The concept of bacterial therapy dates back over a century to clinical observations that incidental infections could induce tumor regression. Recent advances in genetic engineering and synthetic biology have since transformed bacteria into versatile living therapeutics with significant preclinical potential against diseases such as cancer, inflammatory disorders, and metabolic conditions. However, clinical translation faces considerable hurdles. Here, we provide a clinically oriented perspective on the translational gap in bacterial therapy. Drawing inspiration from the success of antibody-drug conjugates in achieving precise payload delivery, we highlight an emerging paradigm of "precision living therapeutics" enabled by bacterial surface engineering. we propose the concept of "Tumor accessibility" for the first time, and identify its insufficiency as a critical bottleneck in current therapeutic applications. We then systematically summarize recent advances in bacterial surface engineering, encompassing physical, chemical, and biological strategies, with a focus on their capacity to evade immune clearance, enhance tumor colonization, and improve therapeutic performance. Chemical approaches primarily involve covalent conjugation, including the SpyTag/SpyCatcher system and bioorthogonal click chemistry-based metabolic labeling. Physical strategies center on cell membrane encapsulation and surface coatings such as layer-by-layer encapsulation. Biological strategies include cell camouflage and genetic modulation of surface structures, display of functional biomolecules, and affinity-based systems such as biotin-streptavidin interactions. Finally, we discuss integrative strategies that combine surface-engineered bacteria with conventional treatment modalities, including physical therapy, chemotherapy, and immunotherapy. We propose that future clinical translation of bacterial therapy should shift from localized modification design to a holistic consideration of systemic accessibility.
Peptide therapeutics have emerged as a versatile class of biomolecules bridging the gap between small-molecule drugs and large biologics. Advantages of such molecules include high target specificity, potent bioactivity and reduced off-target toxicity. Despite these, broader clinical translation remains constrained by inherent limitations like poor metabolic stability, rapid renal clearance, limited membrane permeability and scalable synthesis. This review aims to systematically integrate advances in peptide science across natural discovery, synthetic methodologies, structural engineering, and translational delivery systems, while identifying critical research gaps hindering clinical adoption. We highlight diverse natural sources of bioactive peptides, including plant- (lunasin), animal- (Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP)), microbial- (nisin and cyclosporine), marine- (dolastatins) and venom-derived (chlorotoxin and ω-conotoxin MVIIA (ziconotide)) agents. Advances in solid-phase peptide synthesis (SPPS), green chemistry, and catalytic strategies are discussed alongside emerging in silico approaches, including artificial intelligence-driven sequence design and molecular modeling. Structural modifications such as cyclization, hydrocarbon stapling, PEGylation, and lipidation are critically evaluated for their role in enhancing pharmacokinetic and pharmacodynamic properties. Furthermore, nanoformulation strategies, including self-assembling peptides and cell-penetrating systems, are examined for their potential to overcome biological barriers. Importantly, this review identifies key unresolved challenges, including the lack of predictive models for peptide delivery systems, safety concerns associated with long-term modifications, and limited in vivo validation of naturally derived peptides. Addressing these gaps through integrated computational and experimental approaches will be essential for advancing next-generation peptide therapeutics. Collectively, this work provides a comprehensive framework for the rational design and translation of peptide-based precision medicines.
Membrane-associated biomolecules, primarily proteins, are key enablers of communication, responsiveness, and complexity in natural living cells. Aiming to mimic these capabilities, there is growing interest in equipping bottom-up synthetic cells with membrane-associated biomolecular components. In this review, we focus on how proteins and nucleic acids have been associated with synthetic cell membranes, particularly lipid vesicles, to enable the transmission of signals across the membrane. We discuss strategies for anchoring these biomolecules into lipid bilayers and review how they can enable essential signalling mechanisms in synthetic cells, including cell tethering, the generation and fusion of vesicles, and signal transmission and transduction. We highlight how proteins offer native biological functionality, while nucleic acids may bring more modularity and control. Advancing this area will be essential for realising synthetic systems capable of studying natural communication mechanisms and unlocking applications in biosensing, therapeutics, and synthetic tissue engineering.
