Therapeutic biomolecules are widely used to treat various diseases, but their clinical efficacy is often limited by short in vivo half-lives due to rapid renal filtration, proteolytic degradation, and immune clearance. Short half-life leads to frequent dosing, fluctuation in blood drug concentration, and increased treatment costs, severely limiting clinical efficacy. To address these challenges, various half-life extension strategies have been developed, including chemical conjugation (e.g., PEGylation), physical delivery systems (e.g., microspheres), and genetic/fusion approaches. This review provides a comprehensive narrative analysis of molecular engineering methods, discussing the rational design to directly optimize the drug molecule itself or fuse it with long-acting carriers to significantly extend its circulation time. A systematic comparison of several approaches is presented to guide rational strategy selection. By synthesizing current knowledge and recent advances, this review serves as a practical resource for researchers and drug developers navigating half-life extension technologies.
Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by cognitive failure, memory impairment, and behavioral disturbances. The disease is associated with complex pathological mechanisms including amyloid-β (Aβ) plaque deposition, tau hyperphosphorylation, oxidative stress, mitochondrial dysfunction, and chronic neuroinflammation. Despite extensive research, currently available therapeutic options provide only symptomatic relief and fail to halt disease progression. Consequently, increasing attention has been directed toward natural bioactive compounds with multi-target therapeutic potential. Marine ecosystems represent a vast reservoir of structurally unique biomolecules, among which marine-derived polysaccharides have emerged as promising candidates for neuroprotection. Polysaccharides such as fucoidan, alginate, carrageenan, chitosan, ulvan, chondroitin sulfate, and hyaluronic acid exhibit diverse biological activities, including antioxidant, anti-inflammatory, anti-amyloidogenic, and neuroprotective effects. These biomolecules can modulate several critical intracellular signaling pathways implicated in AD pathology, including the NF-κB, MAPK, PI3K/Akt/GSK-3β, Nrf2/ARE, STAT3, and NLRP3 inflammasome pathways. By regulating these pathways, marine polysaccharides can reduce oxidative stress, suppress neuroinflammatory responses, inhibit amyloid aggregation, attenuate tau pathology, and promote neuronal survival. Additionally, certain polysaccharides such as chitosan and alginate have demonstrated significant potential as nanocarriers for targeted drug delivery across the blood-brain barrier. This review summarizes recent advances in understanding the signaling pathways associated with AD and highlights the emerging therapeutic potential of marine-derived polysaccharides as multi-target neuroprotective agents. Overall, these marine biomolecules represent promising candidates for developing novel therapeutic strategies to mitigate neurodegeneration and improve cognitive function in Alzheimer's disease.
The effective management and treatment of cancer have become complex and challenging because of its rapid, uncontrollable growth, ability to evade the immune system, and the development of resistance to treatments. Biogenically synthesized selenium nanoparticles (SeNPs) have gained attention owing to their anticancer properties. The synthesis and development of nanoparticles (NPs) using fungi offer a promising approach to creating new products in nanobiotechnology. Various mycological cultures and their derived substances are promising because they are easy to handle, environmentally friendly, and are considered safe, tolerant, and capable of impressive intracellular metal accumulation or uptake. Fungi are exceptional in their ability to secrete enzymes both intracellularly and extracellularly. Their ability to produce a diverse array of metabolites, including enzymes, polysaccharides, proteins, and other biomolecules, makes them valuable bioreactors for nanoparticle synthesis. Myco-synthesis of NPs has demonstrated effective mechanisms in producing different metal and metalloid NPs. Several studies have confirmed the practical applications of these NPs spanning copious fields, comprising agriculture, medicine, and industry, benefiting goods, services, and mankind. This work presented a comprehensive and critical review of the myco-synthesis of SeNPs and their anticancer potentials. It explores the exceptional properties of fungi as programmed bio-factories by systematically providing a comparative appraisal linking multiple fungal species, their synthesis condition, and secreted biomolecules to SeNPs physiological features. It provides a mechanistic insight into myco-synthesized SeNPs extracellular versus intracellular pathways and expounds how physiological properties influence the anticancer efficacy and biosafety. Finally, the work integrates the in vitro and in vivo evidence, offering a translational insight into the therapeutic potentials of myco-synthesized SeNPs.
