Search Results (29)

Pacific pygmy octopus Paroctopus digueti is a promising model for cephalopod research and aquaculture; its feeding and nutritional biology remain poorly understood. The anterior salivary glands (ASG), posterior salivary glands (PSG), and digestive gland (DG) are central to these processes, but molecular comparisons are lacking. To address this gap, we performed a transcriptomic study to explore the enzymatic repertoire and functional specialization of these tissues. Total RNA was extracted from ASG, PSG, and DG of three pre-adult individuals collected in La Paz Bay, Mexico. RNA-Seq libraries were sequenced, and a non-redundant multi-tissue transcriptome was assembled. The ASG displayed high expression of neuropeptides, playing a role in neuroendocrine regulation. The PSG showed elevated protease expression, supporting its function in extracellular digestion, alongside toxins that reinforce its role as a venom gland. The DG was enriched in proteins linked to biomolecule catabolism and antimicrobial peptides, alluding to metabolic specialization and immune defense. These results were validated by qPCR, and target genes were also amplified in Octopus maya and O. hubbsorum, showing some similarities in expression patterns. Overall, our findings suggest strong glandular specialization in P. digueti, providing insights into cephalopod digestive physiology and supporting its value as a model species. Full article

The growing global demand for clean energy and sustainability has increased interest in lignocellulosic biomass as a viable alternative to conventional fossil fuels. Among the various biomass resources, sugarcane bagasse, an abundant agro-industrial by-product, has emerged as a promising feedstock to produce renewable fuels and value-added chemicals. Its high carbohydrate content offers significant potential for bioconversion. However, its complex and recalcitrant lignocellulosic matrix presents significant challenges that necessitate advanced pretreatment techniques to improve enzymatic digestibility and fermentation efficiency. This review consolidates recent developments in the valorization of sugarcane bagasse focusing on innovative pretreatment and fermentation strategies for sustainable bioethanol production. It emphasizes the synergistic benefits of integrating various pretreatment and fermentation methods to improve bioethanol yields, reduce processing costs and enhance overall process sustainability. This review further explores recent technological advancements, the impact of fermentation inhibitor, and emerging strategies to overcome these challenges through microbial strains and innovative fermentation methods. Additionally, it highlights the multi-faceted advantages of bagasse valorization, including waste minimization, renewable energy production and the promotion of sustainable agricultural practices. By evaluating the current state of research and outlining future perspectives, this paper serves as a comprehensive guide to advancing the valorization of sugarcane bagasse in the transition towards a low-carbon economy. The novelty of this review lies in its holistic integration of technological, economic, and policy perspectives, uniquely addressing the scalability of integrated pretreatment and fermentation processes for sugarcane bagasse, and outlining practical pathways for their translation from laboratory to sustainable industrial biorefineries within the circular bioeconomy framework. Full article

The extensive use of antibiotics in animal husbandry leads to the release of unmetabolised residues and the dissemination of antimicrobial resistance genes (ARGs) in manure, posing environmental and public health challenges. Conventional treatment technologies, including hydrolysis, photodegradation, and phytoremediation, are often constrained by incomplete mineralisation, high cost, and environmental variability. Biocatalytic and microbially mediated processes are increasingly recognised as sustainable alternatives. Enzymes, which in clinical contexts confer resistance, can, in environmental matrices, catalyse the dismantling of antibiotic scaffolds, attenuating bioactivity and promoting detoxification. Catalytic classes such as hydrolases, transferases, and oxidoreductases mediate diverse transformations, including hydrolytic cleavage, functional group transfer, and oxidative modification. Microbial consortia and bioaugmentation further enhance biodegradation, while biochar and other amendments reduce ARG persistence. Advances in multi-omics, enzyme engineering, and immobilisation have expanded catalytic repertoires, improved stability, and enabled integration with composting, anaerobic digestion, and hybrid bioprocesses. Nonetheless, incomplete degradation, recalcitrant intermediates, and horizontal gene transfer remain challenges. Importantly, since degradation products may leach into soils and aquatic systems, optimising these processes is critical to prevent residues from entering the water cycle. This review synthesises advances in microbial and enzymatic degradation strategies, highlighting opportunities for sustainable manure management while mitigating water pollution risks. Full article