Colorectal cancer (CRC) remains a leading cause of cancer-related mortality worldwide, driven largely by pronounced molecular heterogeneity and delayed clinical detection. Although high-throughput sequencing technologies have substantially advanced the understanding of CRC biology, their routine clinical implementation remains constrained by high costs, infrastructural requirements, and limited accessibility. This review addresses these translational barriers by systematically synthesizing circulating transcriptomic and proteomic biomarkers within a clinically scalable framework. Particular emphasis is placed on biomolecules detectable using reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), two widely accessible platforms that underwent extensive global optimization during the COVID-19 pandemic and are readily adaptable to liquid biopsy workflows. Through a stage-resolved analysis, we identify 9 genomic and 7 proteomic biomarkers associated with early-stage (I-II) CRC, alongside 9 genomic and 4 proteomic biomarkers linked to advanced-stage (III-IV) disease progression. Beyond biomarker cataloging, these molecules are integrated with Cancer Hallmark pathways, clinical-stage associations, and available clinical trial evidence to evaluate their biological relevance and translational readiness. In addition, we summarize standardized operating procedure (SOP) considerations and multiplex detection strategies to improve assay reproducibility, scalability, and cross-border clinical implementation. Collectively, this review bridges molecular discovery with clinically deployable laboratory workflows and provides a translational roadmap for the development of affordable, liquid biopsy-based diagnostic strategies aimed at improving CRC detection, patient stratification, longitudinal monitoring, and early therapeutic intervention. Stage-resolved biomarker framework: Provides an integrated catalog of circulating transcriptomic and proteomic biomarkers stratified across CRC stages I–IV.Mechanistic and functional integration: Links circulating biomarkers with Cancer Hallmark pathways, highlighting biomolecules that function as both diagnostic indicators and molecular regulators of disease progression.Clinically scalable diagnostic focus: Prioritizes RT-PCR– and ELISA-based detection strategies to support affordable, high-throughput, and clinically accessible liquid biopsy applications.Translational readiness: Integrates clinical trial evidence, assay feasibility, and SOP considerations to strengthen the clinical applicability and reproducibility of candidate biomarkers.Multi-analyte precision diagnostics: Supports the development of combined transcriptomic–proteomic biomarker panels to improve diagnostic sensitivity, prognostic stratification, and disease monitoring.Global clinical applicability: Proposes a practical framework for biomarker implementation and validation across diverse healthcare systems, including resource-limited settings.
Neurological illnesses continue to be a major global health challenge and require novel and safe therapeutic strategies. Blue foods like marine fish, algae, and invertebrates consist of many bioactive proteins/peptides that might provide multiple benefits for maintenance of a healthy brain, including neuroprotection, reduction of inflammation, and reduction of oxidative stress. This review looks at both recent advances made with blue food bio-molecules and their potential future roles in therapeutic targeting for neurological disorders. This study is designed as a narrative review, aiming to comprehensively summarize and critically analyze existing literature on marine-derived bioactive peptides and their neuroprotective potential. Literature searches were conducted from January 2009 to March 2025 using PubMed and other biomedical databases. Available preclinical evidence suggests that marine-derived proteins and peptides may exert neuroprotective effects through multiple mechanisms, including activation of antioxidant defense pathways via Nrf2-mediated signaling, modulation of NF-κB-associated neuroinflammatory responses, regulation of serotonergic and dopaminergic neurotransmission, and attenuation of amyloid-β aggregation. Experimental studies conducted in animal models have reported improvements in cognitive performance, reductions in oxidative stress biomarkers, and decreased production of pro-inflammatory cytokines following administration of marine protein hydrolysates. Clinical translation of marine-derived bioactive peptides remains challenging due to the following factors: standardization of extraction, bioavailability, batch-to-batch variability, allergenic potency, and complex regulatory requirements. Ongoing new approaches using nano-formulation, synthetic biology, and Artificial Intelligence (AI) in the discovery of peptides hold the promise of enhancing their stability, targeting, and scalability. However, in order to validate the therapeutic utility of these approaches, it will require large, well-designed clinical studies.
