasma-derived FVIII (pdFVIII) and rhFVIII reference products, treating hemophilic mice with rhFVIII-H6A induced lower levels of anti-FVIII antibody formation, including those with inhibitory neutralizing activity, while no difference was observed in the functional activity of rhFVIII-H6A in reverting the in vivo hemophilia phenotype. In addition, our results indicate that deleting the major part of the B-domain from the HCh might lower the immunogenicity of novel rhFVIII products. Full article

Haemonchus contortus is a blood-feeding gastrointestinal nematode that significantly impacts the health and productivity of small ruminants. The burden of parasitism and the escalating incidence of anthelmintic resistance necessitate alternative control methods. Here, we characterize the enzymatic activities of five mammalian cell-expressed recombinant H. contortus cysteine proteases (HcCPs), which include two cathepsin B-like proteins (HcCBP1 and HcCBP2) and three cysteine protease 1 proteins (HcCP1a, HcCP1b, and HcCP1c). We hypothesize that these enzymes degrade host blood proteins, thereby facilitating the parasite’s nutrient acquisition and survival. Using synthetic cathepsin (cat) substrates, we show that HcCBP2 was the only protein that exhibited high catB/L but low catB or catK activity, which was inhibited by the cysteine protease inhibitor E-64. All mHcCPs degraded fibrinogen (Fg), which led to delayed plasma clotting, reduced clot density, and lysed plasma clots. All HcCPs partially degraded hemoglobin (Hb), except for mHcCBP2, which degraded Hb and bovine serum albumin completely and bovine IgG partially in the presence of a reducing agent. In conclusion, by sustaining blood feeding and facilitating immune evasion and nutrient acquisition, the HcCPs may play an essential role in the parasite’s survival. Thus, vaccines or cysteine protease inhibitors targeting these parasitic enzymes may mitigate or prevent infections. Full article

Cymbidium sinense is a highly valued ornamental orchid renowned for its strong floral fragrance. In this study, dihydro-β-ionone was identified as a major volatile compound in C. sinense ‘Qi Hei’. Its emission increased progressively during flower development and was predominantly released from the sepals and petals. Transcriptome analysis of flowers at three developmental stages led to the identification of four double-bond reductase genes, designated CsDBR1–CsDBR4. Spatiotemporal expression profiling demonstrated that transcript levels of CsDBRs were highest in sepals and petals, showing a significant positive correlation with dihydro-β-ionone accumulation (p < 0.05). Heterologous expression in Escherichia coli and subsequent in vitro enzymatic assays confirmed that recombinant CsDBR1, CsDBR2, and CsDBR4 proteins catalyze the conversion of β-ionone to dihydro-β-ionone, whereas CsDBR3 exhibited no detectable activity. Transient expression in Nicotiana benthamiana leaves further verified the in planta function of CsDBR1, CsDBR2, and CsDBR4, resulting in elevated production of dihydro-β-ionone upon infiltration of β-ionone. Substrate specificity assays revealed that CsDBR2 and CsDBR4 also reduced 1-octen-3-one, 3-nonen-2-one, and pulegone. Collectively, these findings demonstrate that CsDBR1, CsDBR2, and CsDBR4 are key enzymes responsible for dihydro-β-ionone biosynthesis in C. sinense, providing a genetic foundation for molecular breeding aimed at improving floral fragrance in orchids. Full article

Background/Objectives: To evaluate the cost-effectiveness of recombinant tumor necrosis factor receptor Fc fusion protein compared with methotrexate as first-line therapy for active rheumatoid arthritis in China using evidence from a Chinese head-to-head randomized trial. Methods: A Markov model with 6 months per cycle was developed to estimate costs and health utilization in the lifetime of patients with RA from the Chinese healthcare system. The analysis data were derived from the randomized clinical trial in China. The primary cost includes drug and other medical costs. The health utilities quality-adjusted life years (QALYs) were derived using EQ-5D-5L mapping from disease-specific health assessment questionnaire (HAQ) scores obtained in clinical trials. The cost-effectiveness analysis was conducted by calculating the incremental cost-effectiveness ratio (ICER) values for rhTNFR:Fc and MTX. One-way and probabilistic sensitivity analyses were conducted to test the robustness of the base-case result. Results: In the base case, rhTNFR:Fc yielded 8.20 QALYs versus 7.46 with methotrexate, resulting in an ICER of CNY 12,783.56 per QALY. Scenario ICERs for bDMARD group combination treatment were 11,776.31 per QALY. Scenario ICERs were CNY 8079.04 per QALY for the patient perspective and CNY 7630.34 per QALY for the medical insurance perspective. One-way analysis highlighted utility inputs as the main drivers, and probabilistic analysis indicated a high probability of cost-effectiveness across common willingness-to-pay thresholds. Conclusions: The fusion protein strategy achieved an incremental cost-effectiveness ratio far below the 2024 China per capita gross domestic product threshold of CNY 95,749 per quality-adjusted life year. As first-line therapy for active rheumatoid arthritis, it is cost-effective relative to methotrexate in the Chinese setting. Full article