This study addressed the challenges posed by wet-grade maize distiller’s dried grains with solubles (DDGS), which are characterized by high moisture and complex fibers that limit their storage and utilization in poultry feed. Three experiments were conducted to enhance their nutritional value through enzymatic and solid-state fermentation treatments. In vitro pre-digestion using multiple enzymes significantly improved dry matter solubility (DMS) and reducing sugar yield for maize DDGS and the ingredient maize germ meal (MGM). Using optimized parameters, wet-based DDGS-MGM was subjected to solid-state fermentation with 500 mg/kg of cellulase and 200 mg/kg of the X1 enzyme (a laboratory-developed multi-enzyme complex), and this treatment enhanced both DMS and reducing sugar yield, and the resulting fermented product was subsequently applied in further experiments. In the broiler trial, forty 22-day-old Arbor Acres broilers with similar body weights were randomly assigned to five treatment groups, including the control group, (50% DDGS + 50% MGM) unfermented group, (62.5% DDGS + 37.5% MGM) unfermented group, (50% DDGS + 50% MGM) fermented group, and (62.5% DDGS + 37.5% MGM) fermented group, with eight replicates per treatment (one broiler per replicate). Replacement of 30% of the basal diet with fermented 50:50 DDGS-MGM material significantly increased apparent metabolizable energy (AME) and nitrogen-corrected AME by 2.74 MJ/kg and 2.73 MJ/kg, respectively, corresponding to improvements of 39.60% and 40.81% compared to the unfermented control (p < 0.05). Economic analysis indicated that using 5% fermented DDGS-MGM in feed reduced cost by 20.45 RMB per metric ton. These findings demonstrate that bioprocessing can improve the utilization and economic value of maize processing by-products, although further validation under practical conditions is needed. Full article

Gerronema lapidescens (Lei Wan), a valued medicinal basidiomycete traditionally employed for antiparasitic and digestive ailments, faces severe conservation threats due to unsustainable wild harvesting and the absence of reliable cultivation protocols. To address this crisis and unlock its pharmacotherapeutic potential, we present the first chromosome-scale genome assembly and comprehensive methylome profile for the wild strain G. lapidescens QL01, domesticated from the Qinling Mountains. A multi-platform sequencing strategy (Illumina and PacBio HiFi) yielded a high-quality 82.23 Mb assembly anchored to 11 chromosomes, exhibiting high completeness (98.4% BUSCO) and 46.03% GC content. Annotation predicted 15,847 protein-coding genes, with 81.12% functionally assigned. Genome-wide analysis identified 8.46 million high-confidence single-nucleotide polymorphisms (SNPs). Notably, methylation profiling revealed 3.25 million methylation events, with elevated densities on chromosomes 4, 9, and 10, suggesting roles in gene silencing and environmental adaptation. Phylogenomic analyses clarified the evolutionary status of G. lapidescens, whilst gene family evolution indicated moderate dynamics reflecting niche adaptation. Carbohydrate-Active enzymes (CAZymes) analysis identified 521 enzymes, including 211 Glycoside Hydrolases (GHs), consistent with organic matter degradation. Additionally, 3279 SSRs were catalogued as molecular markers. This foundational resource elucidates G. lapidescens’s genetic architecture, epigenetic regulation, evolutionary history, and enzymatic toolkit, underpinning future research into medicinal compound biosynthesis, environmental adaptation, germplasm conservation, and sustainable cultivation. Full article