The ability to access atomically tailored complex peptides and proteins provides powerful opportunities for dissecting molecular functions and advancing applications in chemical biology, therapeutics, and (bio)-materials science. Robust precision-engineering strategies are essential to construct well-defined protein architectures while preserving native folding and activity. To do so, chemoselective bioconjugation techniques have been developed to modify specific side chains of amino acids. This allowed for the selective introduction of functionalities on predetermined amino acids. However, ultimate control can be achieved only through site-selective modifications that precisely define both the nature of the linkage and the exact position of conjugation on elongated peptide sequences or fully assembled proteins. Cysteine residues are of particular interest, as their highly nucleophilic thiols offer excellent chemoselectivity and typically occur in low abundance in their reduced form. Here, we examine chemoselective transformations targeting cysteine residues that have been further refined to occur exclusively at predefined positions within a peptide or protein, thereby achieving a high degree of site-selectivity. This review focuses exclusively on chemical strategies for cysteine modification, offering guidance for future synthetic developments within the field of precision chemistry. Achieving this level of precision requires advanced chemical strategies that exploit the local environment of the targeted cysteine. One approach involves leveraging neighboring functional groups, for example, engaging the thiol together with the α-amine or carboxylate to enable selective N- or C-terminal modification, respectively. In such designs, the cysteine side chain may contribute through transient interactions, direct incorporation into the covalent linkage, or the stabilization of the desired product. Recently, a promising strategy has attracted increasing attention in which site-selectivity is enabled by temporary interaction with a proximal amine, thus being applicable to differentiate also between internal cysteines. Together, these strategies highlight that site-selective protein modification has evolved into a powerful tool for the rational design and functional control of complex biomolecules, redefining what is achievable in chemical biology, therapeutics, and biomaterials science. We anticipate that increasingly routine or user-friendly approaches such as the programmable TriTEx method will further accelerate the adoption of precision biomolecule conjugates in both research and industrial settings.
Sulfhydryl functionality is useful for the chemoselective and site-specific conjugation of biomolecules for medical diagnosis and therapeutic purposes. Several types of thiol-reactive conjugation chemistry have been developed, including a maleimide-based addition reaction. Our previous study showed that activated 6-[18F]-fluoronicotinic acid ([18F]-FNA) ester chemoselectively forms an S-acylated product with peptide ACooP (H-ACGLSGLGVA-NH2) bearing both a free amino group and a sulfhydryl group for positron emission tomography applications. The aim of this work is to better understand the chemoselectivity of S-acylation and explore its potential use for site-specific peptide radiolabeling. Accordingly, three new peptide variants of the decapeptide ACooP were designed as model sequences bearing both free amino and sulfhydryl functionalities, with the cysteine residue located at different positions in the sequences. The peptide variants C@3 (H-AGCLSGLGVA-NH2), C@4 (H-AGLCSGLGVA-NH2), and C@5 (H-AGLSCGLGVA-NH2) were conjugated with FNA to prepare the corresponding nonradioactive reference compounds. The conjugated products were characterized using one- and two-dimensional nuclear magnetic resonance analysis and high-resolution mass spectrometry. Peptide radiolabeling tests were performed using [18F]-FNA 4-nitrophenyl ester as the prosthetic group at pH 8.6 and 7.4. The radiolabeled products were S-acylated compounds with high or exclusive chemoselectivity (>95%) in all three cases. To demonstrate the utility of this type of chemoselective S-acylation for radiotracer preparation, [18F]-FNA-S-C@5 was prepared with a radiochemical purity of 97.0% ± 0.8 (n = 3) and a decay-corrected radiochemical yield of 16.1% ± 3.5. A batch size of the end product with hundreds of MBq was easily achieved, which is sufficient for PET imaging applications. [18F]-FNA-S-C@5 showed limited in vitro stability in rat plasma, with intact tracer observed at 13.1% ± 4.2 (n = 3) after 15 min of incubation. In conclusion, chemoselective S-acylation-based peptide radiolabeling was achieved using [18F]-FNA 4-nitrophenyl ester, and this reaction holds promise for highly chemoselective and site-specific radiolabeling of other types of biomolecules.
