Search Results (50)

Lactobacillus-enriched yogurt is in increasingly high demand due to its health benefits, but the product stability requires an understanding of the microbial dynamics during fermentation and storage. This study investigated the interactions between probiotic pairs (L. paracasei L9 and L. acidophilus LAC) and starter culture (HYY) through fermentation kinetics, microbial viability, organic acid profiles, and metabolomics. The results demonstrated that L. paracasei L9 significantly increased the titratable acidity from 25.20 ± 7.01 °T to 36.56 ± 3.47 °T at 3 h and reduced the fermentation time by 0.5 h, whereas L. acidophilus LAC showed minimal effects. L. paracasei L9 achieved higher viability (8.4 lg CFU/g) via the high-affinity lactose transport and Leloir pathway, whereas the L. acidophilus LAC growth remained limited (6.9 lg CFU/g). The metabolomic investigation revealed the L9 + HYY upregulated glycerophospholipid metabolism and pantothenate/CoA biosynthesis to support rapid biomass accumulation. In contrast, LAC + HYY modulated the arginine and branched-chain amino acid metabolism for acid tolerance. During 21 days of storage, there were no significant differences in final TA values and lactic acid content among the probiotic supplementation groups. L9 + HYY remained stable (>9.0 lg CFU/g) by upregulating the aromatic amino acid biosynthesis and suppressing the purine/sulfur metabolism, whereas L. acidophilus LAC decreased to 6.02 lg CFU/g. These findings demonstrate the dual role of L. paracasei L9 in accelerating the fermentation and maintaining the microbial stability through metabolic reprogramming, which guides the development of improved probiotic yogurts. Full article

Background/Objectives: Alzheimer’s disease (AD), a leading cause of dementia, lacks effective long-term treatments. Current therapies offer temporary relief or fail to halt its progression and are often inaccessible due to cost. AD involves multiple pathological processes, including amyloid beta (Aβ) deposition, insulin resistance, tau protein hyperphosphorylation, and systemic inflammation accelerated by gut microbiota dysbiosis originating from a leaky gut. Given this context, exploring alternative therapeutic interventions capable of addressing the multifaceted components of AD etiology is essential. Methods: This study suggests D-Pinitol (DPIN) as a potential treatment modifier for AD. DPIN, derived from carob pods, demonstrates insulin-sensitizing, tau hyperphosphorylation inhibition, and antioxidant properties. To test this hypothesis, we studied whether chronic oral administration of DPIN (200 mg/kg/day) could reverse the AD-like disease progression in the 5×FAD mice. Results: Results showed that treatment of 5×FAD mice with DPIN improved cognition, reduced hippocampal Aβ and hyperphosphorylated tau levels, increased insulin-degrading enzyme (IDE) expression, enhanced pro-cognitive hormone circulation (such as ghrelin and leptin), and normalized the PI3K/Akt insulin pathway. This enhancement may be mediated through the modulation of cyclin-dependent kinase 5 (CDK5). DPIN also protected the gut barrier and microbiota, reducing the pro-inflammatory impact of the leaky gut observed in 5×FAD mice. DPIN reduced bacterial lipopolysaccharide (LPS) and LPS-associated inflammation, as well as restored intestinal proteins such as Claudin-3. This effect was associated with a modulation of gut microbiota towards a more balanced bacterial composition. Conclusions: These findings underscore DPIN’s promise in mitigating cognitive decline in the early AD stages, positioning it as a potential disease modifier. Full article

