Search Results (94)

Thermophilic cyanobacteria are key models for thermotolerance and a promising source of thermophilic bioresources. Yet the subcellular basis of their stress resilience remains poorly resolved. Here, we focus on intracellular polyphosphate (polyP)-rich granules, termed “stabilisomes,” which have been implicated in stress adaptation. The lack of a high-purity, structure-preserving isolation method has been a major technical bottleneck hindering the elucidation of this resilience mechanism. This study describes a robust, structure-preserving purification strategy, boosting the granule-to-protein yield by over 10,000-fold compared with conventional methods. The specificity and structural integrity of this method are supported by the specific enrichment of complex proteomic (937 proteins) and metabolomic (1076 metabolites) signatures. Building on this, subsequent quantitative analysis across cyanobacteria at 7 hot spring sampling sites revealed a conserved core chemical composition dominated by polyphosphate (~21–36%), proteins (~10–20%), amino acids (~7–18%), and lipid components (~12–21%). The variability in abundance across species suggests a dynamic adjustment of these stabilizing components consistent with specific micro-environmental conditions. This work provides a robust bioseparation platform for prokaryotic organelles, offering a critical tool for investigating cyanobacterial resilience and developing novel biomaterials. Full article

Oral squamous cell carcinoma (OSCC) is an aggressive disease with a glycoproteomically unmapped progression and a low five-year survival rate. Thus, the aim of this pilot study was to explore the N-glycosylation pattern differences in malignant, adjacent mucosal and healthy tissues in the context of OSCC. Oral mucosal soft tissue samples was obtained by incisional biopsy from five patients with OSCC, both from the malignant and the opposite healthy gingival sides, and from seven age-sex-matched healthy controls. The collected tissues were homogenized, followed by N-glycan profiling of the endoglycosidase-released and fluorophore-labeled carbohydrates using capillary electrophoresis with ultra-sensitive laser-induced fluorescent detection (CE-LIF). Six out of the twenty-two identified N-glycan structures, including glycogens, showed significant (p < 0.05) differences between the malignant tissue samples of the OSCC patients and the healthy controls. Comparing the healthy and the positive control oral mucosal samples, differences in four N-glycan structures were revealed, while only one alteration was observed between the N-glycan profiles of the malignant tumor and positive control samples. However, the results are presented descriptively, reflecting the limited sample size of the pilot study, it shows the potential of high-resolution CE-LIF-based glyocoanalytical protocol to be highly efficient and sensitive for glycobiomarker-based molecular diagnostics of oral malignant lesions. Full article

In the last half-century, capillary gel electrophoresis (CGE) became a versatile and high-performance analytical platform for the separation of complex biomolecular mixtures featuring rapid separations, high efficiency, and small sample consumption. Integrating a pore-size gradient mechanism in CGE makes it possible to achieve enhanced selectivity of polyionic macromolecules such as SDS-proteins and nucleic acids. This review provides a comprehensive overview of the theoretical foundations and operational principles of capillary pore-size gradient gel electrophoresis (CGGE), including the physicochemical basis of gradient formation, the influence of pore-size distributions on analyte mobility, and the challenges of generating stable, reproducible gradients in narrow-bore capillaries. Instrumental considerations such as capillary surface treatment, gradient filling and polymerization strategies, temperature and voltage control, detection modalities, and method-development frameworks are discussed in detail, emphasizing their critical impact on analytical performance and reproducibility. Key application areas in bioanalytical chemistry are highlighted, covering nucleic acid analysis and peptide/protein characterization. CGGE offers unique analytical advantages where fine molecular discrimination, tunable selectivity, and high resolution in a broad molecular weight range are required. Full article

Rambutan seed waste from fruit processing remains underutilized, while conventional biodiesel routes face high feedstock costs and food-versus-fuel concerns. This study investigated a novel catalyst-free process for biodiesel production from rambutan seed waste using supercritical ethanol and ethyl acetate as renewable reactants to valorize fruit by-products. Batch reactions on the semi-solid fraction of rambutan seed oil (RSO) were conducted at 15 MPa to evaluate the effects of temperature (275–375 °C), reactant-to-oil molar ratio (20:1–40:1), and reaction time (15–50 min) on fatty acid ethyl ester (FAEE) yield. Under optimal conditions, FAEE yields of 59.92 and 41.92% were obtained using ethanol (350 °C, 40:1, 30 min) and ethyl acetate (350 °C, 30:1, 40 min), respectively. However, severe conditions degraded unsaturated esters, revealing a conversion–stability trade-off. The ethanol system exhibited faster reaction kinetics and lower activation energy than ethyl acetate. Applying the optimized ethanol-based conditions to the liquid fraction of RSO, which contained a lower proportion of saturated fatty acids, resulted in a markedly improved FAEE yield of 94.16%. This study demonstrated a catalyst-free supercritical route for converting rambutan seed waste into biodiesel, advancing waste-to-energy strategies and circular bioeconomy. Full article