Liposomal or polymeric nanoparticles have been instrumental in improving the delivery of poorly soluble chemotherapeutics and those with dose limiting toxicity such as doxorubicin (DOX). More recently, nanoformulations have been shown to enable simultaneous delivery of emerging biomolecules such as siRNA. However, for larger nucleic acids such as mRNA, this remains challenging. In this study, we developed a poly(β-amino ester) (PBAE) based platform, capable of co-formulating mRNA and doxorubicin into nanoparticles. To demonstrate proof of concept using therapeutically relevant cargo, immunomodulatory interleukin-12 (IL-12) was selected as a model mRNA. IL-12 is a pro-inflammatory cytokine that promotes anti-tumour immunity partly through amplifying effector cytokines such as interferon-γ (IFNγ). We found that PBAE complexed DOX and mRNA into positively charged nanoparticles of 120 nm and size-exclusion chromatography indicated a DOX loading efficiency of over 97%. Co-association of both DOX and mRNA was characterised at a single nanoparticle level by nano-flow cytometry. Following delivery to B16F10 murine melanoma cells, more than 95% of cells were double-positive for DOX and Cy5-labelled mRNA, and confocal microscopy confirmed co-localised regions of DOX with mRNA. Interestingly, nanoformulated DOX had increased nuclear accumulation by 1.7-fold relative to free DOX, which correlated with a significantly reduced cell viability of 12.9% with PBAE-DOX/mRNA, compared to 26.6% for free DOX at the same dose. Moreover, despite this strong cytotoxic effect, reporter mRNA translation remained robust, with luciferase expression approximately two orders of magnitude above non-transfected controls at the highest DOX doses. Co-formulation of IL-12 mRNA and DOX with PBAE demonstrated effective IL-12 protein secretion in transfected B16F10 cells with a simultaneous DOX dose dependent reduction in viability. Secreted IL-12 was bioactive, inducing dose-dependent STAT4 phosphorylation and IFNγ secretion in primary mouse splenocytes. Furthermore, in a syngeneic melanoma mouse model, intratumoural administration of PBAE-DOX/IL-12 mRNA achieved significantly elevated levels of IL-12 and IFNγ in the tumour compared to the saline control, confirming delivery of DOX, as well as IL-12 protein secretion, and immunostimulatory activity in vivo. These findings demonstrate that PBAE is a promising platform for co-delivery of cytokine encoded mRNA with DOX in a single formulation, establishing feasibility for advanced chemoimmunotherapy approaches.
Large poly(ethylene) glycol (PEG) chains are often conjugated to proteins or biomolecules to inhibit proteolytic degradation, mask immunogenic response, reduce clearance rates, and improve biodistribution of therapeutics, vaccines, drug delivery systems, and gene therapy formulations. The PEG macromolecular chain can also be used as a noninvasive reporter to track biologics in vivo by magnetic resonance spectroscopy (MRS). Rapid internal dynamics of PEG render the transverse 1H spin relaxation time to be comparable to water (~0.5 s) and amenable to imaging through traditional pulsed field gradient techniques. While water signal grossly exceeds that of PEG it is possible to filter 1H MRS signal of PEGylated conjugates through one of two ways-(1) stimulated echo acquisition mode (STEAM) MRS, which leverages huge differences in the diffusion of water versus PEGylated constructs, and (2) 13C-edited 1H MRS of fully 13C-enriched PEGylated constructs. Here, we compare both approaches. A 15 kDa 13C-enriched PEG chain was prepared alone, conjugated to bovine serum-albumin (BSA), and incorporated into a 52-nm-diameter PEG-poly(lactic acid) (PLA) nanoparticle. These three PEG constructs were then separately monitored in real time by 13C-edited 1H MRS, after introducing them into rat animal models intravenously. A 13C-editing scheme was employed to monitor 1H MRS PEG signal in the vasculature via a radiofrequency coil placed around the tail. An observed two-component decay of the PEG signal is attributed to perfusion and early equilibration (alpha phase) and slow clearance (beta phase). 13C-PEG alone, 13C-PEG-BSA, and 13C-PEG-PLA nanoparticles exhibited half-lives of 38.6 min, 23.4 h, and 11.9 h, respectively. The relatively rapid clearance rates associated with the PEG-PLA nanoparticles is expected to arise from enzymatic degradation of the PLA chain. Using STEAM-based editing schemes, we then evaluated sensitivity and water suppression in diffusion-edited 1H MRS for (12C)-PEGylated BSA contrasting 2-, 20-, and 40-kDa PEG chains, in imaging phantom samples. Larger molecular weight PEG chains (i.e., 40 kDa) proved far superior to smaller PEG chain reporters due to reduced inhomogeneities and longer T2, upon employing either a 13C-HQMC filter or a STEAM-based diffusion filter.
