Search Results (351)

Porcine endogenous retroviruses (PERVs), their transmembrane envelope protein p15E and peptides corresponding to a domain in p15E that is highly conserved among retroviruses, the immunosuppressive (isu) domain, demonstrated immunosuppressive properties. They inhibited in vitro immune reactions, and induced release of IL-6 and IL-10 in human peripheral blood mononuclear cells (PBMCs). We recently showed that p15E of PERV expressed on 293T cells reduced MHC expression, induced cytokine release in co-incubated human PBMCs, and inhibited cytotoxic cells. The cell-surface expression of p15E was chosen to simulate an artificially introduced expression on a transplant. Here, a new construct with a higher expression of p15E and consequently higher effects on cytokine release was designed and used. An intracellular cytokine assay was newly developed and applied, whereas a commercial cytokine array was used to analyze the release of 105 cytokines into the supernatant. The differential gene expression was analyzed by sequencing the RNA of PBMCs incubated with p15E-expressing and wild-type 293T cells. PERV p15E induced an elevated expression of IL-10, IL-6 and 24 other cytokines and modulated the expression of hundreds of genes. Furthermore, coating porcine L23 cells with the synthetic isu peptide of the PERV p15E protein and subsequently co-incubating them with human PBMCs induced IL-10 production, whereas direct addition of the peptide to PBMCs alone did not induce cytokine production. These results demonstrate that the PERV p15E protein can modulate cytokine release by human immune cells and suppress their cytotoxic activity. This immunomodulatory property may have potential applications in protecting transplanted tissues from immune-mediated rejection. Full article

Native chemical ligation (NCL) combined with post-ligation desulfurization is a powerful strategy for the chemical joining of unprotected peptide segments to form larger polypeptides or to synthesize head-to-tail cyclized peptides. However, a simple, robust, and safe desulfurization protocol suitable for high-throughput synthesis workflows has yet to be developed. Here, we present a photochemical strategy in which desulfurization is achieved using 270 nm ultraviolet (UV) irradiation in the presence of tris(2-carboxyethyl)phosphine (TCEP). Optimization of reaction conditions resulted in reaction efficiency comparable with current chemical desulfurization methods. Desulfurization of a combinatorial peptide library confirmed that the photochemical desulfurization strategy is compatible with all canonical amino acids as well as thiol-protected cysteines. Implementation of the optimized conditions in a 96-well plate format using a custom-built light-emitting diode (LED) array confirmed that product formation is dependent on local light exposure. Increasing the distance between the plate and the light source enabled more uniform irradiation, giving homogenous product formation. Altogether, this study establishes UV-mediated desulfurization as a safe and scalable alternative for high-throughput peptide synthesis workflows. Full article

Background: Mechanosensitive ion channels PIEZO1 and PIEZO2 are key mediators of mechanotransduction, which converts physical forces into cellular signals involved in proprioception, touch, vascular function, and other physiological processes. Mutations in human PIEZO proteins are linked to various diseases, such as hereditary xerocytosis, lymphatic dysplasia, and proprioceptive dysfunction. However, the role of intrinsic disorder in the regulation of these proteins and their susceptibility for disease-associated mutations remains unclear. Methods: We analyzed canonical human PIEZO1 and PIEZO2 protein sequences using machine learning, neural network, and energy-based disorder predictors, together with the prediction of disorder-mediated binding regions, phase separation propensity, interaction networks, evolutionary conservation, clinically annotated human variants, and peptide structural modeling. Results: Both proteins showed moderate intrinsic disorder, with PIEZO2 having slightly greater disorder propensity and higher predicted phase separation potential. Intrinsically disordered regions frequently overlapped binding-prone segments and post-translational modification sites, supporting regulatory functions. Evolutionary comparisons showed strong conservation of PIEZO proteins, while selected disordered regions retained disorder propensity despite greater sequence variability. Disease-causing variants mainly affected the ordered regions of both proteins, whereas disordered regions contained proportionally more benign variants and relatively few pathogenic mutations. The modeling of mutations within disordered hotspots showed altered local conformational tendencies, indicating that some disease variants may disrupt dynamic interaction interfaces rather than global structure. Interaction network analysis linked both proteins to enriched mechanotransduction, ion transport, and cytoskeletal pathways. Conclusions: Overall, our findings identify intrinsic disorder as an underappreciated feature of PIEZO channel biology and provide a framework for interpreting PIEZO-associated channelopathies. PIEZO proteins also perfectly illustrate the proteoform concept, where one gene yields a highly diverse kit of mechanosensitive molecular tools. While humans only have two primary PIEZO genes (PIEZO1 and PIEZO2), the body generates a vast array of functional variations. Full article