The tomato leafminer (Tuta absoluta) is a globally invasive pest that causes severe yield losses in tomato crops. Nanotechnology-based strategies offer promising alternatives to conventional insecticides. This study examines the physiological, biochemical, and demographic responses of T. absoluta following exposure to mesoporous silica nanoparticles (MSNs) applied to tomato leaves at concentrations of 0, 3, 30, and 300 mg L⁻¹. Comprehensive assessments were conducted, including digestive and detoxifying enzyme activities in the insect, neurotoxicity indicators, life table parameters, and antioxidant responses in the host plant. At 30 mg L⁻¹, MSNs significantly impaired larval development, fecundity, and survival of T. absoluta without inducing phytotoxicity. Tomato plants treated at this concentration exhibited enhanced antioxidant enzyme activity (SOD, CAT, POD) and a reduced malondialdehyde (MDA) content, indicating an active oxidative defense. These plant responses were significantly correlated with changes in insect fitness traits, suggesting a plant-mediated effect on pest physiology. Digestive enzyme disruption, decreased acetylcholinesterase activity, and extended developmental periods contributed to suppressed population growth, as evidenced by reductions in the intrinsic rate of increase (r), net reproductive rate (R₀), and fecundity. At 300 mg L⁻¹, however, severe phytotoxicity and enzymatic collapse were observed in both plant and insect systems. These findings highlight moderate concentration of MSNs (30 mg L⁻¹) as a promising dose for sustainable and host-safe pest management, offering multi-targeted suppression of T. absoluta through combined plant and insect biochemical pathways. Full article

Scorpionism is a growing public health concern in Brazil, with the Amazon region presenting the highest mortality rates but remaining understudied, especially regarding local scorpion venoms composition. This study presents the first comprehensive biochemical characterization of venoms from three Amazonian species—Tityus metuendus (TmetuV), Tityus silvestris (TsilvV), and Brotheas amazonicus (BamazV)—using an integrated approach combining Multi-Enzymatic Limited Digestion (MELD)-based bottom-up proteomics, high-resolution LC-MS/MS, chromatography, zymography, and enzymatic assays. Tityus serrulatus venom was included as a reference. Significant biochemical differences were observed: TsilvV was rich in 20–30 kDa proteins and showed strong metalloprotease activity; BamazV exhibited high molecular weight proteins and potent phospholipase A₂ (PLA₂) activity but lacked proteolytic and fibrinogenolytic activities; TmetuV showed the highest hyaluronidase activity and abundance of α-KTx neurotoxins. Zymography revealed a conserved ~45 kDa hyaluronidase in all species. Three novel components were partially characterized: BamazPLA₂ (Group III PLA₂), Tmetu1 (37-residue α-KTx), and TsilvMP_A (a metalloprotease homologous to antarease). This is the first application of MELD-based proteomics to Amazonian scorpion venoms, revealing molecular diversity and functional divergence within Tityus and Brotheas, emphasizing the need for region-specific antivenoms. These findings provide a foundation for future pharmacological studies and the discovery of bioactive peptides with therapeutic potential. Full article

This study evaluated the effects of replacing SBM with CWYWEP on in vitro rumen fermentation, nutrient degradability, and gas production kinetics. Citric waste was co-fermented with yeast waste and a multi-enzyme complex for 14 days, then sun-dried and pelleted. The final CWYWEP product contained 50.4% crude protein (DM basis). A completely randomized design tested seven diets in which SBM was replaced by CWYWEP or non-enzymatic citric waste–yeast waste pellets (CWYWP) at 0%, 33%, 66%, or 100% inclusion. Replacing SBM with CWYWEP significantly increased cumulative gas production at 96 h, with the 100% CWYWEP group achieving 93.7 mL/0.5 g DM—a 14% increase over the control (p < 0.01). Microbial lag time was reduced to 0.17 h vs. 0.28 h in the control (p < 0.05), suggesting faster microbial colonization. The highest in vitro DM degradability (IVDMD) at 48 h was observed in the 100% CWYWEP group (64.5%), outperforming both the SBM control and all CWYWP treatments (p < 0.01). Notably, CWYWEP increased total volatile fatty acids by 5% at 4 h and propionate by 9% at 2 h, while reducing methane production by 5% (p < 0.05). Other parameters, including pH, ammonia nitrogen, organic matter digestibility, and protozoal counts, were unaffected (p > 0.05). In contrast, CWYWP without enzymes showed minimal improvement. These findings indicate that CWYWEP is a promising high-protein alternative to SBM, enhancing fermentation efficiency and reducing methane under in vitro conditions. Further in vivo studies are warranted to validate these effects. Full article