Carbon dots (CDs), zero-dimensional carbon-based nanomaterials, have significantly advanced laboratory medicine due to their high photoluminescence quantum yield, excellent biocompatibility, and versatile surface functionalization. This review systematically summarizes CDs' synthesis strategies (top-down and bottom-up approaches), structural/optical characteristics, and diverse applications in the field including the sensitive detection of biomolecules (proteins, nucleic acids, and small biomolecules), microbial identification, cell/tissue imaging, and diagnosis of cancer, infectious diseases, and other disorders. Despite challenges such as poor reproducibility, lack of standardized protocols, and hurdles in clinical translation, CDs hold great promise for enabling sensitive, rapid, and personalized diagnostic solutions. Future directions focus on developing multifunctional probes, advancing clinical translation, integrating with artificial intelligence and cutting-edge technologies (microfluidics), and enabling single-cell analysis, positioning CDs to revolutionize precision diagnostics and personalized medicine.
Acoustic, electric, and magnetic external fields have been extensively investigated as a promising non-invasive approach to control nucleation from solution. Among these approaches, light fields leveraging lasers such as non-photochemical laser-induced nucleation (NPLIN) have seen a recent surge of interest. NPLIN involves an interaction between the solute-solvent system and laser light, but takes place at wavelengths and intensities not known to invoke any photochemical phenomena. Promising applications include direct enhancement of the nucleation rate, as well as providing polymorphic control and spatial precision in initiating crystallisation. This chapter mainly reviews the current state of NPLIN research focusing on biomolecules. Secondly, this chapter discusses other external-field-based methods, albeit in much less detail. The chapter concludes with the authors' perspective on future directions of research dedicated towards biomolecule crystal nucleation using such field-based methods.
Extracellular vesicles (EVs) are small nanometric particles surrounded by a lipid bilayer and actively secreted by different cell types. EVs play a key role in cell-to-cell communication, and the vast array of biomolecules that EVs transport reflects the molecular profile of the originating cells. In cancer, EVs are key components of the tumor microenvironment (TME); meanwhile once released into the peripheral circulation, EVs can travel systemically and transmit signals beyond the primary tumor site, making EVs ideal candidates for liquid biopsy. Notably, EVs can be isolated from a blood sample and analyzed to obtain real-time information on tumor biology, enabling early diagnosis, monitoring of treatment response, and evaluation of disease evolution, with high sensitivity and specificity. This review examines the biological significance and clinical utility of EVs expressing the stemness-associated glycoproteins CD44 and CD133 in gastrointestinal (GI) malignancies. Cancer stem cells (CSCs) expressing these surface markers are known to exhibit enhanced tumorigenic potential, metastatic capacity, and therapy resistance. In particular, we focus on the increasing evidence that EVs enriched in CD44+ and CD133+ populations play critical roles in key aspects of tumor progression in cholangiocarcinoma, pancreatic, colorectal, and gastric cancers. Following internalization by recipient cells, CD44+ and CD133+ EVs drive phenotypic reprogramming, foster more aggressive cellular states, and promote chemoresistance by delivering specific molecular cargo. Mechanistically, CD44 isoforms, particularly CD44v6 and CD44v9, activate key oncogenic signaling pathways, including Wnt/β-catenin and phosphoinositide 3-kinase (PI3K)/serine/threonine kinase AKT (AKT). In parallel, CD133-enriched EVs help maintain stemness and contribute to TME reorganization, thereby facilitating tumor progression. Despite ongoing challenges in EV isolation and standardization, EVs positive for stemness markers show great potential as liquid biopsy analytes for noninvasive disease monitoring, prognostic evaluation, and patient stratification. This review summarizes the expanding body of knowledge on cancer stem cell (CSC)-derived EVs in GI tumors, underscoring the potential of these particles for early diagnosis, prognosis, and the development of targeted therapies to overcome treatment resistance.
