Search Results (29)

The objective of this study was an assessment of the anti-biofilm properties of fluoride non-thermal atmospheric plasma (FNTAP) generated using argon and hydrocarbon fluoride gas 1,1,1,2-tetrafluoroethane (TFE). These properties were evaluated by measuring the destruction and recovery of in vitro dual-species biofilms of Streptococcus mutans and Streptococcus sanguinis exposed to FNTAP at 5 or 10 standard cubic centimeters per minute (sccm) or argon non-thermal atmospheric plasma (ArNTAP) for 1 or 2 min, using resazurin-based reagent viability assays, colony forming units (CFU), culture media pH and live/dead staining. Both ArNTAP and FNTAP resulted in significant immediate reductions in bacterial load as compared to the control. Although ArNTAP did not significantly reduce biofilm regrowth, FNTAP treatment showed a bacterial load reduction of more than 5 log units of biofilm regrowth. FNTAP treatments significantly reduced the acidification of the culture medium after recovery incubation, indicating reduced living bacteria, with a pH of 6.92 ± 0.02 and 6.90 ± 0.03, respectively, for the 5 sccm and 10 sccm FNTAP treatments, as compared to a pH of 5.83 ± 0.26 for the ArNTAP treatment, and a significantly acidic pH of 4.76 ± 0.04 for the no-treatment groups. Our results suggest that FNTAP has exceptional anti-biofilm effects, and future directions of our research include the assessment of potential applications of FNTAP in clinical settings. Full article

Tissue acidification resulting from dysregulated cellular bioenergetics accompanies various inflammatory states. GPR68, along with other members of proton-sensing G protein-coupled receptors, responds to extracellular acidification and has been implicated in chronic inflammation-related diseases such as ischemia, cancer, and colitis. The present study examined the role of extracellular acidification on human pulmonary endothelial cell (EC) permeability and inflammatory status per se and investigated potential synergistic effects of acidosis on endothelial dysfunction caused by bacterial lipopolysaccharide (LPS, Klebsiella pneumoniae). Results showed that medium acidification to pH 6.5 caused a delayed increase in EC permeability illustrated by a decrease in transendothelial electrical resistance and loss of continuous VE-cadherin immunostaining at cell junctions. Likewise, acidic pH induced endothelial inflammation reflected by increased mRNA and protein expression of EC adhesion molecules VCAM-1 and ICAM-1, upregulated mRNA transcripts of tumor necrosis factor-α, IL-6, IL-8, IL-1β, and CXCL5, and increased secretion of ICAM-1, IL-6, and IL-8 in culture medium monitored by ELISA. Among the GPCRs tested, acidic pH selectively increased mRNA and protein expression of GPR68, and only the GPR68-specific small molecule inhibitor OGM-8345 rescued acidosis-induced endothelial permeability and inflammation. Furthermore, acidic pH exacerbated LPS-induced endothelial permeability and inflammatory response in cultured lung macrovascular as well as microvascular endothelial cells. These effects were suppressed by OGM-8345 in both EC types. Altogether, these results suggest that GPR68 is a critical mediator of acidic pH-induced dysfunction of human pulmonary vascular endothelial cells and mediates the augmenting effect of tissue acidification on LPS-induced endothelial cell injury. Full article

