Search Results (68)

The ancestral method of sparkling wine production requires no sugar addition and is based on a single alcoholic fermentation that finishes in the bottle. It has recently gained popularity among both natural and conventional producers due to its capacity to expand sparkling wine portfolios and its advantages for sparkling wine production in warm regions such as the Mediterranean basin. However, some technological and other key aspects of the process remain poorly defined. This study investigates how must settling (using pectolytic enzymes) and pre-bottling filtration influence the physicochemical composition, colloidal structure, foaming properties, volatile profile, and sensory attributes of ancestral sparkling wines. Macabeo grapes (2023 vintage) were vinified under four treatment combinations of these two operations, and the wines were analysed after eighteen months of lees ageing. Significant differences were observed in total polyphenol index, titratable acidity, macromolecular composition, and colloidal particle concentration. Non-settled wines exhibited higher colloidal loads and lower foamability, whereas settling and filtration improved foam height and stability. Volatile (GC-Headspace) analysis showed that settled wines contained higher concentrations of fruity and floral esters, while non-settled and non-filtered wines had greater levels of volatile phenols and volatile acids associated with unpleasant aromas. Sensory assessment confirmed perceptible differences between treatments, particularly in phenolic, fruity/floral, and Maillard reaction aromaticity. These findings provide insights into the impact of two critical oenological practices on wine stability and aroma and provide technologically relevant guidance for optimizing quality in this emerging sparkling-wine category. Full article

Background: Although prolonged inflammatory symptoms are an infrequent and problematic adverse effect of vaccination that can occur in some people, the transient activation of acute phase reactants (APRs) is expected with adjuvanted vaccines and helps to potentiate immune responses. Methods: This experiment examined the association between vaccine reactogenicity and immunogenicity in monkeys immunized with an adjuvanted recombinant protein including a receptor binding domain–human IgG1-Fc fusion protein (RBD-Fc) sequenced from the ancestral Wuhan strain of SARS-CoV-2. The acute inflammatory reaction to immunization was assessed by determining the decline in serum iron levels at 24 h and the increase in the neutrophil-to-lymphocyte ratio (NLR) as the adherent neutrophil pool trafficked into circulation. Results: Robust primary and secondary antibody responses were elicited. Larger decreases in serum iron and higher NLRs were associated with a stronger inhibition of RBD binding with angiotensin-converting enzyme (ACE2) when five early viral variants of SARS-CoV-2 were tested, including Wuhan, Alpha, Beta, Gamma and Delta. Inhibition of ACE2-RBD binding was less evident when the Omicron variant was tested. Individual variation in the APR was also predictive of the persistence of cell-mediated immunity based on the number of interferon-expressing mononuclear cells activated by viral antigen in ELISpot assays. Conclusions: Rapid antibody responses to primary immunization and large secondary responses to booster immunizations were elicited by this adjuvanted recombinant RBD-Fc vaccine, and our analysis affirmed the view that a transient APR can enhance antibody binding with antigen proteins. Full article

γ-Aminobutyric acid (GABA), a vital bioactive component, is biosynthesized via the decarboxylation of L-glutamate (L-Glu) catalyzed by glutamate decarboxylase (GAD). However, the GADs from various sources commonly suffer from low thermal stability, which hampers their industrial applications. In this work, four ancestral sequences of GAD (Anc19, Anc20, Anc28, and Anc30) were designed via an ancestral sequence reconstruction (ASR) approach. Thereafter, the genes were synthesized and heterologously expressed in the probiotic Escherichia coli strain Nissle 1917 (EcN). Among all variants tested, Anc28 exhibited the highest catalytic performance. The K_m and k_cat values were determined to be 26.80 mM and 57.41 s⁻¹, respectively, yielding a catalytic efficiency (k_cat/K_m) of 2.14 s⁻¹mM⁻¹, which was 2.71-fold higher than that of the wild-type enzyme. Meanwhile, compared with the wild-type GAD, Anc28 exhibited a 6.74 °C increase in T₅₀¹⁵ and a 4.1-fold extension in t_1/2 at 60 °C. Furthermore, the GABA synthesis system using dormant Escherichia coli Nissle (T7)/pET28a-gadB_Anc28 cells as the biocatalyst and pure water as a sole medium was also constructed. Upon completion of the 4 h reaction, the GABA titer reached 307.53 g/L with a conversion ratio of 99.36%. The resulting engineered strains were successfully employed for the efficient biosynthesis of GABA. Full article