Obesity is a chronic epidemic caused by abnormal fat metabolism. As a key digestive enzyme, pancreatic lipase (PL) is an important target for regulating fat metabolism. The inhibitory potential of 5,10,15,20-Tetrakis (4-aminophenyl) porphyrin (TAPP), 5,10,15,20-Tetrakis (4-hydroxyphenyl) porphyrin (THPP), meso-Tetra (4-carboxyphenyl) porphine (TCPP), Cu (II) meso-Tetra (4-carboxyphenyl) porphine (Cu-TCPP) on PL was studied by enzymatic kinetics, multi-spectral, and molecular simulation technology. THPP, TCPP, TAPP, and Cu-TCPP all had good PL inhibitory activity (IC₅₀ range: 97.49–248.70 μM) and were uncompetitive inhibitors. The order of inhibitory ability was: THPP > TCPP > TAPP > Cu-TCPP. The fluorescence quenching mechanism of THPP to PL was a mixed quenching dominated by static quenching, while TCPP, TAPP, and Cu-TCPP were static quenching. The binding of THPP, TCPP and TAPP to PL was mainly driven by hydrogen bonds and van der Waals forces, while Cu-TCPP was mainly driven by a hydrophobic interaction. Four porphyrin compounds changed the conformation of PL, affected the microenvironment of Tyr and Trp residues, and induced changes in the secondary structure of PL, thereby reducing the stability and catalytic activity of PL. Hydrogen bonds played an important role in the binding stability of THPP, TCPP, TAPP, and PL. Full article

Snakebite envenoming constitutes a significant global health issue, particularly in Africa, where venomous species such as Echis vipers and Dendroaspis mambas pose substantial risks to human health. This study employs a standardized venomics workflow to comprehensively characterize and comparatively quantify the venom composition of nine medically relevant snake species chosen from among the deadliest in Africa. Utilizing shotgun venom proteomics and venom gland transcriptomics, we report detailed profiles of venom complexity, highlighting the relative abundance of dominant toxin families such as three-finger toxins and Kunitz-type proteins in Dendroaspis, and metalloproteinases and phospholipases A₂ in Echis. We delineate here the relative abundance and structural diversity of venom components. Key to our proteomic approach is the implementation of Multi-Enzymatic Limited Digestion (MELD), which improved protein sequence coverage and enabled the identification of rare toxin families such as hyaluronidases and renin-like proteases, by multiplying the overlap of generated peptides and enhancing the characterization of both toxin and non-toxin components within the venoms. The culmination of these efforts resulted in the construction of a detailed toxin database, providing insights into the biological roles and potential therapeutic targets of venom proteins and peptides. The findings here compellingly validate the MELD technique, reinforcing its reproducibility as a valuable characterization approach applied to venomics. This research significantly advances our understanding of venom complexity in African snake species, including representatives of both Viperidae and Elapidae families. By elucidating venom composition and toxin profiles, our study paves the way for the development of targeted therapies aimed at mitigating the morbidity and mortality associated with snakebite envenoming globally. Full article