Bacterial leaching is a well-known green technology proposed for the extraction of valuable metals into solution. However, this biotechnology has some “bottle neck” problems too. Arsenopyrite, a gold-bearing ore, is a refractory mineral material that is hardly soluble and contains toxic arsenic compounds which decrease any bioleaching production. The most common biotechnology used for this process is provided with the species Acidithiobacillus ferrooxidans: autotrophic and acidophilic bacterial strains including ones resistant to inorganic arsenic compounds. Common attempts to dissolve arsenopyrite with increasing volumes of sulfuric acid provoke acidification of the environment and its pollution with toxic compounds. In our research, we compared two A. ferrooxidans strains of different origin: TFBk isolated from arsenopyrite ore (pre-adopted to arsenic), the Republic of Kazakhstan, and ShA-GNK isolated from silicate nickel-ferrous ore (laterite, without arsenic), the Russian Federation. The studied genomes of both strains showed the presence of the same genes providing defense against arsenic compounds, but the resistance to toxic compounds was higher in the strain that had never been exposed to any high As concentration under the natural conditions. Both strains showed a weak oxidation of the arsenopyrite flotation concentrate (AFC). In accordance with the published data, supplementation of the medium with formate stimulated bacterial growth in the culturing medium. However, this supplementation to the leaching solution decreased the arsenopyrite oxidation during the first stage of the AFC leaching because formate was used as an alternative energy substrate, but subsequently gave a higher iron yield later. Full article

The selection of starter cultures with different technological profiles and suitable microclimatic conditions is among the main tools used to improve the technological quality and safety of dry-cured salami. The aim of this study is to evaluate the effect of two different starter cultures [fast (SR) and medium (SM) acidification] during the process and on the quality of typical Italian dry-cured “Salame Napoli”. The ripening process was evaluated in dry-cured salami made with different cultures: Euroferment Medium (Staphylococcus xylosus, Lactobacillus plantarum) in SM and Euroferment Rapid (Staphylococcus carnosus, Staphylococcus xylosus, Lactobacillus sakei) in SR. The salami was stuffed in artificial casings, dried for 5 days and then ripened for 28 days at a controlled temperature of 12–14 °C and 80–90% RH. During the ripening process, an evaluation of the appearance, the pH, and the weight loss of the salami were conducted. For each finished product, the physical–chemical, microbiological, rheological, and sensory characteristics were evaluated. The results showed that the different starter cultures influenced the pH descent, which was faster in SR, reaching a pH value of 4.80 in three days. This influenced the consistency profile of the SR salami, which showed higher hardness (46.04 ± 6.53 in SR vs. 35.60 ± 2.62 in SM; p < 0.05) and gumminess (19.21 ± 3.44 in SR vs. 11.89 ± 0.71 in SM; p < 0.05) values. SR salami revealed a higher count of yeasts and a lower malondialdehyde concentration than SM. The presence of the starter in SM has positively affected the intensity of the aroma. The outcomes indicated the importance of selecting starter cultures to not only ensure food safety but also to obtain the desired sensorial characteristics of the product. Full article

The integration of sensing devices into cell culture systems is a topic of great interest in the study of pathologies and complex biological mechanisms in real-time. In particular, the fit-for-purpose microfluidic devices called organ-on-chip (OoC), which host living engineered organs that mimic in vivo conditions, benefit greatly from the integration of sensors, enabling the monitoring of specific chemical-physical parameters that can be correlated with biological processes. In this context, copper is an essential trace element whose total concentration may be associated with specific pathologies, and it is therefore important to develop reliable analytical techniques in cell systems. Copper can be determined by using the anodic stripping voltammetry (ASV) technique, but its applicability in cell culture media presents several challenges. Therefore, in this work, the performance of ASV in cell culture media was evaluated, and an acidification protocol was tested to improve the voltammetric signal intensity. A Transwell^® culture model with Caco-2 cells was used to test the applicability of the developed acidification protocol by performing an off-line measurement. Finally, a microfluidic device was designed in order to perform the acidification of the cell culture medium in an automated manner and then integrated with a silicon microelectrode to perform in situ measurements. The resulting sensor-integrated microfluidic chip could be used to monitor the concentration of copper or other ions concentration in an organ-on-chip model; these functionalities represent a great opportunity for the non-destructive strategic experiments required on biological systems under conditions close to those in vivo. Full article