One potential strategy to mitigate the detrimental effects of heat stress on corals is upwelling, which brings deep, cold, nutrient-rich water to the reef surface, creating transient cooling. However, cold temperatures can also stress corals, and it is, therefore, important to understand the mechanisms of both cold and heat stress responses in corals. Similar to how mammals activate thermogenic and adaptive metabolic pathways, corals may also regulate energy and redox metabolism under fluctuating environmental conditions. Guanosine monophosphate reductase (GMPR), a conserved enzyme in purine metabolism, plays a critical role in maintaining intracellular adenine and guanine nucleotide balance. To study this enzyme in corals, we expressed Pocillopora damicornis (PD) GMPR heterologously in Escherichia coli and purified the recombinant protein using nickel–NTA affinity chromatography. SDS-PAGE analysis showed a single band corresponding to the expected molecular weight, indicating high purity. Sequence alignment revealed ~70% identity with mammalian GMPR2 orthologs, suggesting evolutionary conservation of function. Structural modeling and phylogenetic analysis positioned PD GMPR between the GMPR1 and GMPR2 clades, suggesting it may represent an ancestral or functionally intermediate variant. Kinetic analysis determined Km values of 33.76 ± 6.44 μM for GMP and 17.71 ± 0.99 μM for NADPH under fixed substrate concentrations. This study provides the first biochemical characterization of GMPR, which may open the door to uncovering mechanisms of cold tolerance in corals and inform strategies to enhance coral resilience in the face of climate change. Full article

Tocosh, an ancestral fermented potato product, relies on spontaneous processes near freshwater springs under extreme high-altitude conditions and represents an underexplored reservoir of microbial diversity with significant potential for the discovery of probiotics. This study provides, for the first time, a comprehensive probiogenomic characterization of 19 lactic acid bacteria (LAB) isolated from tocosh, in the Peruvian Andes, at three distinct altitudes—2992, 3882, and 4451 m above sea level (m.a.s.l.)—using whole genome sequencing (WGS) and bioinformatic profiling. A total of six species were identified: Lactiplantibacillus plantarum and Levilactobacillus brevis at all three study sites, Lacticaseibacillus paracasei and Lentilactobacillus buchneri at the lowest altitude (2992 m.a.s.l.), and Latilactobacillus curvatus and Latilactobacillus sakei at the highest altitudes (3882 and 4451 m.a.s.l.). Our results reveal that the extreme Andean environment is associated with stability in L. plantarum (genome sizes from 3.36 to 3.38 Mb) across all altitudinal levels. Functional analysis using CAZymes determined that L. brevis and L. buchneri act as primary degraders (high percentage of glycosyl hydrolases/carbohydrate binding) while L. curvatus and L. sakei function as primary builders through exopolysaccharide biosynthesis, likely a cryoprotective adaptation preventing cell damage during cold temperatures at high altitudes. Additionally, L. sakei and L. plantarum exhibited unique auxiliary activity (AA) enzymes, suggesting an oxidative mechanism to breach recalcitrant starch surfaces. All isolates were confirmed as genomically safe, lacking transferable antibiotic resistance genes and virulence factors. Pathogenic risk potential scores (PPRS) were consistently ≤2.0, fulfilling qualified presumption of safety (QPS) criteria. These findings provide the first genomic characterization of tocosh-associated LAB, establishing a basis for tocosh standardization, enabling the rational design of starter cultures that preserve ancestral traits and ensure microbiological safety in modern food applications. Full article