This study explored IgG N-glycosylation pattern differences in maternal and infant serum in the context of gestational diabetes mellitus (GDM). Serum samples from 15 mother–infant pairs were collected at 12 weeks postpartum and categorized according to maternal body mass index (BMI) and GDM status. The N-glycosylation patterns of the isolated IgG pools were analyzed by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). Descriptive comparison of the relative area percentage of IgG N-glycan structures revealed differences between the groups. Comparison of the maternal and infant sialo-form/neutral-form ratio (SF/NF) of the N-glycans suggested differences between control mothers and their children, as well as between obese mothers and their children. The maternal SF/NF ratio of IgG varied between the obese and normal-weight GDM mothers. The SF/NF ratios of IgG from the infants showed variation between infants of control mothers and infants of obese mothers, between infants of obese and infants of obese GDM mothers, and between infants of GDM with normal-weight and GDM with obese mothers. The observed differences in maternal and infant IgG N-glycosylation profiles suggest potentially selective placental transfer mechanisms. Full article

When iron oxide nanoparticles are incubated together with a biological broth, the biomolecules compete for the binding sites at the solid–liquid interface. At the same time, the biomass rearranges in suspension, building agglomerated structures. Despite general knowledge of the forces involved in bio–nano interactions, gaps remain in the understanding of how biomolecules organize themselves in solution and onto surfaces. This work examines biomolecule adsorption onto metal oxide surfaces with the goal of strengthening this understanding, essential in industrial and natural processes. We demonstrate nearly complete separation of proteins from a biotechnological suspension for non-oxidized and highly oxidized magnetic nanoparticles. Varying the nanoparticle-to-biomass ratio, we find, can lead to different separation patterns, i.e., that selectivity using bare, low-cost materials is possible. Furthermore, we explore how preliminary “passivation” with a biological corona only partially reduces the ability to separate total protein mass from a new suspension in subsequent incubation steps. The study underscores the crucial role of concentration gradients with regard to targets and binding sites as the primary determinant of separation capacity and of biomolecule behavior in solution, highlighting the potential for using bio–nano coronae as biomolecule carriers across diverse fields, including environmental, biomedical, pharmaceutical and nutritional applications. Full article

Poly-β-hydroxybutyrate (P(3HB)) represents a suitable alternative for plastic replacement, since it consists of intracellularly produced polyesters by different microorganisms including Bacillus thuringiensis (Bt). P(3HB) conserves most of the properties of petroleum-derived plastics; however, some drawbacks are the production costs, processing times, and bioseparation techniques, limiting its extended use. Bt has production advantages over other microorganisms, such as those growing in conventional or non-conventional substrates, with short periods of fermentation, which make it an interesting candidate to develop optimized production processes. In this work, we identified P(3HB) producers from 72 isolates of Bt, from which we selected four potential candidates. These isolates were cultivated under different carbon:nitrogen (C:N) ratios of 3, 7, 30, and 50 in a complex medium named (CM). Here, the best conditions for growth in Bt isolates were C:N 3 and 7 ratios, whereas for P(3HB) production they were C:N 7 and 30. Following this, an experiment in a bioreactor was conducted with isolate 81C with the selected C:N ratio of 30, where the produced P(3HB) achieved a maximum at 10 h. Fourier transform infrared spectroscopy (FTIR)was used to characterize flask and bioreactor cultures. It must be mentioned that although a higher concentration of medium was used, this did not improve P(3HB) accumulation. This research demonstrates that C:N ratios can differentially influence growth and P(3HB) accumulation in Bt isolates, which can serve as a reference to develop P(3HB) production processes using Bt as a microbial production platform. Full article

The development of ion exchange chromatography to polish biopharmaceuticals requires extensive experimental benchmarking. As part of the Design of Experiments (DoE), statistical models increased efficiency somewhat and are still state of the art; however, the capability to predict process conditions is limited due to their nature as interpolating models. Applying the DoE still requires numerous experiments and is constrained to the design space, posing a risk of missing the potential optimum. To make a leap in model-based process development, applying extrapolating models can tremendously extend the design space and also allow for process understanding and knowledge transfer. While existing chromatography modeling software explains experimental data, it often lacks predictive power for new conditions. In academic–industrial cooperation, we demonstrate a new high-fidelity model based on biophysics for developing ion-exchange chromatography in biomanufacturing, making it a general tool in rationalizing process development for the present demand of recombinant proteins and monoclonal antibodies and the emerging demand of new modalities. Using the new computational tool, we achieved predictability and attained high accuracy; with minimal experimental effort to calibrate the system, the mathematical model predicted sensitive process conditions, and even described product-related impurities, antibody charge variants. Thus, the computational tool can be deployed for process-by-design and material-by-design approaches. Full article