Membrane processes are one of the most promising separation and extraction technologies in modern industries due to their multiple advantages, including low energy consumption, operational simplicity, and scalability for commercial applications. Currently, these processes, particularly affinity polymer membranes, are a significant research area for selective separations. This study investigates a novel affinity polymer membrane containing Dibenzo-18-crown-6 for the extraction and separation of alanine and tryptophan from aqueous solutions. The membrane was characterized and evaluated for these amino acids individually and in mixtures. In addition to quantitative evaluation, qualitative analysis indicated that the incorporation of DB18C6 modifies the membrane structure and enhances specific interactions between the carrier and amino acids, which influence the extraction process and selectivity. Thermodynamic and activation parameters, including enthalpy (ΔHth), activation energy (Ea), transition state enthalpy (ΔH≠), and transition state entropy (ΔS≠), were determined to elucidate the substrate extraction mechanisms. Notably, this work demonstrates a higher selectivity for alanine over tryptophan in the mixture separation process, highlighting the novelty of selective separation based on specific extractive agent-substrate interactions. These findings provide valuable insights into the design and optimization of affinity membranes for efficient and selective separation of biomolecules, with strong potential for industrial and commercial applications in pharmaceuticals, food, and chemical processing sectors.
Traditional agricultural delivery methods, such as foliar spray and soil application, suffer from low uptake efficiency, environmental contamination, and short-term effects, whereas nanoparticle-mediated delivery platforms face issues of stability, cytotoxicity, and regulatory concerns. Recently, microneedle (MN) technology has emerged as a promising alternative for precise, minimally invasive delivery of agrochemicals and biomolecules. In this opinion article, we explore the evolution of MN-based delivery systems in agriculture. We discuss the structure-function relationships of MNs (solid, hollow, dissolving, and coated MNs) and highlight their applications in nutrient delivery, pathogen control, and genome editing for plants. We conclude with challenges and future directions for integrating MNs into precision agriculture to improve crop productivity, sustainability, and genetic manipulation.
Various marine protists inhabiting high-salinity environments can convert organic nutrients into high-value biomolecules or biomass, making them suitable agents for valorizing saline organic residue from fermentation waste streams, such as condensed molasses fermentation solubles (CMS). This study established a screening method to identify mixotrophic or heterotrophic marine protists suitable for CMS valorization and to assess their bioconversion potential. Growth-performance and nutrient-replacement assays were evaluated based on relative colony coverage on agar plates and final cell density in liquid culture. When applied to the selected strain, Aurantiochytrium limacinum, CMS supplementation was shown to enhance glucose consumption and biomass production; however, it did not increase the production of fatty acids such as docosahexaenoic acid (DHA). Reducing medium salinity did not rescue the impaired lipid biosynthesis, whereas supplementation with vitamins B1, B7, and B12 restored fatty acid production in a dose-dependent manner. These results indicate that vitamin-associated cofactors are major determinants of fatty acid biosynthesis under CMS-based cultivation. Our findings demonstrate that molasses-derived residues can serve as substrates for the production of highly unsaturated fatty acids. They also provide evidence that B-vitamin cofactors play an important role in the fatty acid biosynthetic pathway of thraustochytrids. KEY POINTS: • CMS supported robust growth but did not promote proportional lipid synthesis in Aurantiochytrium limacinum. • B-vitamin insufficiency (B₁, B₇, and B₁₂) was identified as the primary factor limiting the biosynthesis of fatty acids. • Supplementation with B-vitamin cofactors restored fatty-acid and DHA biosynthesis in a dose-dependent manner.
Thermophilic cyanobacteria and microalgae have a set of coordinated structural and molecular changes that allow them to survive under elevated temperatures. All these features make are their thermostable enzymes, strong stress response systems, and high-capacity carbon-fixation systems that make these organisms interesting candidates of biotechnological use and sustainability. Cyanobacteria and thermophilic microalgae are a unique group of extremophiles that can survive high temperatures and complex environments. Their morphological, physiological, and evolutionary characteristics enable them to survive in hot springs, arid soils, geothermal environments, and hydrophilic ecosystems. This involves production of heat-stable enzymes, osmolytes, pigments, and protective biomolecules, and increased thermostability of phycobilisomes, reliable repair of photosystem II components, and structural changes of photosystems. Microalgae and cyanobacteria exhibit remarkable morphological plasticity, transforming between unicellular, colonial, and filamentous forms while producing specialized cells like heterocysts, spores, and dormant vegetative cells to survive in various environments. Further, their ecological resilience is enhanced by adaptations to oxidative stress, nutrient limitation, UV radiation, and desiccation. These organisms have great potential for industrial biotechnology, particularly biofuels, bioprocessing, carbon capture, bioremediation, and the synthesis of high-value compounds, due to their unique thermostable enzymes, heat-stable pigments, and carbon fixation efficiency. This review highlights current understanding of the phylogeny, stress adaptation mechanisms, and ecological significance of thermophilic microalgae and cyanobacteria, emphasizing their growing importance in sustainable biotechnology.