The α-gal epitope is synthesized in non-primate mammals and New-World monkeys by the glycosylation enzyme α1,3galactosyltransferase (α1,3GT), encoded by the GGTA1 gene. Ancestral Old-World monkeys and apes synthesizing α-gal epitopes underwent extinction 20–30 million years ago. Their mutated offspring, with the inactivated GGTA1 gene, survived and produced the natural anti-Gal antibody, specifically binding α-gal epitopes. Anti-Gal protected the surviving offspring from lethal viruses presenting α-gal epitopes, which killed α-gal-synthesizing parental primates. Anti-Gal constitutes ~1% of human immunoglobulins and is also produced in Old-World monkeys and apes. α-Gal epitopes can serve as therapeutic agents in several clinical disciplines: 1. Cancer immunotherapy: Engineering cancer cells to express α-gal epitopes results in anti-Gal binding to these cells and localized activation of the complement system that kills these cancer cells and recruits the antigen-presenting cells (APCs) dendritic cells and macrophages. Anti-Gal bound to cancer cells targets them for robust uptake by APCs, which process internalized tumor antigens (TAs) and transport them to lymph nodes for activation of cytotoxic T-cells. These T-cells kill TA-presenting metastatic tumor cells. Clinical trials demonstrated that such engineering is achieved by intra-tumoral injection of α-gal glycolipids, the use of recombinant α1,3GT, or the use of oncolytic viruses containing the GGTA1 gene. 2. Viral vaccines: Inactivated whole-virus vaccines presenting α-gal epitopes bind anti-Gal, which targets them for extensive uptake by APCs, thereby increasing their immunogenicity by ~100-fold. 3. Injured-tissue regeneration: Anti-Gal binding to α-gal-presenting nanoparticles administered to wounds, into the post-myocardial infarction (MI) injured myocardium and into injured spinal cord, activates the complement system that recruits pro-regenerative macrophages, which orchestrate regeneration by recruiting stem cells and the secretion of pro-regenerative cytokines. All these findings suggest that α-gal/anti-Gal antibody interaction can serve as a novel therapeutic approach, applicable to various clinical settings. Full article

The first forms of photosynthetic life date back to more than 3.2–3.5 billion years ago. They performed photosynthesis in an anoxic world of the early earth and formed large mass accumulations on any kind of surface and in shallow waters. During evolution they diverged into several major phyla with differences in structure and function of the photosynthetic machinery including two different types of photosynthetic reaction centers. The combination of both of these types eventually led to the establishment of oxygenic photosynthesis present in cyanobacteria and chloroplast-containing plants. Common to all photosynthetic life is the presence of phylogenetically related reaction center proteins and of various chlorophyll molecules as mediators of light energy transformation into biochemical forms of energy. Therefore, we used phylogenetic analyses of reaction center proteins and representative enzymes of (bacterio)chlorophyll biosynthesis in addition to synteny of genome organization to unravel early divergencies of all known phyla of photosynthetic prokaryotes: Cyanobacteria, Chloroflexi, Chlorobi, Heliobacteria, Chloracidobacteria, and phototrophic purple Proteobacteria including Gemmatimonas. It was concluded that photosynthesis evolved only once, and all known forms diverged by various mechanisms from one primordial principal ancestor with properties resembling an ancestral cyanobacterium driving anoxygenic photosynthesis with a Type I photosystem and reduced inorganic electron donors. Full article

The bifunctional glutathione synthase (GshF) is able to catalyze glutathione synthesis and is favored for industrial application due to its lack of product inhibition. However, its practical use is limited by moderate catalytic efficiency and poor thermostability. Here, we applied ancestral sequence reconstruction (ASR) to engineer a more robust ancestral GshF (Anc427) with thermal denaturation temperature of 56.2 ± 0.2 °C, representing an increase of 10.8 ± 0.2 °C over the probe enzyme (St-GshF). Additionally, Anc427 exhibited a thermal half-life (t_1/2) of 3465.7 min at 40 °C, representing a 20-fold increase over that of St-GshF. Under optimal conditions (pH 7.0, 37 °C), Anc427 displayed a specific activity of 3.3 ± 0.02 U·mg⁻¹, representing a 20% enhancement compared to St-GshF. Structural modeling and molecular dynamics simulations indicated that the improved stability can be attributed to increased structural rigidity in Anc427. These findings demonstrate that ASR effectively enhances both thermostability and catalytic activity of GshF, significantly advancing its potential for industrial biocatalysis. Full article

Ticks transmit diseases and harm animals worldwide, and their control primarily relies on pesticides. Resistance to these pesticides has developed consistently over centuries. Arginine Kinase (AK, EC 2.7.3.3) is a conserved, ancestral enzyme that provides reserve energy in emergency situations and a viable target for novel antiparasitic drugs. Our aim was to evaluate six carbamoyl carboxylic acid analogues (CCAs) as potential lead compounds by investigating their interaction with the active site of Rhipicephalus sanguineus AK (RsAK) using a structural modeling approach. The binding was characterized using fluorescence quenching (Stern–Volmer analysis) and molecular dynamics simulations. The simulations, performed with GROMACS using the CHARMM 26 force field over 100 ns, provided atomic-level insight into the ligand–protein interactions and stability. CCA4 exhibited the lowest dissociation constant (K_D~13·10⁻⁶ M) among the analogues, which we attribute to its end moieties (carboxylate and a pyridine on the ends). Purely aromatic ends (CCA1) or those with dual carboxylates (CCA6) showed lower affinity, suggesting that electrostatic complementarity and steric fit are processes involved in the binding. Despite requiring optimization, the CCA scaffold represents a novel strategy for tick control. These compounds provide a foundation for developing synergistic agents to enhance the efficacy of sustainable acaricides. Full article