Background/Objectives: The detection of anti-Coxiella antibodies using serological methods is essential for identifying exposed ruminants and preventing this important zoonotic disease in livestock. In recent years, numerous attempts have been made to increase diagnostic performance as well as simplify the production of serological assays. Commercially available tests often use whole-cell antigens, which can decrease specificity and require high-level biosafety facilities for manufacturing. The aim of this work was to produce three Coxiella burnetii (C. burnetii) antigens in recombinant form and assess them for the detection of anti-Coxiella antibodies in ruminants. Methods: Three recombinant C. burnetii antigens (Com-1, MceB, AdaA) were selected among immunodominant antigens and produced in a heterologous system (Escherichia coli). Following purification, the proteins were utilized to coat ELISA plates and evaluated for seroreactivity against sera from both negative and positive cattle. Results: Com-1 demonstrated the greatest agreement with the commercial test, albeit moderate. MceB exhibited nonspecific reactivity against a large number of sera, while the AdaA showed reactivity against only a few positive sera. Conclusions: Our findings are consistent with previous research, indicating that utilizing a single antigen to identify exposed animals is unfeasible with current knowledge, most likely due to the complex immunological response following C. burnetii infection in cattle. Consequently, it is critical to continue testing and identifying immunoreactive antigens in order to further investigate them and, potentially, select the most appropriate. Full article

Adipose tissue and skeletal muscle are the foremost energy depots and locomotor organs; they orchestrate metabolic homeostasis through the secretion of cytokines via autocrine, paracrine, and endocrine pathways. This intricate interplay is pivotal in the pathogenesis of numerous metabolic disorders, encompassing obesity and muscle atrophy, as well as influencing meat quality in animal production. Despite its significance, unraveling the molecular mechanisms underlying muscle–adipose crosstalk remains a major challenge. Recent advancements in multi-omics technologies have facilitated the identification of a multitude of cytokines derived from adipose tissue and muscle, including adipokines, lipokines, myokines, and myogenic exosomes and adipose-derived exosomes containing various biomolecules. The functional roles of these cytokines have been elucidated through meticulous studies employing trans-well cultures and recombinant proteins. In this comprehensive review, we summarize the bidirectional roles of adipokines and myokines in key biological processes—such as muscle satellite cell differentiation, mitochondrial thermogenesis, insulin sensitivity, and lipid metabolism. By synthesizing these findings, we aim to provide novel insights into the treatment of metabolic diseases and the improvement of animal production. Full article

The limited heat stability of skim milk powder (SMP) constrains its application in high-temperature processes. While dry heating can improve its thermal resistance, it often accelerates the advanced Maillard reaction, compromising protein quality. This study applied low relative humidity conditions (<10% RH) during dry heating to modulate the Maillard reaction, aiming to enhance the heat resistance of SMP and derive recombined filled evaporated milk emulsions with fewer undesirable changes in colour and solubility. SMP was subjected to dry heating at 80, 100, and 120 °C for durations ranging from 2 to 20 min (at 120 °C) and up to 16 h (at 80 °C). The progression of the Maillard reaction and associated protein modifications were evaluated. The results indicate that the advanced Maillard reaction was retarded, evidenced by minimal colour development and well-preserved protein solubility (90–97%, n = 3), determined using the Lowry assay on the supernatants. The hydroxymethylfurfural and protein carbonyl contents increased only moderately with temperature and time. Moreover, the sulfhydryl group content remained largely stable, consistent with limited disulfide-mediated aggregation. Heat treatment of SMP at 120 °C for 10 min greatly improved its heat stability, as reflected by a 25-fold reduction in the volume-weighted average diameter (D_4,3; 95% CI = 3 to 47) and a 108-fold reduction in the consistency coefficient (K; 95% CI = 12 to 200) of the SMP-derived sterilised recombined filled evaporated milk (RFEM) compared to the control. These findings demonstrate that dry heating under low RH helps to improve the functional properties of SMP without inducing the detrimental effects associated with advanced Maillard products. Full article

Collagen types I and III are primary structural proteins that maintain human skin integrity, and their ratio is disrupted during aging. In this study, we developed a biomimetic 3D skin model with vascularization potential to evaluate the effects of nano-zinc oxide (nano-ZnO) in a physiologically relevant context. The model used methacrylated recombinant human collagen (RHC-MA) bioinks with tunable collagen I/III ratios that mimic the skin of children and adults. The bioinks exhibited excellent printability and mechanical properties that enabled the 3D bioprinting of full-thickness skin products, including dermal layers with human skin fibroblasts (HSFs) and human umbilical vein endothelial cells (HUVECs), as well as epidermal layers with keratinocytes (KCs). The model recapitulated native skin architecture, and key markers such as keratin 10 (K10), keratin 14 (K14), and cluster of differentiation 31 (CD31) were determined. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed that nano-ZnO significantly modulated genes associated with apoptosis, inflammation, and oxidative stress in skin cells. Full article