Epithelial and endothelial barriers play essential roles in maintaining compartmentalization within the body through specialized intercellular junctional complexes. Botulinum neurotoxin type E (BoNT/E), a ~150 kDa protein toxin produced by Clostridium botulinum, is capable of traversing the intestinal epithelial barrier despite its large molecular size. BoNT/E is produced in association with several neurotoxin-associated proteins (NAPs), which are believed to facilitate toxin stability and absorption. Among these, an approximately 80 kDa protein (P80), corresponding to OrfX2, has been identified as a BoNT/E-associated protein; however, its functional role in epithelial transport remains poorly understood. P80 was purified from the BoNT/E progenitor toxin complex and characterized using circular dichroism spectroscopy and thermal denaturation analysis. Its biological activity was evaluated using hemagglutination assays, cytotoxicity studies, confocal microscopy, atomic force microscopy (AFM), immunofluorescence imaging, tight junction protein analysis, and transcytosis assays in Caco-2 and HT-29 intestinal epithelial cell models. Structural analyses revealed that P80 is a stable, predominantly α-helical protein with a well-defined tertiary structure. P80 exhibited no detectable hemagglutination activity and showed minimal cytotoxicity toward intestinal epithelial cells. Cellular studies demonstrated that P80 interacts with epithelial monolayers, induces actin cytoskeletal remodeling, increases claudin-1 phosphorylation, and alters tight junction organization. AFM and confocal microscopy revealed changes consistent with transient modulation of epithelial barrier integrity. Functional transcytosis studies showed that P80 significantly enhanced the transport of co-incubated macromolecular cargo across polarized Caco-2 monolayers, resulting in approximately a fivefold increase in translocation compared with untreated controls. These findings demonstrate that P80 functions as a non-toxic modulator of epithelial barrier permeability capable of enhancing paracellular transport of large biomolecules. Unlike classical hemagglutinin-associated neurotoxin proteins, P80 lacks hemagglutination activity while retaining the ability to reversibly alter tight junction dynamics. The results suggest that P80 contributes to BoNT/E intestinal absorption and may serve as a promising bioenhancer for improving mucosal delivery of therapeutic proteins and other hydrophilic macromolecules. Further mechanistic and in vivo studies are warranted to evaluate its translational potential.
The escalating global cancer burden, particularly in low- and middle-income countries, necessitates safer and more effective therapeutic strategies. The toxicity and environmental issues of traditional heavy-metal-based quantum dots (QDs) have been addressed by green-synthesised QDs, which have become a promising platform for nanomedicine. In photodynamic treatment (PDT), photothermal therapy (PTT), and theranostic applications, the anticancer effectiveness of green-synthesized QDs that are derived from plant extracts, microbes, biomolecules, and biomass waste is critically assessed. Green synthesis techniques, such as hydrothermal and microwave-assisted methods, provide biocompatible QDs with good photostability, tunable optical characteristics, and decreased cytotoxicity. Mechanistically, these QDs generate reactive oxygen species (ROS), induce mitochondrial dysfunction, produce localized hyperthermia upon near-infrared irradiation, and activate apoptotic pathways (such as p53, Bax/Bcl-2, and caspase cascade), leading to selective cancer cell death. Preclinical in vitro and in vivo studies demonstrate potent tumor ablation through passive (EPR effect) and active targeting strategies. Despite these advances, some key setbacks hinder their clinical translation: lack of standardized synthesis protocols, batch-to-batch variability, limited long-term biosafety data, suboptimal targeting efficiency, and regulatory hurdles. Future perspectives include AI-driven optimization, smart theranostic platforms integrating multimodal imaging and therapy, and sustainable circular economy approaches using biowaste. Addressing these challenges through harmonized protocols and rigorous preclinical validation will be essential to realize the full potential of green-synthesized QDs as safe, multifunctional, and effective cancer nanomedicines.
The present study re-examines the efficacy of prevalent methods of Thymoquinone (TQ) estimation in the biomass of Nigella sativa L. using RP-HPLC. The differential efficacy in terms of magnitude, accuracy and cognate correlation with metabolically affiliated groups of biomolecules, phenolics, and antioxidants opens new vistas.  The study was conducted to develop a more accurate and sensitive method for quantitative assessment and correlation among TQ content, total phenolics, and flavonoids in six different plant parts of Nigella sativa, which are used in culinary applications, therapeutic formulations, nutraceuticals, and biowaste. The re-examination was based on the extraction of TQ-rich fractions with solvents of differential polarity (methanol and benzene), followed by quantitative assessment by RP-HPLC using methanol: water (80:20) as the mobile phase. RP-HPLC data revealed that TQ in methanolic extracts was highest in root (0.00959 ± 0.00032 mg/g FW), followed by flower (0.00882 ± 0.00013 mg/g FW), and lowest in fruit (0.00488 ± 0.00014 mg/g FW). Benzene extracts yielded the same pattern but with substantially higher magnitude, particularly in flower (0.0634 ± 0.0272 mg/g FW ) and fruit (0.0616 ± 0.0416 mg/g FW), making the system more resolvable. With the objective to understand the correlation with compounds of antioxidant nature, total phenolic content (TPC) and total flavonoid content (TFC) were determined and correlated with TQ content estimated using two different solvents. TPC was highest in leaf (0.208 ± 0.010 mg GAE/g) and lowest in fruit. TFC was highest in bud (0.344 ± 0.004 mg QE/g) and lowest in root. Pearson correlations using triplicate values showed very weak associations between methanolic TQ and TPC (r = 0.008; p < 0.05) and between methanolic TQ and TFC (r = -0.330; p < 0.05). Benzene-extracted TQ also showed a negligible positive correlation with TPC (r = 0.110; p < 0.01) and a very poor negative correlation with TFC (r = -0.016; p< 0.01). TPC and TFC remained moderately positively correlated (r = 0.450; p = 0.05). The results indicate that TQ and phenols accumulate largely independently in different plant parts of N. sativa. The TQ estimation using benzene extracts appears far superior to the method relying on methanolic extracts, given the large-scale and magnitude differences, making it more resolvable during screening.
