Search Results (9,354)

The precipitation of minerals, in particular carbonates, is a widespread phenomenon in all ecosystems, where it assumes a high relevance both from a geological and biogeochemical standpoint. Most carbonate rocks are of biological origin and made in an aquatic environment. In particular, bioprecipitation of carbonates is believed to have started in the Mesoproterozoic Era, thanks to a process often driven by photosynthetic microorganisms. Nevertheless, an important contribution to carbonate precipitation is also due to the metabolic activity of heterotrophic bacteria, which is not restricted to specific taxonomic groups or to specific environments, making this process a ubiquitous phenomenon. In this framework, the relationship between carbonatogenic microorganisms and other living organisms assumes a particular interest. This study aims to isolate and identify the culturable heterotrophic bacterial component associated with the coenosarc of Corallium rubrum in order to evaluate the occurrence of strains able to precipitate carbonates. In particular, the study was focused on the identification and characterisation of bacterial strains isolated from a coral coenosarc showing a high carbonatogenic capacity under laboratory conditions. Samples of C. rubrum were taken in the coastal waters of three Italian regions. The concentration of the aerobic heterotrophic microflora colonising C. rubrum coenosarc samples spanned from 3 to 6∙10⁶ CFU/cm². This variation in microbial populations colonising the C. rubrum coenosarc, spanning over 6 orders of magnitude, is not mirrored by a corresponding variability in the colony morphotypes recorded, with the mean being 5.1 (±2.1 sd). Among these bacteria, the carbonatogenic predominant species was Staphylococcus equorum (93% of the isolates), whereas Staphylococcus xylosus and Shewanella sp. accounted only for 3% of isolates each. All these strains showed a remarkable capacity of precipitating calcium carbonate, in the form of calcite crystals organised radially as well crystalised spherulites (S. equorum) or coalescing spherulites (Shewanella sp.). S. xylosus only produced amorphous precipitates of calcium carbonate. All bacterial strains identified were positive both for the production of urease and carbon anhydrase in vitro at 30 °C. It seems that they potentially possess the major biochemical abilities conducive to Ca²⁺ precipitation, as they showed in vitro. In addition, all our carbonatogenic isolates were able to hydrolyse the phytic acid calcium salt and then were potentially able to induce precipitation of calcium phosphates also through such a mechanism. Full article

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder characterized by impaired mucociliary clearance due to defects in motile cilia. This study investigates the impact of loss-of-function mutations in the CFAP300 gene on the ciliary structure and function in three PCD patients. Using a multimodal approach, we integrated molecular genetic testing, transmission electron microscopy, the high-speed video microscopy assay and immunofluorescence staining to analyze ciliary motility and protein expression in both ex vivo and in vitro-obtained ciliary cells. Our results revealed that the pathogenic variant c.198_200delinsCC (p.Phe67ProfsTer10) in CFAP300 led to the absence of the functional CFAP300 protein, the complete loss of outer and inner dynein arms and immotile cilia. Air–liquid interface (ALI)-cultured cells from patients exhibited no ciliary beating, contrasting with healthy controls. Immunostaining confirmed the absence of CFAP300 in patient-derived cilia, underscoring its critical role in dynein arm assembly. These findings highlight the diagnostic utility of ALI cultures combined with functional and protein analyses for PCD, offering a clinically actionable framework that can be readily incorporated into standard diagnostic workflows. Full article