Background: Glioblastoma (GBM) uses Glut3 and/or Glut14 and the Leloir pathway to catabolize D-Galactose (Gal). UDP-4-deoxy-4-fluorogalactose (UDP-4DFG) is a potent inhibitor of the two key enzymes, UDP-galactose-4-epimerase (GALE) and UDP-Glucose 6-dehydrogenase (UGDH), involved in Gal metabolism and in glycan synthesis. The Gal antimetabolite 4-deoxy-4-fluorogalactose (4DFG) is a good substrate for Glut3/Glut14 and acts as a potent glioma chemotherapeutic. Methods: Primary GBM cell cultures were used to examine toxicity and alterations in glycan composition via lectin binding in fixed cells and by Western blots. Toxicity/efficacy in vivo data was performed in mouse flank and intracranial models. The effect of 4DFG on D-glucose (Glc) metabolism in GBM cells was assessed by using ¹³C NMR-based tracer studies. Results: 4DFG is moderately potent against GBM cells (IC₅₀: 125–300 µM). GBM glycosylation is disrupted by 4DFG. Survival analysis in an intracranial mouse model showed that treatment with 4DFG (6 × 25 mg/kg of 4DFG, intravenously) improved outcomes by three-fold (p < 0.01). Metabolic flux analysis revealed that both glycolytic and mitochondrial metabolic fluxes of [U-¹³C]Glc were significantly decreased in the presence of 4DFG in GBM cells. Conclusion: A functional Gal-scavenging pathway in GBM allows Gal-based antimetabolites to act as chemotherapeutics. 4DFG is metabolized by GBM in vitro and in vivo, is lethal to GBM tumors, and is well tolerated in mice. Full article

PROTEIN ARGININE METHYLTRANSFERASES (PRMTs) catalyze arginine (R) methylation that is critical for transcriptional and post-transcriptional gene regulation. In Arabidopsis, PRMT5 that catalyzes symmetric R dimethylation is best characterized. PRMT5 mutants are late-flowering and show altered responses to environmental stress. Among PRMT5 targets are Arabidopsis thaliana GLYCINE RICH RNA BINDING PROTEIN 7 (AtGRP7) and AtGRP8 that promote the transition to flowering. AtGRP7 R141 has been shown to be modified by PRMT5. Here, we tested whether this symmetric dimethylation of R141 is important for AtGRP7’s physiological role in flowering time control. We constructed AtGRP7 mutant variants with non-methylable R141 (R141A, R141K). Genomic clones containing these variants complemented the late-flowering phenotype of the grp7-1 mutant to the same extent as wild-type AtGRP7. Furthermore, overexpression of AtGRP7 R141A or R141K promoted flowering similar to overexpression of the wild-type protein. Thus, flowering time does not depend on R141 and its modification. However, germination experiments showed that R141 contributes to the activity of AtGRP7 in response to abiotic stress reactions mediated by abscisic acid during early development. Immunoprecipitation of AtGRP7-GFP in the prmt5 background revealed that antibodies against dimethylated arginine still recognized AtGRP7, suggesting that additional methyltransferases may be responsible for modification of AtGRP7. Full article

The COVID-19 pandemic highlighted testing inequities in developing countries. Lack of lateral flow test (LFT) manufacturing capacity was a major COVID-19 response bottleneck in low- and middle-income regions. Here we report the development of an open-access LFT for SARS-CoV-2 detection comparable to commercial tests that requires only locally available supplies. The main critical resource is a locally developed horse polyclonal antibody (pAb) whose sensitivity and selectivity are greatly enhanced by affinity purification. We demonstrate that these Abs can perform similarly to commercial monoclonal antibodies (mAbs), as well as mAbs and other pAbs developed against the same antigen. We report a workflow for test optimization using nasopharyngeal swabs collected for RT-qPCR, spiked with the inactivated virus to determine analytical performance characteristics as the limit of detection, among others. Our final prototype showed a performance similar to available tests (sensitivity of 83.3% compared to RT-qPCR, and 90.9% compared to commercial antigen tests). Finally, we discuss the possibility and the challenges of utilizing affinity-purified pAbs as an alternative for the local development of antigen tests in an outbreak context and as a tool to address inequalities in access to rapid tests. Full article