The Langmuir monolayer technique has proven to be an effective method for constructing lipid-based models of cell membranes. Compared to other artificial membrane systems, such as liposomes or supported lipid bilayers, it offers a relatively simple and versatile approach to reconstructing lipid components of biological membranes and systematically modifying their composition. The technique allows precise control over experimental parameters such as surface pressure and temperature, which influence the physical state and organization of lipid monolayers. Lipid monolayer models are widely used to investigate molecular interactions at membrane interfaces, including the effects of biomolecules or xenobiotics on membrane properties, identification of potential molecular targets of drugs, and evaluation of mechanisms underlying their pharmacological activity or toxicity. While the successful application of the monolayer technique in lipid membrane modeling has been extensively reported in the literature, comprehensive discussions of lipid compositions for modeling various membrane types-such as eukaryotic, prokaryotic, viral, and pathological membranes-remain limited. In particular, systematic descriptions of lipid mixtures used to model membranes characteristic of eukaryotic cells, prokaryotes, viruses, or pathological states are limited. The aim of this review is to address this gap by summarizing lipid compositions used in Langmuir monolayer models designed to mimic different biological membrane types.
Nuclear transport is a vital system that mediates movement of essential biomolecules between the nucleus and cytoplasm. It is tightly regulated by the Importin (IMP) superfamily to maintain separation of cellular compartments. Cellular stress in various forms, particularly oxidative, can suspend nuclear transport and lead to cell death. Prolonged oxidative stress manifests in myriad conditions, including cancer, viral infection and metabolic disease. An IMP protein, Importin-13 (IMP13), retains function under stress, while all other IMP family members tested to date do not. Phylogenetic and structural analysis revealed Transportin-3 (TNPO3) as the closest homologue of IMP13, suggesting it may also retain its function under stress. Subcellular localisation studies indicated that TNPO3 maintained its typical subcellular localisation, even in the presence of stress, unlike most IMP family members. Also, fluorescence recovery after photobleaching (FRAP) demonstrated that TNPO3 shuttling is unaffected under stress. Co-immunoprecipitation studies examining cargo binding revealed the capacity of TNPO3 to bind its cargo in the presence of stress. This demonstrated for the first time that TNPO3 retains functionality under stress conditions, in contrast to other IMPs, but similar to IMP13. Furthermore, both IMP13 and TNPO3 appear to protect against the potentially critical mislocalisation of Ran, a key molecule involved in the maintenance of the nuclear transport system.
Anti-arboviral peptides are biomolecules capable of interfering with key stages of the arboviral lifecycle. We present a dataset compiling 270 peptides composed of standard amino acids with reported activity against arboviruses, along with their lengths, sequences, target specificities, peptide entry mechanisms, and interaction mechanisms. The dataset was structured to support the training of machine learning models designed to identify anti-arboviral peptides. Therefore, it facilitates the development and validation of predictive tools against arboviruses. As a carefully assembled and expert-reviewed resource, this dataset aims to serve as a reference standard for evaluating new prediction models or comparing them with automatically compiled peptide datasets. This resource provides a comprehensive overview of the antiviral mechanisms currently being explored and can serve as a foundation for designing next-generation peptides targeting arboviral infections.