Chronic infection with the hepatitis B virus (HBV) results in over 1 million deaths annually. Although currently licensed treatments, including pegylated interferon-α and nucleoside/nucleotide analogs, can inhibit viral replication, they rarely eradicate covalently closed circular DNA (cccDNA) reservoirs. Moreover, vaccination does not offer therapeutic benefit to already infected individuals or non-responders. Consequently, chronic infection is maintained by the persistence of cccDNA in infected hepatocytes. For this reason, novel therapeutic strategies that permanently inactivate cccDNA are a priority. Obligate heterodimeric transcription activator-like effector nucleases (TALENs) provide the precise gene-editing needed to disable cccDNA. To develop this strategy using a therapeutically relevant approach, TALEN-encoding mRNA targeting viral core and surface genes was synthesized using in vitro transcription with co-transcriptional capping. TALENs reduced hepatitis B surface antigen (HBsAg) by 80% in a liver-derived mammalian cell culture model of infection. In a stringent HBV transgenic murine model, a single dose of hepatotropic lipid nanoparticle-encapsulated TALEN mRNA lowered HBsAg by 63% and reduced viral particle equivalents by more than 99%, without evidence of toxicity. A surveyor assay demonstrated mean in vivo HBV DNA mutation rates of approximately 16% and 15% for Core and Surface TALENs, respectively. This study presents the first evidence of the therapeutic potential of TALEN-encoding mRNA to inactivate HBV replication permanently. Full article

Recent studies have demonstrated that the coumarin derivative 4-Methylumbelliferone (4MU) has an antidiabetic effect in rodent models. 4MU is known to decrease the availability of hyaluronan (HA) substrates and inhibit the activity of different HA synthases. Nevertheless, it has been observed that 4MU may also affect cellular metabolism. In this study, we utilize the rat insulinoma beta cell line (INS-1E) cultured in both two-dimensional (2D) and three-dimensional (3D) experimental settings (pseudo islets), as an in vitro model to study beta cell functionality. For the first time, we observed that treating INS1E cells with 4MU results in improved insulin secretion. Additionally, we discovered that 4MU treatment elicited morphological changes from multilayer to monolayer conditions, along with a varied distribution of insulin granules and cell adhesion properties. Notably, we found that insulin secretion is not correlated with HA production. The same result was observed in co-culture experiments involving INS-1E cells and stromal vascular fraction (SVF) from adipose tissue. These experiments aim to investigate the effects of 4MU on beta cells in the context of its potential use in early-stage type 1 diabetes and in enhancing islet transplantation outcomes. Full article

One of the important traits of many plant growth-promoting rhizobacteria (PGPR) is the biocontrol of phytopathogens. Some PGPR metabolize phytohormone abscisic acid (ABA); however, the role of this trait in plant–microbe interactions is scarcely understood. Phytopathogenic fungi produce ABA and use this property as a negative regulator of plant resistance. Therefore, interactions between ABA-producing necrotrophic phytopathogen Botrytis sp. BA3 with ABA-metabolizing rhizobacterium Rhodococcus sp. P1Y were studied in a batch culture and in gnotobiotic hydroponics with sunflower seedlings. Rhizobacterium P1Y possessed no antifungal activity against BA3 and metabolized ABA, which was synthesized by BA3 in vitro and in associations with sunflower plants infected with this fungus. Inoculation with BA3 and the application of exogenous ABA increased the root ABA concentration and inhibited root and shoot growth, suggesting the involvement of this phytohormone in the pathogenesis process. Strain P1Y eliminated negative effects of BA3 and exogenous ABA on root ABA concentration and plant growth. Both microorganisms significantly modulated the hormonal status of plants, affecting indole-3-acetic, salicylic, jasmonic and gibberellic acids, as well as cytokinins concentrations in sunflower roots and/or shoots. The hormonal effects were complex and could be due to the production of phytohormones by microorganisms, changes in ABA concentrations and multiple levels of crosstalk in hormone networks regulating plant defense. The results suggest the counteraction of rhizobacteria to ABA-producing phytopathogenic fungi through the metabolism of fungal ABA. This expands our understanding of the mechanisms related to the biocontrol of phytopathogens by PGPR. Full article

Adult trees of pedunculate oak (Quercus robur L.) are recalcitrant to vegetative propagation. In this study, we investigated the micropropagation of five oak genotypes corresponding to trees aged 60–800 years in a liquid medium. We used commercial RITA bioreactors to study the influence of the explant type, the culture medium, shoot support and number of immersions. Variables evaluated included the number of normal and hyperhydric shoots, shoot length, multiplication coefficient and number of rootable shoots per explant. All genotypes could be cultured in temporary immersion. Basal stem sections attached to callus grew better than apical sections and developed less hyperhydricity. For long-term cultivation, Gresshoff and Doy medium was the best of the three media evaluated. All genotypes produced vigorous shoots suitable for rooting and acclimation. This is the first protocol to proliferate adult oak trees in bioreactors, representing significant progress towards large-scale propagation of this and other related species. Full article