Abscisic acid (ABA) and gibberellic acid (GA₃) are regulators of fruit color and sugar levels, and the application of these hormones is a common practice in commercial vineyards dedicated to the production of table grapes. However, the effects of exogenous ABA and GA₃ on wine cultivars remain unclear. We investigated the impact of ABA and GA₃ application on Malbec grapevine berries across three developmental stages. We found similar patterns of berry total anthocyanin accumulation induced by both treatments, closely associated with berry H₂O₂ levels. Quantitative proteomics from berry skins revealed that ABA and GA₃ positively modulated antioxidant defense proteins, mitigating H₂O₂. Consequently, proteins involved in phenylpropanoid biosynthesis were downregulated, leading to decreased anthocyanin content at the almost ripe stage, particularly petunidin-3-G and peonidin-3-G. Additionally, we noted increased levels of the non-anthocyanins E-viniferin and quercetin in the treated berries, which may enhance H₂O₂ scavenging at the almost ripe stage. Using a linear mixed-effects model, we found statistical significance for fixed effects including the berry H₂O₂ and sugar contents, demonstrating their roles in anthocyanin accumulation. In conclusion, our findings suggest a common molecular mechanism by which ABA and GA₃ influence berry H₂O₂ content, ultimately impacting anthocyanin dynamics during ripening. Full article

Inherited macular dystrophies (iMDs) are a group of genetic disorders, which affect the central region of the retina. To investigate the genetic basis of iMDs, we used single-molecule Molecular Inversion Probes to sequence 105 maculopathy-associated genes in 1352 patients diagnosed with iMDs. Within this cohort, 39.8% of patients were considered genetically explained by 460 different variants in 49 distinct genes of which 73 were novel variants, with some affecting splicing. The top five most frequent causative genes were ABCA4 (37.2%), PRPH2 (6.7%), CDHR1 (6.1%), PROM1 (4.3%) and RP1L1 (3.1%). Interestingly, variants with incomplete penetrance were revealed in almost one-third of patients considered solved (28.1%), and therefore, a proportion of patients may not be explained solely by the variants reported. This includes eight previously reported variants with incomplete penetrance in addition to CDHR1:c.783G>A and CNGB3:c.1208G>A. Notably, segregation analysis was not routinely performed for variant phasing—a limitation, which may also impact the overall diagnostic yield. The relatively high proportion of probands without any putative causal variant (60.2%) highlights the need to explore variants with incomplete penetrance, the potential modifiers of disease and the genetic overlap between iMDs and age-related macular degeneration. Our results provide valuable insights into the genetic landscape of iMDs and warrant future exploration to determine the involvement of other maculopathy genes. Full article

This work aims to clarify the effect of dietary polyunsaturated fatty acid (PUFA) intake on the adult brain affected by amyloid pathology. McGill-R-Thy1-APP transgenic (Tg) rat and 5xFAD Tg mouse models that represent earlier or later disease stages were employed. The animals were exposed to a control diet (CD) or an HFD based on corn oil, from young (rats) or adult (mice) ages for 24 or 10 weeks, respectively. In rats and mice, the HFD impaired reference memory in wild-type (WT) animals but did not worsen it in Tg, did not cause obesity, and did not increase triglycerides or glucose levels. Conversely, the HFD promoted stronger microglial activation in Tg vs. WT rats but had no effect on cerebral amyloid deposition. IFN-γ, IL-1β, and IL-6 plasma levels were increased in Tg rats, regardless of diet, while CXCL1 chemokine levels were increased in HFD-fed mice, regardless of genotype. Hippocampal 3-nitrotyrosine levels tended to increase in HFD-fed Tg rats but not in mice. Overall, an HFD with an elevated omega-6-to-omega-3 ratio as compared to the CD (25:1 vs. 8.4:1) did not aggravate the outcome of AD regardless of the stage of amyloid pathology, suggesting that many neurobiological processes relevant to AD are not directly dependent on PUFA intake. Full article