Background/Objectives: In Ecuador, the prevalence of type 2 diabetes mellitus (T2DM) is the second leading cause of death after ischemic heart disease. Genetic variability in protein-coding genes, single nucleotide variants (SNVs), influences the response to antidiabetic drugs. The frequency of SNVs varies among different populations, so studying the ancestral proportions among SNVs is important for personalized medicine in the treatment of T2DM. This study aimed to evaluate the distribution of Native American, European, and African (NATAM, EUR, and AFR) ancestry in 23 allelic variants of the seven genes that encode the relevant enzymes that metabolize antidiabetic drugs in an Ecuadorian population. Methods: Twenty-three allelic variants of seven genes were analyzed in 297 patients with T2DM from Ecuador, and the molecular ancestry of the samples was analyzed considering three ancestral groups, NATAM, EUR, and AFR using 90 ancestry informative markers (AIMs). Allele and ancestry distributions were analyzed using Spearman’s correlation. Results: The Ecuadorian population presents NATAM (61.33%), EUR (34.48%), and AFR (2.60%) ancestry components. CYP2C8*1 and CYP2C9*1 were positively related to NATAM ancestry, while CYP2C8*4 and CYP2C9*2 were positively related to EUR ancestry. CYP2C19*17 was positively correlated to AFR ancestry. The correlation of SLC22A1 variants such as A in rs594709 was positively correlated with NATAM, while GAT in rs72552763 was positive for EUR. The G variant of rs628031 of the SLC22A1 gene was positively correlated with NATAM and negatively correlated with EUR. The C variant of rs2076828 of the SLC22A3 gene was positively correlated with NATAM ancestry. Conclusions: In the Ecuadorian population, a predominance of Native American ancestry has been observed. Among the allelic variants related to enzymes that metabolize antidiabetic drugs, a relationship has been observed between this ancestral component and variants of the CYP2C8*1, CYP2C9*1, SLC22A1 (rs594709 and rs628031), and SLC22A3 (rs2076828) genes. This information is fundamental for the development of strategies for the implementation of personalized medicine programs for Latin American patients. Full article

Xyloglucan endotransglucosylase/hydrolases (XTHs) are a class of cell wall-associated enzymes involved in the construction and remodeling of cellulose/xyloglucan crosslinks. However, knowledge of this gene family in the model monocot Brachypodium distachyon is limited. A total of 29 BdXTH genes were identified from the whole genome, and these were further divided into three subgroups (Group I/II, Group III, and the Ancestral Group) through evolutionary analysis. Gene structure and protein motif analyses indicate that closely clustered BdXTH genes are relatively conserved within each group. A highly conserved amino acid domain (DEIDFEFLG) responsible for catalytic activity was identified in all BdXTH proteins. We detected three pairs of segmentally duplicated BdXTH genes and five groups of tandemly duplicated BdXTH genes, which played vital roles in the expansion of the BdXTH gene family. Cis-elements related to hormones, growth, and abiotic stress responses were identified in the promoters of each BdXTH gene, and when roots were treated with two abiotic stresses (salinity and drought) and four plant hormones (IAA, auxin; GA3, gibberellin; ABA, abscisic acid; and BR, brassinolide), the expression levels of many BdXTH genes changed significantly. Transcriptional analyses of the BdXTH genes in 38 tissue samples from the publicly available RNA-seq data indicated that most BdXTH genes have distinct expression patterns in different tissues and at different growth stages. Overexpressing the BdXTH27 gene in Brachypodium led to reduced root length in transgenic plants, which exhibited higher cellulose levels but lower hemicellulose levels compared to wild-type plants. Our results provide valuable information for further elucidation of the biological functions of BdXTH genes in the model grass B. distachyon. Full article