This study evaluated biomass accumulation and phenolic compound production in seven Hypericum species (H. androsaemum, H. calycinum, H. hirsutum, H. kalmianum, H. olympicum, H. perforatum, and H. triquetrifolium) cultivated in vitro under varying growth regulator treatments and culture periods. Shoots were grown on Murashige and Skoog (MS) medium supplemented with benzyladenine (BA) or meta-topoline (mT) and analyzed after 40 and 60 days. MS medium supplemented with 0.2 mg/L BA was the most effective condition for promoting biomass across all species, with shoot fresh weight increasing significantly at 60 days, particularly in H. olympicum, H. perforatum, and H. triquetrifolium. High-performance liquid chromatography coupled with diode array detection and electrospray ionization mass spectrometry (HPLC-DAD-ESI-MS) identified 13 phenolic compounds, including flavonols, hydroxycinnamic acids, anthocyanins, phloroglucinols, and naphthodianthrones. Phenolic profiles were species-specific and influenced by culture period. H. kalmianum accumulated the highest total phenolic content (37.6 mg/g DW), while H. olympicum was the top producer of hypericin and pseudohypericin. These results highlight the crucial role of culture conditions in regulating both biomass and phytochemical production and provide a promising approach for producing bioactive metabolites in Hypericum species through in vitro systems. Full article

Cystic fibrosis (CF), the most common hereditary lung disease in Caucasians, is caused by dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR). We evaluated CFTR function using a newly developed Ussing chamber system, the Multi Trans Epithelial Current Clamp (MTECC), in an in vitro model of human airway epithelia. Air–liquid interface (ALI) cultures were established from nasal brushings of healthy controls (HC) and CF patients with biallelic CFTR variants. ALI layer thickness was similar between groups (HC: 62 ± 13 µm; CF: 55 ± 9 µm). Immunofluorescence showed apical CFTR expression in HC, but reduced or absent signal in CF cultures. MTECC enabled continuous measurement of transepithelial resistance (Rt), potential difference (PD), and conductance (G_t). G_t was significantly reduced in CF cultures compared to HC (0.825 ± 0.024 vs. −0.054 ± 0.016 mS/cm²), indicating impaired cAMP-inducible ion transport by CFTR. Treatment of CF cultures with elexacaftor, tezacaftor, and ivacaftor (Trikafta^®) increased G_t, reflecting partial restoration of CFTR function. These findings demonstrate the utility of MTECC in detecting functional differences in CFTR activity and support its use as a platform for evaluating CFTR-modulating therapies. Our model may contribute to the development of personalized treatment strategies for CF patients. Full article

In vitro cartilage explant culture has been used to assess nutraceuticals on cartilage responses to inflammatory stimuli. However, applying extracts of nutraceuticals directly to cartilage explants does not account for effects of digestion and hepatic biotransformation, or selective exclusion of product metabolites from joint fluid by the synovial membrane. The current study produced a simulated biological extract of a common nutraceutical (glucosamine; G_sim) by exposing it to a simulated upper gastrointestinal tract digestion, hepatic biotransformation by liver microsomes, and purification to a molecular weight cut-off of 50 kDa. This extract was then used to condition cartilage explants cultured for 120 h in the presence or absence of an inflammatory stimulus (lipopolysaccharide). Media samples were analyzed for prostaglandin E₂ (PGE₂), glycosaminoglycan (GAG), and nitric oxide (NO). Tissue was digested and analyzed for GAG content and stained for viability. Conditioning of explants with G_sim significantly reduced media GAG in stimulated and unstimulated explants and reduced nitric oxide production in unstimulated explants. These data provide evidence for the value of glucosamine in protecting cartilage from deterioration following an inflammatory challenge, and the model improves applicability of these in vitro data to the in vivo setting. Full article