Biogenic amines (BAs) are nitrogenous compounds formed by microbial decarboxylation of amino acids in protein-rich foods. Their accumulation indicates spoilage and poses health risks. Traditional methods like high-performance liquid chromatography (HPLC) and gas chromatography (GC) are sensitive but time-consuming, limiting on-site use. Rapid technologies based on specific recognition molecules offer feasible alternatives for real-time monitoring. This review summarizes five categories of recognition elements: antibodies, aptamers, molecularly imprinted polymers (MIPs), enzymes, and peptides for BA detection in foods. These elements convert BA concentrations into optical, electrical, or colorimetric signals, establishing a complete biosensing chain. Integration with portable platforms (lateral flow assays (LFAs), microfluidic chips, smart labels, and smartphone devices) is also discussed. Recognition-element-based sensing enables high-selectivity and rapid monitoring of BAs in foods. Antibody/aptamer systems excel in specific histamine detection, enzyme platforms in rapid total amine assessment, and MIPs in chemical stability and matrix tolerance. Yet practical application is limited by poor selectivity for similar amines, matrix interference, insufficient real-food validation, and device standardization. Our future focus will be on AI-assisted design, multi-target arrays, smartphone quantification, and IoT-enabled freshness monitoring. Full article

Acinetobacter baumannii, a Gram-negative opportunistic bacterium in the ESKAPE group (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, A. baumannii, Pseudomonas aeruginosa and Enterobacter spp.), has emerged as a leading cause of nosocomial infections worldwide. It is known to possess diverse virulence traits and antimicrobial resistance, making it a critical priority pathogen on the World Health Organization’s 2024 Bacterial Priority Pathogens List. Carbapenem-resistant A. baumannii (CRAB) is currently endemic across several continents, with global carbapenem resistance exceeding 70% in healthcare settings and multidrug-resistant infections being associated with alarming mortality rates. This review comprehensively discusses the molecular underpinnings of A. baumannii pathogenesis and virulence, detailing the array of factors coordinated by complex regulatory networks. The convergence of this pathogen’s virulence and antimicrobial resistance traits, resulting in multidrug resistance, leaves clinicians with only a handful of therapeutic options. The review also discusses upcoming therapeutic strategies, including phage therapy, antimicrobial peptides, monoclonal antibodies, photodynamic therapy, and vaccine candidates in the pipeline. While emerging therapeutics show promise, several challenges remain, and integrated approaches are warranted to efficiently combat A. baumannii’s virulence and resistance armamentarium. Full article

Lifespan is increasing throughout the world leading to a rise in non-communicable diseases in the global population that impacts over 800 million individuals with metabolic disorders, such as diabetes mellitus. Metabolic disease presents a significant challenge for clinical care since multi-organ disease progression ensues despite a broad array of treatment protocols. The pursuit of innovative strategies with mammalian forkhead transcription factors of the “O” class (FoxOs) and intimately related pathways of aging, cellular senescence, telomere integrity, oxidative stress, programmed cell death with apoptosis, autophagy, ferroptosis, pyroptosis, and cuproptosis, Wnt/β-catenin signaling, Wnt1 inducible signaling pathway protein 1, and the gut microbiome becomes vital to address the clinical hurdles of metabolic disorders. Platforms incorporating novel diagnostics with artificial intelligence and machine learning can further address the underlying mechanisms tied to FoxOs that include the mechanistic target of rapamycin, AMP activated protein kinase, silent mating type information regulation 2 homolog 1 (S. cerevisiae), and glucagon-like peptide-1 receptor agonists that can markedly influence biological outcomes. Given the premise that it is essential to comprehend the intimate relationship that FoxO signaling pathways hold, FoxOs offer an exciting and promising approach to address the clinical aspects of disease onset, progression, and treatment with metabolic disorders. Full article