Biotherapeutic antibodies are increasingly being developed and, various strategies have recently been used to maximize their potential therapeutic efficacy. The crystallizable fragment (Fc) region of therapeutic monoclonal antibodies (mAbs) is often engineered to tailor their effector functions and pharmacokinetic (PK) properties by introducing point mutations. Notably, most of these mutations are in the hinge and constant domains of the heavy chain, which may silence antibody effector functions. Several liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods have been published to quantify biotherapeutics with a canonical human Fc portion. This work presents a rapid and sensitive hybrid immunocapture liquid chromatography-tandem mass spectrometry (IC-LC-MS/MS) method for quantifying total antibody concentration, specifically targeting the LALA-mutated peptide (L234A/L235A). The sample preparation process, which includes immunocapture, as well as trypsin and Glu-C digestion, is efficiently completed within two days through automation. The developed method was validated according to the ICH M10 guideline and white papers recommendation, focusing on the following parameters - accuracy, precision, dilution linearity, selectivity, stability, recovery- and using a humanized IgG1 LALA-mutated antibody, teplizumab, as analytical standard. The method demonstrated linearity for total antibody detection in mouse plasma samples, with a dynamic range from 150 ng/mL (lower limit of quantification, LLOQ) to 15,000 ng/mL (upper limit of quantitation, ULOQ). All validation parameters tested in mouse plasma met the predefined acceptance criteria, demonstrating the method's reliability and robustness. Additionally, the qualified method was successfully used to characterize the pharmacokinetic profile in mice of an antibody-drug conjugate (ADC-1) containing the LALA mutation in its Fc region. This work provides a valuable foundation for the quantification of new biological entities (NBEs) and antibody-drug conjugates (ADCs) in pharmaceutical development, as it enables the measurement of engineered Fc biotherapeutics using a unique and highly selective peptide, irrespective of the type of biological matrix and even in the presence of other biomolecules of similar IgG isotype.
Adipose-derived mesenchymal stromal cells (ASCs) are important components of the breast tumor microenvironment (TME) with hemostatic features and immunomodulatory abilities that can either inhibit or promote tumor growth through direct cell-cell contact or the secretion of biomolecules. This study aimed to investigate the effects of ASCs on PD-1 and/or TIM-3 expression by T cells isolated from breast tumor-draining lymph nodes (TDLNs). Lymphocytes were isolated from fresh, uninvolved axillary lymph nodes and cultured either directly with ASCs from the breast adipose tissue of healthy women (N.ASCs) or breast cancer patients (C.ASCs), or in conditioned media (CM) from these ASCs. After 48 and 72 h, the expression of PD-1 and/or TIM-3 on CD3+, CD4+, and CD8+ T cells was analyzed using flow cytometry. Results showed that direct co-culture with both N.ASCs and C.ASCs significantly increased the frequency of CD3+, CD3+CD4+, and CD3+CD8+T cells expressing the exhaustion marker TIM-3 in both 48- and 72-hour cultures. This increase was independent of PD-1 expression, as both TIM-3+PD-1+ and TIM-3+PD-1- T cell populations increased significantly. Interestingly, the frequency of T cell populations that expressed PD-1 without TIM-3 remained unchanged. Furthermore, culturing lymphocytes in CM of ASCs had no significant impact on the frequency of TIM-3- and/or PD-1-expressing T cell populations. Additionally, the effect of N.ASCs on TIM-3 and PD-1 expression showed no significant difference from that of C.ASCs. In conclusion, ASCs can contribute to T cell differentiation into a terminally exhausted phenotype by significantly increasing the expression of TIM-3 on these cells, primarily through direct contact.
Nanopore is a single-molecule technology for sensing biomolecules. Biomolecular interactions are essential biological processes that govern biological functions and therapeutic responses. However, high-resolution nanopore sensing of biomolecular interactions, such as protein-ligand interactions, remains challenging. In this study, we demonstrate that a YaxAB nanopore with LiCl-modulated electrostatic potential enables detection of molecular interactions of the BRD4 protein with histone peptides, as well as diverse small-molecule drugs, at the single-molecule level. Our electrical recordings and molecular dynamics simulations confirm that the oscillating dynamics of BRD4 within the funneled YaxAB nanopore generate two-level current transitions between narrow- and wide-pore regions. Using the parameters derived from dual-level dynamics and their signal decomposition, a YaxAB nanopore sensing approach enables the sensitive discrimination of BRD4-small-molecule drug complexes with a subtle mass difference as small as 2.5 Da. This near-atomic, high-resolution sensing capability of YaxAB nanopores may enable applications in single-molecule-based drug discovery, proteomics, and diagnostics.