Cellulosomes are sophisticated multi-enzyme complexes synthesized and secreted by anaerobic microorganisms, characterized by intricate structural components and highly organized modular assembly mechanisms. These complexes play a pivotal role in the efficient degradation of lignocellulosic biomass, significantly enhancing its bioconversion efficiency, and are thus regarded as invaluable enzymatic molecular machines. Cellulosomes are not only prevalent in anaerobic bacteria from soil and compost environments but are also integral to the digestive systems of herbivorous animals, primates and termites. The cellulosomes produced by digestive tract microbiota exhibit unique properties, providing novel enzymes and protein modules that are instrumental in biomass conversion and synthetic biology, thereby showcasing substantial application potential. Despite their promise, the isolation and cultivation of digestive tract microorganisms that produce cellulosomes present significant challenges. Additionally, the lack of comprehensive genetic and biochemical studies has impeded a thorough understanding of these cellulosomes, leaving them largely underexplored. This paper provides a comprehensive overview of the digestive tract cellulosome system, with a particular focus on the structural and functional attributes of cellulosomes in various animal digestive tracts. It also discusses the application prospects of digestive tract cellulosomes, highlighting their potential as a treasure in diverse fields. Full article

Background/Objectives: Multi-dimensional liquid chromatography coupled with mass spectrometry (mD-LC-MS) has emerged as a powerful technique for the in-depth characterization of biopharmaceuticals by assessing chromatographically resolved product variants in a streamlined and semi-automated manner. The study aims to demystify and enhance the accessibility to this powerful but inherently complex technique by detailing a robust and user-friendly instrument platform, allowing analysts to switch seamlessly between intact, subunit, and peptide mapping workflows. Methods: Starting from a commercially available Two-Dimensional Liquid Chromatography (2D-LC) system, we introduce specific hardware and software extensions leading to two versatile mD-LC-MS setups, in slightly different configurations. The technique’s efficacy is demonstrated through a case study on a cation exchange chromatography method assessing the charge variants of a bispecific antibody, isolating peak(s) of interest, followed by online sample processing, including reduction and enzymatic digestion, and subsequently mass spectrometry analysis. Results: The accuracy and reproducibility of both mD-LC-MS setups proposed in this study were successfully tested. Despite the complex peak patterns in the first dimension, the systems were equally effective in identifying and quantifying the underlying product species. This case study highlights the routine usability of mD-LC-MS technology for the characterization of (ultra) high-performance liquid chromatography (UHPLC) of therapeutic biomolecule. Conclusions: The demonstrated reliability and accuracy underscore the practicality of mD-LC-MS for routine use in biopharmaceutical analysis. Our detailed description of the mD-LC-MS systems and insights simplify access to this advanced technology for a broader scientific community, regardless of expertise level, and lower the entry barrier for its use in various research and industrial settings. Full article

Porous starch has been created through hydrolysis by amyloglucosidase and α-amylase. However, little information is known about the precise evolution of multi-scale structures of starch during digestion. In this study, rice starch and potato starch, containing different crystalline structures, were hydrolyzed by amyloglucosidase and α-amylase for 20 and 60 min, respectively, and their resulting structural changes were examined. The digestion process caused significant degradation of the molecular structures of rice and potato starches. In addition, the alterations in the ordered structures varied between the two starches. Rice starch exhibited porous structures, thicker crystalline lamellae as determined by small-angle X-ray scattering, and enhanced thermostability after digestion using differential scanning calorimetry. For rice starch, the extent of crystalline structures was analyzed with an X-ray diffractometer; it was found to first increase after 20 min of digestion and then decrease after 60 min of digestion. In contrast, potato starch did not display porous structures but exhibited thicker crystalline lamellae and a reduction in ordered structures after digestion. These findings suggest that it is possible to intentionally modulate the multi-scale structures of starch by controlling the digestion time, thereby providing valuable insights for the manipulation of starch functionalities. Full article