The toxic element arsenic (As) has become the major focus of global research owing to its harmful effects on human health, resulting in the establishment of several guidelines to prevent As contamination. The widespread industrial use of As has led to its accumulation in the environment, increasing the necessity to develop effective remediation technologies. Among various treatments, such as chemical, physical, and biological treatments, used to remediate As-contaminated environments, biological methods are the most economical and eco-friendly. Microbial oxidation of arsenite (As(III)) to arsenate (As(V)) is a primary detoxification strategy for As remediation as it reduces As toxicity and alters its mobility in the environment. Here, we evaluated the self-detoxification potential of microcosms isolated from Nakdong River water by investigating the autotrophic and heterotrophic oxidation of As(III) to As(V). Experimental data revealed that As(III) was oxidized to As(V) during the autotrophic and heterotrophic growth of river water microcosms. However, the rate of oxidation was significantly higher under heterotrophic conditions because of the higher cell growth and density in an organic-matter-rich environment compared to that under autotrophic conditions without the addition of external organic matter. At an As(III) concentration > 5 mM, autotrophic As(III) oxidation remained incomplete, even after an extended incubation time. This inhibition can be attributed to the toxic effect of the high contaminant concentration on bacterial growth and the acidification of the growth medium with the oxidation of As(III) to As(V). Furthermore, we isolated representative pure cultures from both heterotrophic- and autotrophic-enriched cultures. The new isolates revealed new members of As(III)-oxidizing bacteria in the diversified bacterial community. This study highlights the natural process of As attenuation within river systems, showing that microcosms in river water can detoxify As under both organic-matter-rich and -deficient conditions. Additionally, we isolated the bacterial strains HTAs10 and ATAs5 from the microcosm which can be further investigated for potential use in As remediation systems. Our findings provide insights into the microbial ecology of As(III) oxidation in river ecosystems and provide a foundation for further investigations into the application of these bacteria for bioremediation. Full article

Arsenic (As) contamination of groundwater is widespread and significantly affects drinking water, posing a threat to public health due to its classification as a human carcinogen. Arsenic (As) can be removed from contaminated water using sustainable technologies (e.g., biotechnological processes). The process of removing Arsenic from water through reactions with iron under acidic and oxidizing conditions in a fungal broth has been proposed alongside the production of bioscorodite (FeAsO₄·2H₂O) crystals by Trichoderma atroviride culture. This ascomycete was selected based on tests with three other fungi (Aspergillus niger, and the basidiomycetes, Postia placenta, and Phanerochaete chrysosporium) because it decreased the pH to 2.2, raised the redox potential (Eh) to 207 mV, and was the quickest to produce 0.39 µg/L of H₂O₂ in a modified Wunder medium. The Eh was further increased to 324.80 mV under improved fungal culture conditions, selected using a 2³⁻¹ fractional factorial design (FFD). The fungal broth was then used for bioscorodite production by adding Fe(III)/As(III) salts and scorodite seeds at 92 °C for 21 h. Scorodite seeds and bioscorodite were characterized by X-ray diffraction (XRD) and scanning electron microscopy (SEM). Arsenic was determined in solution by atomic absorption spectrophotometry (AAS), and a 73% reduction in the initial As concentration (0.30 g/100 mL) was observed after bioscorodite production. Bioscorodite production under appropriate fungal culture conditions could be an option for sustainable As removal from water. The production of H₂O₂ by the fungus resulted in the oxidation of As(III) into As(V) and acidification of the culture broth, which created the necessary conditions for the production of bioscorodite without the need for chemical acids or oxidants. This approach is environmentally friendly and cost effective, making it a promising alternative for the treatment of arsenic-contaminated water. Full article