Breast cancer remains a leading cause of cancer morbidity and mortality worldwide. Although nanomedicines are a promising and rapidly evolving therapeutic platform, their clinical translation remains limited. Depending on the stage and subtype of the disease, systemic treatments and localized strategies are still essential for treating metastatic breast cancer. Metastatic forms are still mostly incurable, even with improvements in early-stage interventions. Low tumor specificity, high systemic toxicity, decreased chemical stability, and multidrug resistance are some of the drawbacks of traditional treatments. Biomimetic nanoparticles (BMNPs) are synthetic cores cloaked with native cell membranes or biomolecules to recapitulate biological surface functionality for improved targeting and immune evasion. They have synthetic structures and artificial antigen-presenting cells in addition to coatings made of biologically derived materials. In preclinical studies, BMNPs often show enhanced tumor accumulation, prolonged circulation, and reduced systemic toxicity compared with uncoated nanoparticles, although reporting heterogeneity limits direct comparisons. By circumventing complex bottom-up synthetic approaches that attempt to replicate biological complexity, biomimetic nanoparticles preserve native biological functions using naturally derived cell membranes.
There is considerable evidence that iron accumulation in the affected brain regions plays a role in the pathology of neurodegenerative diseases, probably because of the ability of iron ions to promote oxidative damage to important biomolecules, such as DNA, RNA, proteins and lipids (leading to lipid peroxidation). Yet recent clinical trials of iron ion-chelating agents in human neurodegenerative diseases have given unimpressive results, in several cases making the diseases worse. In using iron chelators, it is important to remember the basics of their redox chemistry, which could explain their lack of therapeutic effects. This issue is explored here, and the potential of iron ion chelation in the treatment of neurodegeneration is critically evaluated.
Covering: up to 2026Bisretinoids are a chemically distinct class of endogenous natural products formed by the non-enzymatic condensation of visual-cycle retinoids. Derived from dietary provitamin A carotenoids via retinaldehyde intermediates, these pigments form spontaneously within the photoreceptor disc membranes through Schiff base chemistry with phosphatidylethanolamine, generating structurally diverse pyridinium, dihydropyridine and retinal dimer species. In contrast to enzyme-directed biosynthesis, bisretinoid biogenesis is governed by the intrinsic electrophilicity of the conjugated retinaldehydes within a lipid-dense environment. Their extended polyene systems endow them with distinctive excited-state properties, enabling efficient intersystem crossing and photosensitized generation of singlet oxygen under visible light. Subsequent oxidative fragmentation produces reactive electrophilic carbonyl species, including methylglyoxal and glyoxal, which covalently modify biomolecules and contribute to retinal pigment epithelium dysfunction and drusen formation. Despite their well-documented pathological roles, bisretinoids have not been systematically examined within a natural product framework. Here, we integrate the current knowledge of their biogenesis, electronic structure, and photochemical reactivity and consider how factors such as retinaldehyde flux, membrane composition, and iron homeostasis modulate their accumulation and reactivity. By framing bisretinoids as autochthonous natural products governed by intrinsic chemical principles, this review highlights new opportunities for mechanistically informed therapeutic intervention in retinal degeneration.