Light is both the main source of energy and a key environmental signal for plants. It regulates not only gene expression but also the tightly related processes of splicing and alternative splicing (AS). Two main pathways have been proposed to link light sensing with the splicing machinery. One occurs through a photosynthesis-related signal, and the other is mediated by photosensory proteins, such as red light-sensing phytochromes. Here, we evaluated the relative contribution of each of these pathways by performing a transcriptome-wide analysis of light regulation of AS in plants that do not express any functional phytochrome (phyQ). We found that an acute 2-h red-light pulse in the middle of the night induces changes in the splicing patterns of 483 genes in wild-type plants. Approximately 30% of these genes also showed strong light regulation of splicing patterns in phyQ mutant plants, revealing that phytochromes are important but not essential for the regulation of AS by R light. We then performed a meta-analysis of related transcriptomic datasets and found that different light regulatory pathways can have overlapping targets in terms of AS regulation. All the evidence suggests that AS is regulated simultaneously by various light signaling pathways, and the relative contribution of each pathway is highly dependent on the plant developmental stage. Full article

The mammarenavirus Junín (JUNV) is the causative agent of Argentine hemorrhagic fever, a severe disease of public health concern. The most abundant viral protein is the nucleoprotein (NP), a multifunctional, two-domain protein with the primary role as structural component of the viral nucleocapsids, used as template for viral polymerase RNA synthesis activities. Here, we report that the C-terminal domain (CTD) of the attenuated Candid#1 strain of the JUNV NP can be purified as a stable soluble form with a secondary structure in line with known NP structures from other mammarenaviruses. We show that the JUNV NP CTD interacts with the viral matrix protein Z in vitro, and that the full-length NP and Z interact with each other in cellulo, suggesting that the NP CTD is responsible for this interaction. This domain comprises an arrangement of four acidic residues and a histidine residue conserved in the active site of exoribonucleases belonging to the DEDDh family. We show that the JUNV NP CTD displays metal-ion-dependent nuclease activity against DNA and single- and double-stranded RNA, and that this activity is impaired by the mutation of a catalytic residue within the DEDDh motif. These results further support this activity, not previously observed in the JUNV NP, which could impact the mechanism of the cellular immune response modulation of this important pathogen. Full article

More than one million women are diagnosed annually worldwide with a gynecological cancer. Most gynecological cancers are diagnosed at a late stage, either because a lack of symptoms, such as in ovarian cancer or limited accessibility to primary prevention in low-resource countries, such as in cervical cancer. Here, we extend the studies of AR2011, a stroma-targeted and tumor microenvironment responsive oncolytic adenovirus (OAdV), whose replication is driven by a triple hybrid promoter. We show that AR2011 was able to replicate and lyse in vitro fresh explants obtained from human ovarian cancer, uterine cancer, and cervical cancer. AR2011 was also able to strongly inhibit the in vitro growth of ovarian malignant cells obtained from human ascites fluid. The virus could synergize in vitro with cisplatin even on ascites-derived cells obtained from patients heavily pretreated with neoadjuvant chemotherapy. AR2011(h404), a dual transcriptionally targeted derived virus armed with hCD40L and h41BBL under the regulation of the hTERT promoter, showed a strong efficacy in vivo both on subcutaneous and intraperitoneally established human ovarian cancer in nude mice. Preliminary studies in an immunocompetent murine tumor model showed that AR2011(m404) expressing the murine cytokines was able to induce an abscopal effect. The present studies suggest that AR2011(h404) is a likely candidate as a novel medicine for intraperitoneal disseminated ovarian cancer. Full article