The growing demand for food and the environmental impact of conventional agriculture have prompted the search for sustainable alternatives. Phycocyanin (PC) and total phenolic compounds (TPC) extracted from Spirulina platensis have shown potential for the biological control of phytopathogens. The extraction method directly influences the yield and stability of these compounds. This study aimed to establish an efficient extraction protocol for PC and TPC and to evaluate their antimicrobial efficacy in vitro against Colletotrichum orchidearum, Fusarium nirenbergiae, and Alternaria sp. isolated from hydroponically grown lettuce. The phytopathogens were identified based on phylogenetic analyses using sequences from the ITS, EF1-α, GAPDH, and RPB2 gene regions. This is the first report of C. orchidearum in hydroponic lettuce culture in Brazil, expanding its known host range. Extracts were obtained using hydroalcoholic solvents and phosphate buffer (PB), combined with ultrasound-assisted extraction (bath and probe). The extracts were tested for in vitro antifungal activity. Data were analyzed by ANOVA (p < 0.05), followed by Tukey’s test. The combination of the PB and ultrasound probe resulted in the highest PC (95.6 mg·g⁻¹ biomass) and TPC (21.9 mg GAE·g⁻¹) yields, using 10% (w/v) biomass. After UV sterilization, the extract retained its PC and TPC content. The extract inhibited C. orchidearum by up to 53.52% after three days and F. nirenbergiae by 54.17% on the first day. However, it promoted the growth of Alternaria sp. These findings indicate that S. platensis extracts are a promising alternative for the biological control of C. orchidearum and F. nirenbergiae in hydroponic systems. Full article

This review aims to discuss how heat stress affects ovarian follicles and oocytes, steroidogenesis, and embryo development in ruminants. The literature shows that quiescent primordial follicles appear to be less susceptible to heat stress, but from the primary follicle stage onwards, they begin to suffer the consequences of heat stress. These adverse effects are exacerbated when the follicles are cultured in vitro. In antral follicles, heat stress reduces granulosa cell viability and proliferation in both in vivo and in vitro models. Oocyte maturation, both nuclear and cytoplasmic, is also compromised, and embryo quality declines under elevated thermal conditions. These effects are linked to intracellular disturbances, including oxidative imbalance, mitochondrial dysfunction, and altered hormonal signaling. The differences between in vivo and in vitro responses reflect the complexity of the biological impact of heat stress and emphasize the protective role of the physiological microenvironment. A better understanding of how heat stress alters the function of ovarian follicles, oocytes, and embryos is crucial. This knowledge is critical to devise effective strategies that mitigate damage, support fertility, and improve outcomes in assisted reproduction for livestock exposed to high environmental temperatures. Full article

Serial block-face scanning electron microscopy (SBEM) is a powerful technique for three-dimensional ultrastructural analysis of biological samples, though its application to in vitro cultured human cells remains underutilized. In this study, we present an optimized SBEM sample preparation protocol using human dermal fibroblasts and induced pluripotent stem cells (iPSCs). The method includes key modifications to the original protocol, such as using only glutaraldehyde for fixation and substituting the toxic cacodylate buffer with a less hazardous phosphate buffer. These adaptations result in excellent preservation of cellular ultrastructure, with high contrast and clarity, as validated by transmission electron microscopy (TEM). The loss of natural cell morphology resulted from fixation during passage, when cells formed a precipitate, rather than from fixation directly within the culture medium. The protocol is time-efficient, safe, and broadly applicable to both stem cells and differentiated cells cultured under 2D conditions, providing a valuable tool for ultrastructural analysis in diverse biomedical research settings. Full article