Endogenous gene tagging using CRISPR has changed the understanding of the role played by different proteins due to the ability to track and study proteins in their natural state. With CRISPR-based gene tagging, it is possible to insert fluorescent, luminescent, epitope, affinity, and proximity labels into the target protein at its endogenous genomic location without affecting its physiological expression and dynamics. Here, we discuss the DNA-repair mechanisms employed in endogenous gene tagging, including homology-dependent repair, NHEJ-based integration, and alternative approaches that can be used with challenging cell types. Key aspects of efficient CRISPR tagging experiments are also described. Additionally, we review recent advances in the increasing array of protein tag technologies, including fluorescent proteins, split-reporter technologies, NanoLuc/HiBiT, peptide epitopes, and proximity biotinylation enzymes. Lastly, we review the scalability of endogenous tagging approaches using multiplex editing, atlas-scale proteome tagging, iPSC-based disease modeling, and drug discovery platforms for assessing target engagement, protein degradation, phenotype screening, and mechanism of action of compounds. Although difficult in primary and pluripotent cells, new methods based on avoiding double-strand breaks, such as prime editing, PASTE, and CRISPR associated transposases, will drive the future expansion of endogenous tagging approaches. Such developments firmly set up CRISPR gene tagging as a fundamental technology in quantitative cell biology and translational pharmacology. Full article

Background/Objectives: Obesity is a multifactorial chronic condition characterized by the accumulation of excess adiposity and complex interplay between intrinsic and extrinsic factors. It is an increasingly common condition, closely implicated with the incidence and progression of cardiovascular disease and its risk factors. This narrative review synthesizes and summarizes recent evidence on obesity, with a focus on the diagnosis of obesity, an exploration of both visceral and subcutaneous adipose tissue, available interventions for obesity ranging from dietary modifications to novel anti-obesity medications, and key associations with obesity and cardiovascular diseases. This review is distinct in its integrated focus on obesity definition and diagnosis, imaging modalities, the latest non-pharmacologic and pharmacologic interventions, and also the interplay between obesity and certain cardiovascular conditions as well as their risk factors. Results: The diagnosis of obesity has been evolving with the incorporation of anthropometric measurements and imaging modalities rather than simply the body mass index. There is a wide array of contributors to obesity including genetic factors, behavior, hormonal regulators, the brain–gut axis, and psychosocial stressors. Anti-obesity medications have been evolving rapidly, with current emphasis on glucagon-like peptide 1 receptor agonists. Obesity is closely implicated in cardiovascular conditions such as atherosclerotic disease, heart failure, atrial fibrillation, and hypertension as well as related risk factors such as diabetes mellitus, chronic kidney disease and sleep apnea. Conclusions: Obesity is a widely prevalent, chronic, and complex disease. The use of a variety of anthropometric measurements can help risk-stratify individuals. Imaging techniques are also helpful in evaluating body fat. Evaluating individuals from a holistic perspective is imperative to appreciate the various contributors to obesity. There are a variety of interventions available for obesity management including lifestyle interventions, bariatric surgery, and pharmacologic therapy. Notably, obesity is closely tied with cardiovascular diseases and recent pharmacologic anti-obesity agents may mitigate cardiovascular risk. Full article

Marine macroalgae are widely distributed renewable resources that offer substantial economic and environmental benefits. This review comprehensively examines seaweeds from the phyla Chlorophyta, Heterokontophyta, and Rhodophyta, highlighting key advances and persistent challenges. Global seaweed production is highly concentrated: Asia accounts for 97% of the total, with China as the dominant producer. These seaweeds synthesize a diverse array of bioactive compounds, including sulfated polysaccharides, phlorotannins, terpenoids, proteins, peptides, polyunsaturated fatty acids, and pigments. Notably, brown algae represent the richest source of both phlorotannins and polyunsaturated fatty acids. To recover these valuable compounds efficiently, a range of advanced green extraction techniques have been developed, such as enzyme-assisted, microwave-assisted, ultrasound-assisted, and supercritical fluid extraction, along with natural deep eutectic solvents. These methods consistently outperform conventional approaches in terms of yield, extraction time, and environmental sustainability. The isolated compounds exhibit a broad spectrum of validated pharmacological activities, including immunomodulatory, anti-inflammatory, anti-diabetic, neuroprotective, antitumor, and antiviral effects. Consequently, they have found diverse applications in functional foods, biomedicine, cosmetics, agriculture, aquaculture, and environmental protection. Despite this promise, critical challenges remain in elucidating structure–activity relationships, developing scalable and sustainable extraction protocols, and advancing clinical translation. Future research should prioritize the discovery of novel marine bioactives, the enzymatic production of oligosaccharides, efficient purification of algal proteins and peptides, and the scaling-up of industrial processes to fully realize the pharmaceutical and biotechnological potential of marine macroalgae. Full article