Background/Objectives: The plant species Erophaca baetica (L.) Boiss. (synonym: Astragalus lusitanicus Lam.) is found essentially around the Mediterranean basin, with Morocco as its ancestral territory. The foliage of E. baetica is toxic to small ruminants, and for this reason the plant is often eliminated by farmers, despite its ecological and medicinal potential. The phytochemicals at the origin of the toxicity of E. baetica are not precisely known, but several potentially toxic products have been identified. In particular, aliphatic nitro compounds are present in the aerial parts of the plant, such as 3-nitro-propionic acid (NPA) and its precursor 3-nitro-propanol (NPOH) which are most likely at the origin of the plant toxicity. Results: The present review provides a detailed analysis of the nitrotoxins isolated from E. baetica and their mechanism of action. The covalent targeting of metabolic enzymes such as isocitrate lyase and succinate dehydrogenase by NPA is discussed. The mitochondrial chain blocker NPA is most likely responsible for the brain toxicity of E. baetica, but the presence of other potentially toxic chemicals—such as lusitoxamine and lusitoxamide—is also discussed. Conclusions: This review shed light on the widespread but little-known Mediterranean plant E. baetica and the phytochemicals responsible for the plant’s toxicity. Full article

The evolution of SARS-CoV-2, particularly the emergence of Omicron variants, has raised questions regarding changes in its binding affinity to the human angiotensin-converting enzyme 2 receptor (hACE2). Understanding the impact of mutations on the interaction between the receptor-binding domain (RBD) of the spike protein and hACE2 is critical for evaluating viral transmissibility, immune evasion, and the efficacy of therapeutic strategies. Here, we used molecular dynamics (MD) simulations and binding energy calculations to investigate the structural and energetic differences between the hACE2- RBD complexes of wild-type (WT), Delta, and Omicron subvariants. Our results indicate that the Delta and the first Omicron variants showed the highest and the second-highest binding energy among the variants studied. Furthermore, while Omicron variants exhibit increased structural stability and altered electrostatic potential at the hACE2–RBD interface when compared to the ancestral WT, their binding strength to hACE2 does not consistently increase with viral evolution. Moreover, newer Omicron subvariants like JN.1 exhibit a bimodal conformational strategy, alternating between a high-affinity state for hACE2 and a low-affinity state, which could potentially facilitate immune evasion. These findings suggest that, in addition to enhanced hACE2 binding affinity, other factors, such as immune evasion and structural adaptability, shape SARS-CoV-2 evolution. Full article

The Kelch-like ECH-associated protein 1 (KEAP1)/Nuclear factor E2-related factor 2 (NRF2) pathway is a key mechanism that responds to oxidative stress and xenobiotic stimuli in vertebrates. However, knowledge of its evolutionary origins remains limited. In this study, we identify the ancestral homologues of KEAP1 and NRF (BjKEAP1 and BjNRF) in cephalochordate amphioxus (Branchiostoma japonicum). BjNRF uniquely combines the feature domains of vertebrates NRF1 and NRF2, marking it as an evolutionary intermediate. High expression levels of Bjkeap1 and Bjnrf in the gill, hepatic cecum, and intestine highlight their roles in environmental defense at key interface tissues. Functional studies reveal that BjKEAP1 regulates the cytoplasmic localization of BjNRF. Typical NRF2 activator sulforaphane (SFN) induces its nuclear translocation and significantly elevates the transcriptional expression of BjNRF and phase II detoxification enzymes. Moreover, exposure to the environmental toxin Benzo[a]pyrene (BaP) activates this stress response system. These findings bridge critical gaps in our understanding of this pathway in basal chordates and offer new insights into the evolutionary trajectory of the KEAP1-NRF system. Furthermore, this study highlights crucial implications for the conservation of amphioxus in deteriorating marine environments. Full article

Glycoside hydrolase family 11 (GH11) xylanases are used in various industries, such as biorefining, animal feed production, and baking, making them key industrial enzymes. Operating bioprocesses at elevated temperatures enhances the reaction rate and product yield and thus requires thermostable enzymes to sustain catalytic performance. The limited availability of naturally occurring thermostable GH11 xylanases necessitates targeted modifications via protein engineering to enhance their thermal stability. In this review, we present the key drivers of thermostability, an overview of engineering strategies, and the underlying mechanisms of action. Finally, we investigated state-of-the-art technologies involving artificial intelligence (AI)- and ancestral sequence reconstruction-guided approaches. Full article