Glutamine is one of the most abundant amino acids in the cell. In mitochondria, glutaminases 1 and 2 (GLS1/2) hydrolyze glutamine to glutamate, which serves as the precursor of multiple metabolites. Here, we show that ammonium generated during GLS1/2-mediated glutaminolysis regulates lysosomal pH and in turn lysosomal degradation. In primary human skin fibroblasts BJ cells and mouse embryonic fibroblasts, deprivation of total amino acids for 1 h increased lysosomal degradation capacity as shown by the increased turnover of lipidated microtubule-associated proteins 1A/1B light chain 3B (LC3-II), several autophagic receptors, and endocytosed DQ-BSA. Removal of glutamine but not any other amino acids from the culture medium enhanced lysosomal degradation similarly as total amino acid starvation. The presence of glutamine in regular culture media increased lysosomal pH by >0.5 pH unit and the removal of glutamine caused lysosomal acidification. GLS1/2 knockdown, GLS1 antagonist, or ammonium scavengers reduced lysosomal pH in the presence of glutamine. The addition of glutamine or NH₄Cl prevented the increase in lysosomal degradation and curtailed the extension of mTORC1 function during the early time period of amino acid starvation. Our findings suggest that glutamine tunes lysosomal pH by producing ammonium, which regulates lysosomal degradation to meet the demands of cellular activities. During the early stage of amino acid starvation, the glutamine-dependent mechanism allows more efficient use of internal reserves and endocytosed proteins to extend mTORC1 activation such that the normal anabolism is not easily interrupted by a brief disruption of the amino acid supply. Full article

Calciprotein particles (CPPs) represent an inherent mineral buffering system responsible for the scavenging of excessive Ca²⁺ and PO₄³⁻ ions in order to prevent extraskeletal calcification, although contributing to the development of endothelial dysfunction during the circulation in the bloodstream. Here, we performed label-free proteomic profiling to identify the functional consequences of CPP internalisation by endothelial cells (ECs) and found molecular signatures of significant disturbances in mitochondrial and lysosomal physiology, including oxidative stress, vacuolar acidification, accelerated proteolysis, Ca²⁺ cytosolic elevation, and mitochondrial outer membrane permeabilisation. Incubation of intact ECs with conditioned medium from CPP-treated ECs caused their pro-inflammatory activation manifested by vascular cell adhesion molecule 1 (VCAM1) and intercellular adhesion molecule 1 (ICAM1) upregulation and elevated release of interleukin (IL)-6, IL-8, and monocyte chemoattractant protein-1/ C-C motif ligand 2 (MCP-1/CCL2). Among the blood cells, monocytes were exclusively responsible for CPP internalisation. As compared to the co-incubation of donor blood with CPPs in the flow culture system, intravenous administration of CPPs to Wistar rats caused a considerably higher production of chemokines, indicating the major role of monocytes in CPP-triggered inflammation. Upregulation of sICAM-1 and IL-8 also suggested a notable contribution of endothelial dysfunction to systemic inflammatory response after CPP injections. Collectively, our results demonstrate the pathophysiological significance of CPPs and highlight the need for the development of anti-CPP therapies. Full article

T. asperellum MSCL 309 was used in the study. T. asperellum was grown in the stirred bioreactor under submerged cultivation. The resulting biomass was filtered to obtain a thick biomass. The viability and antifungal activity of T. asperellum biomass samples were determined simultaneously by studying the colonization of the malt extract agar medium surface and its competitiveness with the plant pathogenic fungus Fusarium graminearum using in vitro dual culture experiments. Treatment with starch, alone or in combination with copper (II) sulphate and/or hydrochloric acid did not significantly affect fungal viability compared to control. An important factor in maintaining viability was the addition of hydrochloric acid, which significantly increased the storage life of biomass. In all post-treatments, F. graminearum was overgrown with T. asperellum in seven days, and accordingly, the larger the area occupied by T. asperellum, the smaller the area of F. graminearum colonization. Viability and antifungal activity of T. asperellum persisted throughout the experiment, at least for eight weeks. All the post-treatment methods we studied improved the viability and antifungal activity of Trichoderma, at least in terms of the area of the colonized surface. For the development of long-term viable and active T. asperellum preparations, we recommend the use of acidification of T. asperellum biomass obtained by submerged fermentation. Full article