Heavy metal ions are common environmental contaminants that threaten aquatic ecosystems and human health. However, conventional analytical techniques often require expensive instrumentation and complex sample pretreatment. This work presents a peptide-based multichannel solid-state potentiometric microelectrode array for simultaneous detection of Cu²⁺, Cd²⁺, and Pb²⁺. The array consists of one shared Ag/AgCl reference electrode and three groups of gold indicator microelectrodes (10 μm in diameter), with each group containing three parallel electrodes for replicate measurements. Each group is functionalized with a distinct peptide receptor specific to one target metal ion. The proposed array exhibits near-Nernstian responses and good selectivity against common interfering ions. Practical applicability is demonstrated by analyzing spiked lake water samples under a fixed chloride condition, with recoveries ranging from 94% to 105%. This work provides a miniaturized, reproducible, and versatile platform for simultaneous potentiometric detection of multiple heavy metal ions, and the peptide-based recognition strategy can be extended to other targets by simply replacing the peptide sequences. Full article

Background: Pertussis toxin (Ptx) is a major virulence factor and protective antigen of Bordetella pertussis. Understanding its antigenic landscape is essential for improving vaccine design. This study aimed to compare the linear epitope profiles of Ptx recognized by antibodies from vaccinated children and mice, identifying conserved and species-specific immune targets across subunits S1–S5. Methods: Two libraries of overlapping 14-mer peptides spanning the full-length Ptx sequence were synthesized. Sera from children and mice immunized with the whole-cell pertussis vaccine were analyzed to map antibody-binding regions. Comparative and structural analyses were performed to evaluate epitope distribution and recognition patterns. Results: Murine sera recognized 12 major epitopes, whereas children’s sera identified 24. Eleven epitopes were shared between species, mainly in subunits S1 (Ep3–5, 7, 9, 10), S3 (Ep20, 21, 25, 26), and S5 (Ep32), although minor positional shifts were observed. Eight epitopes were unique to children’s sera, located in S1 (Ep1, 6, 8), S3 (Ep22–24), and S4 (Ep27, 29–30). In the S2 subunit, four distinct epitopes were identified for each species, while only one mouse-specific epitope was detected in S4 (Ep28). Structural analysis revealed non-uniform antibody recognition, with dominant targeting of S3 and conserved antigenic hotspots, as well as selective recognition of the catalytic S1 subunit. Fourteen novel epitopes were identified. Conclusions: These findings highlight both shared and species-specific Ptx epitopes, revealing differences between murine and human immune responses. The identified conserved regions and novel epitopes provide a basis for improved pertussis vaccine design. Full article

The secretome of ESKAPE pathogens, including Klebsiella pneumoniae, comprises a diverse array of bioactive molecules that govern virulence, antibiotic resistance, and the establishment of an immunosuppressive microenvironment. However, the high chemical complexity of the secretome impedes the identification of key metabolites mediating pathogenesis. In this study, we profiled the metabolite composition of cell-free K. pneumoniae supernatant using a combined approach of chromatographic fractionation and Raman spectroscopy. Chromatographic separation enabled the resolution of the complex secretome and revealed fractions with distinct biochemical signatures. A key finding was the identification of Fraction 3, characterized by a unique metabolic profile: it was enriched in nucleic acid fragments, peptides containing tyrosine and methionine, polysaccharides, and stress-response metabolites (e.g., citrate), while notably lacking markers of tryptophan and sterol-like lipids. These spectral signatures suggest a potential role for Fraction 3 metabolites in intercellular communication, biofilm formation, and protection against oxidative stress. The remaining fractions also exhibited distinct biochemical profiles, defined by unique profiles of lipids, nucleotides, and amino acids. Collectively, these data underscore the critical role of specific K. pneumoniae secreted metabolites to pathogen survival and host immune modulation. The combined approach effectively resolves functionally relevant secretome fractions, offering new avenues for identifying diagnostic and therapeutic targets for multidrug-resistant infections. Full article