Ruminant milk-borne extracellular vesicles (EVs) have garnered significant attention as assorted bioactive components of intercellular communication and key regulators of both physiological and stressful conditions. These membrane-bound vesicles transport diverse molecular cargo, contributing to immune modulation, cellular homeostasis, metabolic and stress regulation. These distinctive characteristics of EVs position them as potential indicators of physiological and disease conditions in both human and animal research. Further, thermal stress induced alterations in the biological system of the animal are mirrored in these milk-borne molecular structures, indicating them to be accessible, non-invasive markers of heat stress in ruminant livestock. However, a holistic comprehension of the role of ruminant milk-derived EVs in human and livestock health remains limited. In spite of rapid developments in this field, variability in methodologies, investigations and stated findings demands a comprehensive synthesis of existing knowledge. In this context, this work offers a systematic review of the aspects of ruminant milk-derived EV's isolation, function, cargo profiling, biomarker prospects and potential applications. Relevant articles were screened based on predefined inclusion criteria to identify significant research trends and thematic areas. A total of 124 studies published between 1990 and January 2026 were examined using Scopus® data, following the structured search using the keywords "extracellular vesicle" AND milk, combined with additional terms including "heat stress," "cell culture," "buffalo," "cattle," "sheep," and "goat." Descriptive statistics, along with text mining and topic analysis, were performed. Publications on ruminant milk-derived EVs began in 2012, with marked increase after 2021 and significant peak in 2024 with majority of publications in the International Journal of Molecular Sciences and Journal of Dairy Science. Major proportion of publications originated from China followed by the United States, Australia, Italy and Japan. The most frequent co-occurring terms were "extracellular vesicle," "milk," "exosomes," "bovine milk" and "drug delivery." Text mining results indicated strong research focus on ruminant milk-derived EVs in the context of biomedicine, nutraceuticals and human health compared with the diminished concentration on livestock research aspects like heat stress. The 7 identified topics following topic analysis spanned distinct subjects including ruminant milk EV isolation, characterization, functioning, immune-modulatory role and applications. The analysis also revealed significant challenges in method standardization, characterization protocols clinical validation and supportive regulatory frameworks. Research skew toward miRNA, overlooking other biomolecules was acknowledged; amidst EVs applications in livestock, especially under climate stress, remaining unventured. Further, key gaps in toxicity, safety and bioavailability was also identified demanding inter-disciplinary collaboration for diagnostic and therapeutic deployment across species. Bridging these gaps remains crucial for unlocking the maximum potential of ruminant milk-borne EVs in advancing the One Health framework and for the attainment of sustainable development goals.
Human embryonic kidney (HEK) cell lines are used in the production of diverse biopharmaceuticals, including extracellular vesicles (EVs), due to their scalability and human biocompatibility. EVs are nanosized (30-200 nm), carriers of biomolecules that participate in cellular communication and can deliver therapeutic cargo in a targeted manner. Their use as drug vectors requires high-purity isolates; however, achieving such purity is challenging due to complex host matrices. Size exclusion chromatography (SEC) affects separations based on hydrodynamic radii and is a common isolation method due to its downstream applicability; however, it struggles to yield high-purity isolates. Polyester (PET) capillary-channeled polymer (C-CP) fiber spin-down tips are a novel EV separation platform employing hydrophobic interaction chromatography (HIC) to isolate high-purity EVs. Samples are loaded under high ionic strength conditions, where hydrophobic species adsorb to the fibers, and polar species pass through unretained. Analytes are then eluted in order of increasing hydrophobicity using an inverse salt gradient and adding small amounts of organic solvent at each step. A practical comparison of a commercial SEC column and PET C-CP spin-down tip EV eluates is conducted here via quantitative absorbance response curves, protein assays, nanoflow cytometry, and transmission electron microscopy. PET C-CP fiber platforms isolated 4.3 × 1011 particles mL-1 of HEK supernatant and yielded purities of 3.1 × 1010 particles µg-1 protein, a tenfold improvement for both metrics versus the SEC methodology, demonstrating a rapid, high-purity alternative to that commercial method.
Diabetes mellitus represents a major global health challenge with rapidly increasing prevalence and substantial morbidity driven by metabolic and vascular complications. Extracellular vesicles (EVs) have emerged as critical mediators of intercellular communication and are increasingly implicated in the pathogenesis and progression of diabetes. This review summarizes current knowledge on EV biology, including their classification, cellular sources, biogenesis, uptake mechanisms, and molecular cargo. We discuss the contribution of EV-associated microRNAs to immune dysregulation and β-cell damage in type 1 diabetes mellitus (T1DM), as well as the role of EVs in insulin resistance, metabolic signaling, and vascular dysfunction in type 2 diabetes mellitus (T2DM). Particular emphasis is placed on EV-mediated modulation of endothelial function, angiogenesis, and tissue repair, alongside their involvement in the impairment of insulin receptor integrity. We further explore how lifestyle factors may influence EV composition and function, highlighting their potential integration into preventive strategies. Finally, we evaluate the emerging therapeutic potential of EVs as biomarkers and delivery systems, while addressing current limitations and future directions. Collectively, EVs represent a promising frontier in understanding diabetes pathophysiology and developing innovative diagnostic and therapeutic approaches. Unlike previous reviews that examine EVs separately as biomarkers or therapeutic vehicles, this review integrates emerging evidence supporting EVs as mediators of systemic communication linking pancreatic islets, adipose tissue, immune cells, vascular endothelium, kidney, heart, and retina throughout diabetes progression. We further critically evaluate translational barriers that currently limit clinical implementation of EV-based diagnostics and therapeutics.