Thermo-physical perturbation is expected to weaken protein stability and assembly. Yet, in the biological context, chaperones such as heat shock proteins (HSPs) must counter perturbative effects to preserve proteostasis. Herein, we report that the sHSP14, a small heat shock protein from the extremophile Sulfolobus, inverts this paradigm through thermal reinforcement. Fully atomistic in silico sampling is exploited to reveal that elevated temperatures enhance dimeric stability via enthalpic consolidation that persists even at high salinity. This archaeal chaperone operating with minimal molecular machinery achieves robustness through elegant design: β-strand swapping creates topological architecture, while a strategic charged residue network (Arg17, Asp28, Arg66, Asp41, Arg79, Lys74) and nearly 40 preserved hydrogen bonds maintain stabilization even upon temperature elevation. These findings redefine our understanding of extremophile adaptation and offer blueprints for engineering next-generation thermostable biomolecules.
The resistance to antibiotics and the appearance of super-resistant bacteria have become a serious public health problem all around the world. Antibiotics repositioning through the development of metal-antibiotic complexes could be a solution because they improve antibiotic therapeutic activity by increasing its electronic delocalization and lipophilic nature, easing its access into cells and improving interactions with biomolecules. An example of a commonly used antibiotic is levofloxacin (Levo), a third-generation quinolone which has been coordinated with different metal ions to enhance its antibacterial and anti-tumor properties. This work presents the synthesis, crystallography, and photophysical characterization of three coordination compounds based on Levo with Zn(II), Tb(III) and Eu(III), alongside their physicochemical properties under biological media. The most relevant result of this work is that Levo coordinates with Eu(III) and Tb(III) showing the so-called antenna effect through energy transfer from the drug as a ligand to the lanthanide ions in biological medium. This result opens new avenues for exploring its localization in cells and enabling future therapeutic applications in biomedicine, where the drug could act as an antenna ligand. Furthermore, experimental absorption and emission spectra were obtained, and Density Functional Theory (DFT) and Time-Dependent Density Functional Theory (TD-DFT) calculations were carried out to characterize their electronic and photophysical properties and confirm the sensitization of the lanthanide ions by the Levo drug.
The advantage of Raman spectroscopy (RS) in renal analysis lies in its capacity to non-invasively acquire label-free molecular vibrational fingerprint information from biological samples. It characterizes alterations in diverse biomolecules, including proteins, lipids, nucleic acids, urates, oxalates, and cytochromes, facilitating the detection of molecular and metabolic abnormalities prior to the manifestation of morphological changes. The review explains how RS deciphers the molecular characteristics of various kidney diseases without using dyes. Comparisons are drawn among surface-enhanced Raman spectroscopy (SERS), tip-enhanced Raman spectroscopy (TERS), confocal Raman microscopy (CRM), and coherent Raman techniques to elucidate their applications in molecular mapping of urine, blood, kidney tissue, and individual cells. We also summarize the Raman spectral features of diverse biological samples for the molecular diagnosis of kidney diseases, with attention to the role of advanced analytical methods, including multivariate statistics, machine learning, and deep learning, in spectral interpretation and disease classification. Coupled with high-throughput instrumentation, miniaturized platforms, and artificial intelligence, RS holds potential for precision diagnostics and clinical translation in nephrology.
Recent progress in DNA nanotechnology has shown the isothermal assembly of several DNA nanostructures. Isothermal assembly allows DNA nanostructure construction in a variety of ions while simplifying DNA nanotechnology by avoiding the need for thermal cyclers and expands utility by enabling attachment of guest biomolecules on DNA nanostructures at ambient or physiological temperatures. The paranemic crossover (PX) DNA motif has been used in the construction of DNA nanostructures, paranemic cohesion has been used to connect DNA structures as an alternate to sticky end cohesion, and PX DNA has also been implied to have a biological role in homology recognition. In that context, here we demonstrate the successful isothermal assembly of the PX DNA motif in magnesium (Mg2+), calcium (Ca2+), and strontium (Sr2+) at 20 and 37 °C. Using isothermal titration calorimetry, we show that interhelix hybridization of half-PX molecules is favored at higher temperatures, with a heat capacity (ΔCp) of -1.9 kcal/mol·K. To demonstrate a key advantage of isothermal assembly, we show that PX molecules can be designed to contain thrombin-specific aptamers for binding one or two thrombin molecules site specifically in an entirely isothermal procedure. Our work extends isothermal assembly and the use of different counterions for complex DNA motifs while demonstrating the attachment of guest molecules at constant temperatures.