A wide variety of viruses replicate in liquid-like viral factories. Non-segmented negative stranded RNA viruses share a nucleoprotein (N) and a phosphoprotein (P) that together emerge as the main drivers of liquid–liquid phase separation. The respiratory syncytial virus includes the transcription antiterminator M_2-1, which binds RNA and maximizes RNA transcriptase processivity. We recapitulate the assembly mechanism of condensates of the three proteins and the role played by RNA. M_2-1 displays a strong propensity for condensation by itself and with RNA through the formation of electrostatically driven protein–RNA coacervates based on the amphiphilic behavior of M_2-1 and finely tuned by stoichiometry. M_2-1 incorporates into tripartite condensates with N and P, modulating their size through an interplay with P, where M_2-1 is both client and modulator. RNA is incorporated into the tripartite condensates adopting a heterogeneous distribution, reminiscent of the M_2-1-RNA IBAG granules within the viral factories. Ionic strength dependence indicates that M_2-1 behaves differently in the protein phase as opposed to the protein–RNA phase, in line with the subcompartmentalization observed in viral factories. This work dissects the biochemical grounds for the formation and fate of the RSV condensates in vitro and provides clues to interrogate the mechanism under the highly complex infection context. Full article

Nucleoredoxin (Nrx) belongs to the Thioredoxin protein family and functions in redox-mediated signal transduction. It contains the dithiol active site motif Cys-Pro-Pro-Cys and interacts and regulates different proteins in distinct cellular pathways. Nrx was shown to be catalytically active in the insulin assay and recent findings indicate that Nrx functions, in fact, as oxidase. Here, we have analyzed Nrx in the mammalian retina exposed to (perinatal) hypoxia-ischemia/reoxygenation, combining ex vivo and in vitro models. Our data show that Nrx regulates cell differentiation, which is important to (i) increase the number of glial cells and (ii) replenish neurons that are lost following the hypoxic insult. Nrx is essential to maintain cell morphology. These regulatory changes are related to VEGF but do not seem to be linked to the Wnt/β-catenin pathway, which is not affected by Nrx knock-down. In conclusion, our results strongly suggest that hypoxia-ischemia could lead to alterations in the organization of the retina, related to changes in RPE cell differentiation. Nrx may play an essential role in the maintenance of the RPE cell differentiation state via the regulation of VEGF release. Full article

Root hair cells are important sensors of soil conditions. They grow towards and absorb water-soluble nutrients. This fast and oscillatory growth is mediated by continuous remodeling of the cell wall. Root hair cell walls contain polysaccharides and hydroxyproline-rich glycoproteins, including extensins (EXTs). Class-III peroxidases (PRXs) are secreted into the apoplastic space and are thought to trigger either cell wall loosening or polymerization of cell wall components, such as Tyr-mediated assembly of EXT networks (EXT-PRXs). The precise role of these EXT-PRXs is unknown. Using genetic, biochemical, and modeling approaches, we identified and characterized three root-hair-specific putative EXT-PRXs, PRX01, PRX44, and PRX73. prx01,44,73 triple mutation and PRX44 and PRX73 overexpression had opposite effects on root hair growth, peroxidase activity, and ROS production, with a clear impact on cell wall thickness. We use an EXT fluorescent reporter with contrasting levels of cell wall insolubilization in prx01,44,73 and PRX44-overexpressing background plants. In this study, we propose that PRX01, PRX44, and PRX73 control EXT-mediated cell wall properties during polar expansion of root hair cells. Full article

Class III peroxidases constitute a plant-specific multigene family, where 73 genes have been identified in Arabidopsis thaliana. These genes are members of the reactive oxygen species (ROS) regulatory network in the whole plant, but more importantly, at the root level. In response to abiotic stresses such as cold, heat, and salinity, their expression is significantly modified. To learn more about their transcriptional regulation, an integrative phenotypic, genomic, and transcriptomic study was executed on the roots of A. thaliana Pyrenean populations. Initially, the root phenotyping highlighted 3 Pyrenean populations to be tolerant to cold (Eaux), heat (Herr), and salt (Grip) stresses. Then, the RNA-seq analyses on these three populations, in addition to Col-0, displayed variations in CIII Prxs expression under stressful treatments and between different genotypes. Consequently, several CIII Prxs were particularly upregulated in the tolerant populations, suggesting novel and specific roles of these genes in plant tolerance against abiotic stresses. Full article