The CRISPR/Cas9 genome editing system has emerged as an effective platform to generate loss-of-function gene edits through non-homologous end joining (NHEJ) without a repair template. To verify whether small molecules can enhance the efficiency of CRISPR/ Cas9-mediated NHEJ gene editing in porcine cells, this experiment investigated the effects of six small-molecule compounds, namely Repsox, Zidovudine, IOX1, GSK-J4, YU238259, and GW843682X, on the efficiency of CRISPR/Cas9-mediated NHEJ gene editing. The results showed the optimal concentrations of the small molecules, including Repsox, Zidovudine, IOX1, GSK-J4, YU238259, and GW843682X, for in vitro-cultured PK15 viability. Compared with the control group, the single small molecules Repsox, Zidovudine, GSK-J4, and IOX1 increased the efficiency of NHEJ-mediated gene editing 3.16-fold, 1.17-fold, 1.16-fold, and 1.120-fold, respectively, in the Cas9-sgRNA RNP delivery system. There were no benefits when using YU238259 and GW843682X compared with the control group. In the CRISPR/Cas9 plasmid delivery system, the Repsox, Zidovudine, IOX1, and GSK-J4 treatments increased the efficiency of NHEJ-mediated gene editing 1.47-fold, 1.15-fold, 1.21-fold, and 1.23-fold, respectively, compared with the control group. Repsox can also improve the efficiency of NHEJ-mediated multi-gene editing based on a CRISPR sgRNA-tRNA array. We also explored the mechanism of Repsox’s effect on the efficiency of NHEJ-mediated gene editing. The results showed that Repsox reduces the expression levels of SMAD2, SMAD3, and SMAD4 in the TGF-β pathway, indicating that Repsox can increase the efficiency of CRISPR NHEJ-mediated gene editing in porcine cells through the TGF-β pathway. Full article

Eurasian aspen (Populus tremula L.) is a tree species with recognised ecological and economic importance for both natural and plantation forests. For the fast cloning of selected aspen genotypes, the method of plant propagation through in vitro culture (micropropagation) is often recommended. The efficiency of this method is related to the use of shoot-inducing chemical growth regulators, among which cytokinins, a type of plant hormone, dominate. Although cytokinins can inhibit rooting, this effect is avoided by using cytokinin-free media. This study sought to identify concentrations and combinations of growth regulators that would stimulate one type of P. tremula organogenesis (either shoot or root formation) without inhibiting the other. The investigated growth regulators included cytokinin 6-benzylaminopurine (BAP), auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA), auxins indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA), gibberellin biosynthesis inhibitor paclobutrazol (PBZ), and a gibberellin mixture (GA₄/₇). Both BAP and TIBA increased shoot number per P. tremula explant and decreased the number of adventitious roots, but TIBA, in contrast to BAP, did not inhibit lateral root formation. However, for the maintenance of both adventitious shoot and root formation above the control level, the combination of PBZ and GA_4/7 was shown to be especially promising. Full article

Neuroinflammation is the major contributor to the pathology of neonatal hypoxic–ischemic (HI) brain injury. Our previous studies have demonstrated that microRNA210 (miR210) inhibition with antisense locked nucleic acid (LNA) inhibitor mitigates neuroinflammation and provides neuroprotection after neonatal HI insult. However, the underlying mechanisms remain elusive. In the present study, using miR210 knockout (KO) mice and microglial cultures, we tested the hypothesis that miR210 promotes microglial activation and neuroinflammation through suppressing mitochondrial function in microglia after HI. Neonatal HI brain injury was conducted on postnatal day 9 (P9) wild-type (WT) and miR210 knockout (KO) mouse pups. We found that miR210 KO significantly reduced brain infarct size at 48 h and improved long-term locomotor functions assessed by an open field test three weeks after HI. Moreover, miR210 KO mice exhibited reduced IL1β levels, microglia activation and immune cell infiltration after HI. In addition, in vitro studies of microglia exposed to oxygen–glucose deprivation (OGD) revealed that miR210 inhibition with LNA reduced OGD-induced expression of Il1b and rescued OGD-mediated downregulation of mitochondrial iron–sulfur cluster assembly enzyme (ISCU) and mitochondrial oxidative phosphorylation activity. To validate the link between miR210 and microglia activation, isolated primary murine microglia were transfected with miR210 mimic or negative control. The results showed that miR210 mimic downregulated the expression of mitochondrial ISCU protein abundance and induced the expression of proinflammatory cytokines similar to the effect observed with ISCU silencing RNA. In summary, our results suggest that miR210 is a key regulator of microglial proinflammatory activation through reprogramming mitochondrial function in neonatal HI brain injury. Full article