Haseki tick virus (HSTV) is a recently discovered virus detected in human serum following tick bites, yet its protein repertoire remains uncharacterized. In this study, we applied an integrative approach based first on membrane topology prediction, followed by AI-based structural prediction and experimental validation to annotate the structural part of the HSTV polyprotein. For the first time, we recombinantly expressed one of the putative HSTV structural protein (SP1) and determined its overall architecture using small-angle X-ray scattering (SAXS). Structural comparisons of the AI-predicted HSTV SP1 models revealed only a vague resemblance to the pestiviral Erns and Npro. The strong agreement between experimental SAXS data and the AI-predicted HSTV SP1 model supported the conclusion that HSTV SP1 adopts a distinct spatial architecture in solution, one that is not captured by existing pestiviral structures but is reliably represented by modern AI-based prediction. Our findings indicate that HSTV SP1 adopts a fold not previously observed among characterized members of the Flaviviridae family. This work establishes a methodological pipeline for characterizing highly divergent viral proteins and provides the first insights into HSTV SP1, a virus with emerging zoonotic potential. These results lay the foundation for future functional and structural studies, diagnostic development, and evolutionary analyses of atypical Flaviviridae family members. Full article

Piscine orthoreovirus genotype 1 (PRV-1) is an emerging viral pathogen in salmon aquaculture that causes Heart and Skeletal Muscle Inflammation (HSMI), with high prevalence in salmon-producing countries such as Chile. A significant obstacle in PRV-1 vaccine development is the inability to culture the virus in vitro, which limits the scalability and production of traditional inactivated or DNA-based vaccine strategies. This study describes the development of a novel virus-like particle (VLP)-based vaccine against PRV-1. Recombinant VLP were produced by co-expressing the six structural proteins of PRV-1 (λ1, λ2, μ1, σ1, σ2, σ3) using a baculovirus-based expression system in insect cells. In addition, to enable differentiating infected from vaccinated animals (DIVA) strategies, the σ1 protein was modified by adding of a cmyc epitope tag. The results demonstrated that the native VLP vaccine (VLP6n) significantly reduced viral loads in Atlantic salmon challenged with PRV-1. Moreover, in rainbow trout, the cmyc-tagged VLP-like vaccine (VLP6c) elicited a specific antibody response against the cmyc epitope, allowing differentiation between vaccinated and naturally infected fish. Overall, this VLP-based vaccine platform represents a promising strategy for controlling PRV-1 prevalence in salmon-producing counties, supporting the implementation of serological surveillance programs. Full article

BPM1, a representative of the plant MATH-BTB protein family comprises three conserved domains—MATH, BTB, and BACK—that facilitate diverse protein–protein interactions central to developmental processes. However, recombinant production of BPM1 and its variants in Escherichia coli are frequently constrained by low solubility and poor stability. In this study, we systematically optimized E. coli-based expression strategies to enable soluble production and purification of domain-truncated BPM1 variants (BPM1ΔBTB, BPM1ΔMATH, and BPM1ΔBACK). A combinatorial approach was employed: varying induction temperature, medium composition, affinity tag selection, bacterial strain, and solubility-enhancing supplements. Expression outcomes were highly dependent on specific parameter combinations. Notably, BPM1ΔBTB—previously the most recalcitrant variant—showed a marked solubility improvement when expressed as a GST fusion in E. coli Rosetta (DE3) cultivated in TB medium supplemented with MgCl₂. By contrast, BPM1ΔMATH and BPM1ΔBACK displayed enhanced solubility when expressed in BL21 (DE3) cultivated in 4 × YT medium instead Rosetta (DE3) in 2 × YT medium. Constructs with N-terminal His-tags consistently resulted in poor solubility or failed expression. These results establish a framework for producing otherwise insoluble BPM1 variants and highlight a broadly applicable strategy for handling unstable proteins through tailored E. coli expression systems. Full article

The transcriptional response to alkalinization in Saccharomyces cerevisiae, Aspergillus nidulans and Candida albicans raised the interest of the scientific community many years ago for diverse reasons, and the underlying signaling pathways have been elucidated in these organisms in detail. Within the last few years, transcriptomic data for other fungal species have become available, although in most cases little is known about the molecular basis controlling their adaptive response. The objective of this work is to provide an overview on how different fungi remodel their gene expression in response to environmental alkalinization, highlighting the similitudes and differences among them. Microbial stress-responsive promoters have been considered useful tools for biotechnological applications, such as expression of recombinant proteins of industrial interest. Recent work, emphasizing the usefulness of alkaline pH-inducible promoters for heterologous protein production, will also be discussed. Full article