The Cystic Fibrosis Conductance Transmembrane Regulator gene encodes for the CFTR ion channel, which is responsible for the transport of chloride and bicarbonate across the plasma membrane. Mutations in the gene result in impaired ion transport, subsequently leading to perturbed secretion in all exocrine glands and, therefore, the multi-organ disease cystic fibrosis (CF). In recent years, several studies have reported on CFTR expression in immune cells as demonstrated by immunofluorescence, flow cytometry, and immunoblotting. However, these data are mainly restricted to single-cell populations and show significant variation depending on the methodology used. Here, we investigated CFTR transcription and protein expression using standardized protocols in a comprehensive panel of immune cells. Methods: We applied a high-resolution Western blot protocol using a combination of highly specific monoclonal CFTR antibodies that have been optimized for the detection of CFTR in epithelial cells and healthy primary immune cell subpopulations sorted by flow cytometry and used immortalized cell lines as controls. The specificity of CFTR protein detection was controlled by peptide competition and enzymatic Peptide-N-Glycosidase-F (PNGase) digest. CFTR transcripts were analyzed using quantitative real-time PCR and normalized to the level of epithelial T84 cells as a reference. Results: CFTR mRNA expression could be shown for primary CD4⁺ T cells, NK cells, as well as differentiated THP-1 and Jurkat T cells. In contrast, we failed to detect CFTR transcripts for CD14⁺ monocytes and undifferentiated THP-1 cells, as well as for B cells and CD8⁺ T cells. Prominent immunoreactive bands were detectable by immunoblotting with the combination of four CFTR antibodies targeting different epitopes of the CFTR protein. However, in biosamples of non-epithelial origin, these CFTR-like protein bands could be unmasked as false positives through peptide competition or PNGase digest, meaning that the observed mRNA transcripts were not necessarily translated into CFTR proteins, which could be detected via immunoblotting. Our results confirm that mRNA expression in immune cells is many times lower than in that cells of epithelial origin. The immunoreactive signals in immune cells turned out to be false positives, and may be provoked by the presence of a high-affinity protein with a similar epitope. Non-specific binding (e.g., Fab-interaction with glycosyl branches) might also contribute to false positive signals. Our findings highlight the necessity of accurate controls, such as CFTR-negative cells, as well as peptide competition and glycolytic digest in order to identify genuine CFTR protein by immunoblotting. Our data suggest, furthermore, that CFTR protein expression data from techniques such as histology, for which the absence of a molecular weight or other independent control prevents the unmasking of false positive immunoreactive signals, must be interpreted carefully as well. Full article

Undifferentiated germ cells, including the spermatogonial stem cell subpopulation required for fertility restoration using human immature testicular tissue (ITT), are difficult to recover as they do not easily adhere to plastics. Due to the scarcity of human ITT for research, we used neonatal porcine ITT. Strategies for maximizing germ cell recovery, including a comparison of two enzymatic digestion protocols (P1 and P2) of ITT fragment sizes (4 mm³ and 8 mm³⁾ and multi-step differential plating were explored. Cellular viability and yield, as well as numbers and proportions of DDX4+ germ cells, were assessed before incubating the cell suspensions overnight on uncoated plastics. Adherent cells were processed for immunocytochemistry (ICC) and floating cells were further incubated for three days on Poly-D-Lysine-coated plastics. Germ cell yield and cell types using ICC for SOX9, DDX4, ACTA2 and CYP19A1 were assessed at each step of the multi-step differential plating. Directly after digestion, cell suspensions contained >92% viable cells and 4.51% DDX4+ germ cells. Pooled results for fragment sizes revealed that the majority of DDX4+ cells adhere to uncoated plastics (P1; 82.36% vs. P2; 58.24%). Further incubation on Poly-D-Lysine-coated plastics increased germ cell recovery (4.80 ± 11.32 vs. 1.90 ± 2.07 DDX4+ germ cells/mm², respectively for P1 and P2). The total proportion of DDX4+ germ cells after the complete multi-step differential plating was 3.12%. These results highlight a reduced proportion and number of germ cells lost when compared to data reported with other methods, suggesting that multi-step differential plating should be considered for optimization of immature germ cell recovery. While Poly-D-Lysine-coating increased the proportions of recovered germ cells by 16.18% (P1) and 28.98% (P2), future studies should now focus on less cell stress-inducing enzymatic digestion protocols to maximize the chances of fertility restoration with low amounts of cryo-banked human ITT. Full article