Circulating tumor cell enrichment and enumeration are advancing early detection of cancer, monitoring of therapy response, and even next-generation therapies. Efficiently capturing rare cells from complex biological fluids is essential in both diagnostic and therapeutic applications. EpCAM-positive tumor cells are specifically captured by utilizing covalently immobilized anti-EpCAM monoclonal antibodies onto the surface of chemically modified glass microbeads. To maximize the capture efficiency, bead geometry, immobilization conditions, flow rate, and anticoagulant dosage were systematically optimized. An in vitro flow-capture system was designed and used to evaluate the capture efficiency of the proposed technology by utilizing HTC116 colon cancer cell-spiked model media. The effect of substrate surface pretreatment was characterized by goniometry, while the capture performance was monitored by flow cytometry and fluorescent microscopy. The specific capture ability of the bioactive microbead substrate reached over 130,000 cells in the laboratory-scale cartridge (V(cartridge) = 2.6 cm³; m(bead) = 4 g). This capture efficiency suggests a promising rare-cell capture utilization of the proposed technology and may be used for research, diagnostic, and therapeutic purposes. In this paper, we reported on the development and feasibility test of a high-performance bioactive glass-microbead cell capture substrate. Due to the relevance and novelty of the reported results, with further development, the versatile platform technology presented could be readily implemented to capture tumor cells from complex biological samples and represent an additional complementary tool to existing cancer diagnostics and therapies. Full article

As a porous crystalline material, covalent organic frameworks (COFs) have attracted significant attention due to their extraordinary features, such as an ordered pore structure and excellent stability. Synthesized through the aldehyde amine condensation reaction, TpPa-1 COFs (Triformylphloroglucinol-p-Phenylenediamine-1 COFs) were blended with cellulose acetate (CA) to form a casting solution. The TpPa-1 COF/CA ultrafiltration membrane was then prepared using the non-solvent-induced phase inversion (NIPS) method. The influence of TpPa-1 COFs content on the hydrophilicity, stability and filtration performance of the modified membrane was studied. Due to the hydrophilic groups in TpPa-1 COFs and the network structure formed by covalent bonds, the modified CA membranes exhibited higher hydrophilicity and lower protein adsorption compared with the pristine CA membrane. The porous crystalline structure of TpPa-1 COFs increased the water permeation path in the CA membrane, improving the permeability of the modified membrane while maintaining an outstanding bovine serum albumin (BSA) rejection. Furthermore, the addition of TpPa-1 COFs reduced protein adsorption on the CA membrane and overcame the trade-off between permeability and selectivity in CA membrane bioseparation applications. This approach provides a sustainable method for enhancing membrane performance while enhancing the application of membranes in protein purification. Full article

Introduction: Obesity is a rapidly growing common health problem worldwide that can lead to the development of gestational diabetes mellitus (GDM). However, GDM not only affects women with obesity but can also develop at any time, even after the OGTT test; therefore, an increasing number of complications related to GDM can be seen in both mothers and their children. It is necessary to discover biomarkers capable of indicating the development of GDM or complications during/after pregnancy. Since the N-glycosylation motif of human IgG has been described to change under many physiological and pathological conditions, it is a promising target for biomarker research. In our study, the effects of obesity and GDM were investigated on human serum IgG N-linked glycosylation patterns during human pregnancy. Materials and Methods: The study participants were categorized into four groups according to their body mass index (BMI) and GDM status: normal weight as control, obese (BMI > 30 kg/m²), normal weight with GDM, and obese with GDM. The released N-glycan components of IgG were separated with capillary electrophoresis and detected using a laser-induced fluorescence detector. Results: The result revealed several differences between the N-glycosylation patterns of the four study groups. Of this, 17 of the 20 identified structures differed significantly between the groups. The ratios of sialylated to non-sialylated structures were not changed significantly, but the core fucosylation level showed a significant decrease in the GDM and obese GDM groups compared to the control subjects. The lowest degree of core fucosylation was observed in the GDM group. Conclusions: The findings indicate that obesity in isolation does not have a significant impact on the IgG N-glycosylation pattern in pregnancy. Conversely, alterations in the N-glycan profile of antibodies may serve as biomarkers for the diagnosis of GDM in mothers with a normal BMI, although more evidence is needed. By incorporating glycan-based biomarkers into clinical practice, healthcare providers can improve early detection, personalize management strategies, and potentially mitigate adverse pregnancy outcomes associated with obesity and GDM. Full article

Circulating tumor cells (CTCs) are pivotal in cancer metastasis and serve as valuable biomarkers for diagnosis, prognosis, and treatment monitoring. Traditional CTC capture methods predominantly utilize the epithelial cell adhesion molecule (EpCAM) as a marker for isolation. However, the heterogeneity of these circulating cells and the epithelial-to-mesenchymal transition process (wherein epithelial cells acquire mesenchymal characteristics) limit the efficacy of EpCAM-based capture techniques. In this paper, we critically review the role of the EpCAM in CTC capture, explore the impact of epithelial-to-mesenchymal transition on EpCAM expression, and discuss alternative biomarkers and strategies to enhance CTC isolation. By evaluating the limitations of EpCAM-mediated capture and the challenges posed by epithelial-to-mesenchymal transition, we aim to provide insights into the development of more comprehensive liquid biopsy approaches for cancer management. Full article