Background/Objectives: We developed a telehealth-enabled fiber-bundle endomicroscopy platform and evaluated its preclinical feasibility for targeted fluorescence imaging in cervical cancer models. Methods: The platform integrates a portable fiber-bundle endomicroscopy (FBE) system, fluorescein isothiocyanate (FITC)-labeled candidate peptides, and a secure web-based telehealth platform for remote consultation. The FBE probe achieved a field of view of 1,700 µm and a lateral resolution of 4 µm, enabling cellular-level fluorescence imaging in a compact, portable format. Four FITC-labeled peptides (SHS1*, SHS2*, FPP*, and CRL*) were evaluated in A549, SiHa, and CaSki cell lines. Ex vivo testing was performed on commercial cervical tissue-array samples. The telehealth platform was assessed for secure medical-image/video transmission and end-to-end latency in a simulated remote-consultation setting. Results: Among the tested probes, FPP*-FITC and CRL*-FITC showed higher fluorescence-positive fractions in the p16-overexpressing cervical cancer cell lines than in the A549 comparator line, with the strongest signals observed in CaSki cells. In ex vivo testing, CRL*-FITC generated higher fluorescence intensity in malignant cervical tissue-array samples than in non-malignant comparator tissues, with a reported 4.6- to 7.4-fold difference in mean signal intensity (p < 0.001). The telehealth platform supported the secure transmission of medical images and video and demonstrated an end-to-end latency of <500 ms in a simulated remote consultation setting. Conclusions: These results support the technical and preclinical feasibility of integrating targeted fluorescence imaging, portable fiber-bundle endomicroscopy, and telehealth into a single platform. This study should therefore be interpreted as a preclinical feasibility study evaluating optical, molecular, and telehealth integration, rather than as a clinically validated cervical cancer screening test. Full article

Charge transport underpins essential biological processes, including cellular respiration, photosynthesis, and enzymatic catalysis. Advances in molecular electronics have enabled single-molecule measurements that unequivocally establish redox-active proteins as efficient electron conductors, with their metal cofactors serving as intrinsic redox relays. By contrast, ubiquitous non-redox proteins lacking such redox centers have long been considered poor conductors. However, recent research has challenged this view, demonstrating that efficient charge transport in non-redox proteins can be mediated through polypeptide backbones, aromatic side-chain arrays, and hydrogen bond networks. This review surveys progress in understanding the single-molecule conductance of non-redox proteins. Firstly, we elucidate the fundamental transport mechanisms, highlighting the interplay between coherent tunneling and thermally activated hopping. We then provide an overview of state-of-the-art experimental techniques for single-molecule characterization. Through analysis of diverse systems spanning short peptides to large enzymes, we illustrate how aromatic amino acid networks and dynamic conformational fluctuations govern conductance, enabling emerging applications in label-free biosensing and single-molecule protein/DNA sequencing. Finally, we discuss persistent challenges and outline future opportunities for integrating protein-based conductors into bioelectronic devices. This review aims to stimulate further research and pave the way for novel applications harnessing protein conductance. Full article

This study explores the taxonomic diversity, metabolic profile, and bioactivity of marine-derived actinomycetes isolated from sediments collected off the coast of Lisbon and Setúbal Peninsula, Portugal. The combined use of two sediment pre-treatments (heat shock and dry overnight) and four growth media with varying nutrient concentrations revealed that formulations 10% A1 and SWA were most effective for recovering diverse actinomycetes, including rare Actinomadura, resulting in a total of 142 cultivable strains closely related to 47 phylogenetic distinct species dominated by Streptomyces and Micromonospora. Antimicrobial screening against methicillin-resistant Staphylococcus aureus (MRSA, COL) and Escherichia coli (K12) identified 22 bioactive strains, with strain PTS-083 exhibiting the strongest activity against MRSA (MIC = 1.95 µg/mL) and a 98.30% 16S rRNA gene identity to S. chumphonensis, highlighting it as a strong candidate for further metabolite and genomic studies. Cytotoxicity assays against HCT-116 human colorectal adenocarcinoma cells revealed eight bioactive strains with potent anticancer activity for extracts from strains related to S. sundarbansensis, S. violaceorubidus, and S. aculeolatus (IC₅₀ < 0.005–5.08 µg/mL). Untargeted LC-MS/MS metabolomic analysis uncovered a wide array of secondary metabolites, including macrolides, siderophores, fatty acids, and cyclic peptides. Comparative analyses with other Portuguese coastal studies revealed both shared and distinctive metabolomic profiles, emphasizing the importance of exhaustive sampling, even at nearby locations, since localized environmental conditions can influence metabolic diversity and are crucial for uncovering unique metabolites with potential biotechnological value. These findings highlight Portugal’s coastal sediments as a rich and underexplored source of novel actinomycetes and bioactive compounds with promising pharmaceutical applications. Full article