Gold nanoparticles (AuNPs) are highly versatile nanomaterials due to their exceptional physicochemical and biological properties with promising biomedical applications. This study presents a rapid, eco-friendly biosynthesis of AuNPs using the red alga Jania rubens (Jan-AuNPs). GC analysis of the algal extract revealed a total fatty acid content of 1.41 mg/g DW, dominated by saturated fatty acids primarily methyl palmitate (53.22%) and methyl stearate (27.89%) alongside methyl oleate (9.42%). Phytochemical profiling demonstrated a rich biochemical composition, including a high total phenolic content of 299.82 ± 1.83 mg GAE/g DW, total flavonoid content of 35.61 ± 0.52 mg QE/g DW, and total carbohydrate content (TCC) of 166 ± 1.05 mg GE/g DW, collectively conferring potent reducing, capping and stabilizing capacities of the algal extract in mediating Jan-AuNPs biosynthesis. Formation of Jan-AuNPs was visually confirmed by a color change from pale yellow to ruby red or pinkish-red, which was further confirmed by UV-vis spectroscopy with a surface plasmon resonance (SPR) peak at 549 nm. TEM analysis revealed predominantly spherical particles with an average diameter of 16.26 nm, with a few rod-shaped particles also detected. The crystalline nature of Jan-AuNPs was validated by XRD and SAED analyses. Zeta potential measurements revealed a surface charge of -28 mV, indicating good colloidal stability. FTIR analysis confirmed the active involvement of diverse algal biomolecules in the formation and stabilization of Jan-AuNPs. Biosynthesis conditions were optimized using face-centered central composite design (FCCCD), achieving the highest yield of 289 µg/mL at pH 7, 60 °C, 3 h incubation, and 300 µg/mL gold ion concentration. In silico predictive analysis identified cancer-associated gene targets modulated by AuNPs, thereby predicting prostate cancer (PC) as the malignancy with the highest therapeutic susceptibility for AuNPs-based interventions. Experimental validation confirmed potent and selective antitumor activity against PC3 prostate cancer cells in vitro (IC50 = 6.39 µg/mL; SI = 15.24) with minimal cytotoxicity toward normal HFB4 cells (IC50 = 97.41 µg/mL). In vivo evaluation using the Ehrlich ascites carcinoma (EAC) model in Swiss albino mice demonstrated that combined treatment with Jan-AuNPs and doxorubicin achieved a tumor growth inhibition of 97.33%. These findings establish Jania rubens-mediated biosynthesis as a sustainable and scalable platform for producing bioactive AuNPs and highlight the pivotal role of bioinformatics in guiding experimental cancer nanomedicine research. The results highlight Jan-AuNPs as a promising, safe, and multi-targeted nanotherapeutic candidate with significant potential for the management of prostate cancer.
Adaptive immune receptors (AIRs), including antibodies and T-cell receptors (TCRs), mediate antigen recognition and represent a major class of therapeutic biomolecules. Their unique architecture, combining conserved framework with highly diverse complementarity-determining regions (CDRs), poses challenges for structure prediction and design. Recent advances in deep learning have transformed these fields, yet AIR-antigen interactions remain difficult targets due to limited structural data, weak co-evolutionary signals, and conformational heterogeneity. Herein, we review recent progress in structure-based deep learning approaches for AIRs, including AIR-specific language models, structure prediction, and epitope-conditioned design. We discuss commonly used datasets, evaluation metrics, and sources of bias that complicate cross-study comparisons and highlight the need for improved benchmarks. We also review emerging generative design strategies such as inverse folding, diffusion-based backbone generation, sequence-space diffusion, and sequence-structure co-design, and outline key challenges, including accurate modeling of flexible CDR loops, reliable ranking of AIR-antigen complexes, and scalable epitope-specific AIR design.