Bovine Respiratory Disease (BRD) is a multifactorial condition affecting cattle worldwide resulting in high rates of morbidity and mortality. The disease can be triggered by Bovine Herpesvirus-1 (BoHV-1) infection, stress, and the subsequent proliferation and lung colonization by commensal bacteria such as Mannheimia haemolytica, ultimately inducing severe pneumonic inflammation. Due to its polymicrobial nature, the study of BRD microbes requires co-infection models. While several past studies have mostly focused on the effects of co-infection on host gene expression, we focused on the relationship between BRD pathogens during co-infection, specifically on M. haemolytica’s effect on BoHV-1 replication. This study shows that M. haemolytica negatively impacts BoHV-1 replication in a dose-dependent manner in different in vitro models. The negative effect was observed at very low bacterial doses while increasing the viral dose counteracted this effect. Viral suppression was also dependent on the time at which each microbe was introduced to the cell culture. While acidification of the culture medium did not grossly affect cell viability, it significantly inhibited viral replication. We conclude that M. haemolytica and BoHV-1 interaction is dose and time-sensitive, wherein M. haemolytica proliferation induces significant viral suppression when the viral replication program is not fully established. Full article

Physiological responses of different interspecific Pyrus hybrids and an open pollinated Pyrus communis ‘Williams’ (Pcw) grown under in vitro culture conditions simulating lime induced chlorosis were studied. The hybrids were derived from crosses between the ‘Pyriam’ pear rootstock and four Pyrus species of the Mediterranean region, namely P. amygdaliformis Vill. (Pa), P. amygdaliformis persica Bornme. (Pap), P. communis cordata (Desv.) Hook. (Pcc), and P. elaeagrifolia Pall (Pe), all known for their higher field tolerance to iron-chlorosis than P. communis. Twenty hybrids and one open pollinated Pcw were micropropagated, and plantlets were in vitro characterized for their physiological responses to iron-deficiency conditions. Rooted plantlets were transferred to a culture medium with 2 µM Fe³⁺ DTPA and 10 or 20 mM NaHCO₃. These physiological responses were scored at 1, 3, 7, and 28 days from the start of the in vitro assay. Leaf total chlorophyll content, the capacity of roots to acidify the medium, reduced iron, and exudates of phenolic acids and organic acids were analyzed in each media and time sample. Leaf chlorophyll levels for the clones derived from Pcc were the highest, especially under the highest bicarbonate concentration, followed by those derived from Pap and Pa. The higher chlorophyll content of Pcc clones were related with their higher capacity to acidify the media but not with their iron reduction capacity at the root level. On the other hand, hybrid clones derived from Pe showed a higher Fe³⁺ reduction ability than clones from all the other species during the whole assay but only when the bicarbonate concentration was lower. The exudation of phenolic acids by the roots was higher in Pcw than in the other species, and this response might explain why the total chlorophyll levels in Pcw clones are similar to those of Pe and Pa ones. These results with Pyrus spp. bring more evidence in support of the idea that iron reduction capacity at the root level is not directly related with a higher tolerance to iron deficiency caused by the high pH of calcareous soils. Instead, the ability to acidify the rhizosphere is the trait of choice for the selection of the pear hybrid clones better adapted to lime induced chlorosis. In addition, the in vitro assay to select the Pyrus clones for tolerance to iron chlorosis could be shortened to one week of culture in 10 mM NaHCO₃, measuring the leaf chlorophyll level, acidification of the culture medium, and exudation of phenolic acids as the physiological responses to predict tolerance to lime-induced chlorosis. Full article

Cancer cell culture is routinely performed under superphysiologic O₂ levels and in media such as Dulbecco’s Modified Eagle Medium (DMEM) with nutrient composition dissimilar to mammalian extracellular fluid. Recently developed cell culture media (e.g., Plasmax, Human Plasma-Like Medium (HPLM)), which are modeled on the metabolite composition of human blood plasma, have been shown to shift key cellular activities in several cancer cell lines. Similar effects have been reported with respect to O₂ levels in cell culture. Given these observations, we investigated how media composition and O₂ levels affect cellular energy metabolism and mitochondria network structure in MCF7, SaOS2, LNCaP, and Huh7 cells. Cells were cultured in physiologic (5%) or standard (18%) O₂ levels, and in physiologic (Plasmax) or standard cell culture media (DMEM). We show that both O₂ levels and media composition significantly affect mitochondrial abundance and network structure, concomitantly with changes in cellular bioenergetics. Extracellular acidification rate (ECAR), a proxy for glycolytic activity, was generally higher in cells cultured in DMEM while oxygen consumption rates (OCR) were lower. This effect of media on energy metabolism is an important consideration for the study of cancer drugs that target aspects of energy metabolism, including lactate dehydrogenase activity. Full article

The characterized 10 Streptomyces isolates were previously selected by their abilities to solubilize phosphates. To investigate whether these isolates represent multifaceted plant growth-promoting rhizobacteria (PGPR), their potassium-solubilizing, auxin-producing and inhibitory activities were determined. The 10 Streptomyces spp. yielded a variable biomass in the presence of insoluble orthoclase as the sole potassium (K) source, indicating that they were able to extract different amounts of K from this source for their own growth. Three strains (AZ, AYD and DE2) released soluble K from insoluble orthoclase in large amounts into the culture broth. The production levels ranged from 125.4 mg/L to 216.6 mg/L after 5 days of culture. Only two strains, Streptomyces enissocaesilis (BYC) and S. tunisiensis (AI), released a larger amount of soluble K from orthoclase and yielded much more biomass. This indicated that the rate of K released from this insoluble orthoclase exceeded its consumption rate for bacterial growth and that some strains solubilized K more efficiently than others. The results also suggest that the K solubilization process of AZ, AYD and DE2 strains, the most efficient K-solubilizing strains, involves a slight acidification of the medium. Furthermore, these 10 Streptomyces spp. were able to secrete indole acetic acid (IAA) in broth medium and ranged from 7.9 ± 0.1 µg/mL to 122.3 ± 0.1 µg/mL. The results of the antibiosis test proved the potential of the 10 tested strains to limit the growth of fungi and bacteria. In dual culture, S. bellus (AYD) had highest inhibitory effect against the three identified fungal causal agents of root rot of sugar beet: Fusarium equiseti and two F. fujikuroi at 55, 43 and 36%, respectively. Streptomyces enissocaesilis (BYC), S. bellus (AYD) and S. saprophyticus (DE2) exhibited higher multifaceted PGPR with their potassium-solubilizing, auxin-producing and inhibitory activities, which could be expected to lead to effectiveness in field trials of sugar beet. Full article

Among the methods used to remove metals and their compounds from landfill leachates with low application costs and high efficiency are bioleaching and biosorption. The most effective bacteria used in the metal removal process are Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans. The aim of the study was to determine the usefulness of the A. ferrooxidans and A. thiooxidans population in removing heavy metals from landfill leachate. In addition, development opportunities for bacterial population using landfill leachate as growth medium were identified. The substrate for the research was the raw leachate before the reverse osmosis process. In order to increase the efficiency of trace elements removal and recovery from leachate, variable combinations have been used which differ by the addition of sulfuric acid, A. ferrooxidans culture, A. thiooxidans culture, mixed culture containing populations of both bacteria, and elemental sulfur. Based on the research, it was found that the removal of heavy metals from leachate was a selective process. High bioleaching efficiency, from 80% to 90%, was obtained for all metals for which the sample acidification or sulfur addition was used. The simultaneous combination of both these additives turned out to be the most advantageous. The A. thiooxidans culture was the most effective in bioleaching reverse osmosis effluents. For the A. ferrooxidans culture used, much lower efficiencies were obtained, while by contrast, the use of mixed culture of two bacterium species had no significant effect. Full article