A DedA Family Membrane Protein in Indium Extrusion in Rhodanobacter sp. B2A1Ga4

Indium (In) is a critical metal widely used in electronic equipment, and the supply of this precious metal is a major challenge for sustainable development. The use of microorganisms for the recovery of this critical high-tech element has been considered an excellent eco-friendly strategy. The Rhodanobacter sp. B2A1Ga4 strain, highly resistant to In, was studied in order to disclose the bacterial mechanisms closely linked to the ability to cope with this metal. The mutation of the gene encoding for a DedA protein homolog, YqaA, affected drastically the In resistance and the cellular metabolic activity of strain Rhodanobacter sp. B2A1Ga4 in presence of this metal. This indicates that this protein plays an important role in its In resistance phenotype. The negative impact of In might be related to the high accumulation of the metal into the mutant cells showing In concentration up to approximately 4-fold higher than the native strain. In addition, the expression of the yqaA gene in this mutant reverted the bacterial phenotype with a significant decrease of In accumulation levels into the cells and an increase of In resistance. Membrane potential measurements showed similar values for native and mutant cells, suggesting that there was no loss of proton-motive force in the mutant cells. The results from this study suggest a potential role of this DedA family protein as a membrane transporter involved in the In efflux process. The mutant strain also has the potential to be used as a biotool in bioaccumulation strategies, for the recovery of In in biomining activities.

Imaging Flow Cytometry to Study Biofilm-Associated Microbial Aggregates

The aim of the research was to design an advanced analytical tool for the precise characterization of microbial aggregates from biofilms formed on food-processing surfaces. The approach combined imaging flow cytometry with a machine learning-based interpretation protocol. Biofilm samples were collected from three diagnostic points of the food-processing lines at two independent time points. The samples were investigated for the complexity of microbial aggregates and cellular metabolic activity. Thus, aggregates and singlets of biofilm-associated microbes were simultaneously examined for the percentages of active, mid-active, and nonactive (dead) cells to evaluate the physiology of the microbial cells forming the biofilm structures. The tested diagnostic points demonstrated significant differences in the complexity of microbial aggregates. The significant percentages of the bacterial aggregates were associated with the dominance of active microbial cells, e.g., 75.3% revealed for a mushroom crate. This confirmed the protective role of cellular aggregates for the survival of active microbial cells. Moreover, the approach enabled discriminating small and large aggregates of microbial cells. The developed tool provided more detailed characteristics of bacterial aggregates within a biofilm structure combined with high-throughput screening potential. The designed methodology showed the prospect of facilitating the detection of invasive biofilm forms in the food industry environment.

Posttreatment Strategy Against Hypoxia and Ischemia Based on Selective Targeting of Nonnuclear Estrogen Receptors with PaPE-1

Newly synthesized Pathway Preferential Estrogen-1 (PaPE-1) selectively activates membrane estrogen receptors (mERs), namely, mERα and mERβ, and has been shown to evoke neuroprotection; however, its effectiveness in protecting brain tissue against hypoxia and ischemia has not been verified in a posttreatment paradigm. This is the first study showing that a 6-h delayed posttreatment with PaPE-1 inhibited hypoxia/ischemia-induced neuronal death, as indicated by neutral red uptake in mouse primary cell cultures in vitro. The effect was accompanied by substantial decreases in neurotoxicity and neurodegeneration in terms of LDH release and Fluoro-Jade C staining of damaged cells, respectively. The mechanisms of the neuroprotective action of PaPE-1 also involved apoptosis inhibition demonstrated by normalization of both mitochondrial membrane potential and expression levels of apoptosis-related genes and proteins such as Fas, Fasl, Bcl2, FAS, FASL, BCL2, BAX, and GSK3β. Furthermore, PaPE-1-evoked neuroprotection was mediated through a reduction in ROS formation and restoration of cellular metabolic activity that had become dysregulated due to hypoxia and ischemia. These data provide evidence that targeting membrane non-GPER estrogen receptors with PaPE-1 is an effective therapy that protects brain neurons from hypoxic/ischemic damage, even when applied with a 6-h delay from injury onset.

The Impact of Intraspecies Variability on Growth Rate and Cellular Metabolic Activity of Bacteria Exposed to Rotating Magnetic Field

Majority of research on the influence of magnetic fields on microorganisms has been carried out with the use of different species or different groups of microorganisms, but not with the use of different strains belonging to one species. The purpose of the present study was to assess the effect of rotating magnetic fields (RMF) of 5 and 50 Hz on the growth and cellular metabolic activity of eight species of bacteria: Staphylococcus aureus, Pseudomonas aeruginosa, Proteus mirabilis, Klebsiella pneumoniae, Enterococcus faecalis, Enterobacter cloacae, Moraxella catarrhalis, and Bacillus cereus. However, contrary to the research conducted so far, each species was represented by at least four different strains. Moreover, an additional group of S. aureus belonging to a single clonal type but representing different biotypes was also included in the experiment. The results showed a varied influence of RMF on growth dynamics and cellular metabolic activity, diversified to the greatest extent in dependence on the bacterial strain exposed to the RMF and to a lesser extent in dependence on the frequency of the generated magnetic field. It was found that, with regard to the exposed strain of the same species, the effect exerted by the RMF may be positive (i.e., manifests as the increase in the growth rate or/and cellular metabolic activity) or negative (i.e., manifests as a reduction of both aforementioned features) or none. Even when one clonal type of S. aureus was used, the results of RMF exposure also varied (although the degree of differentiation was lower than for strains representing different clones). Therefore, the research has proven that, apart from the previously described factors related primarily to the physical parameters of the magnetic field, one of the key parameters affecting the final result of its influence is the bacterial intraspecies variability.

Photodynamic Inactivation Using Natural Bioactive Compound Prevents and Disrupts the Biofilm Produced by Staphylococcus saprophyticus

Staphylococcus saprophyticus, the food-borne bacteria present in dairy products, ready-to-eat food and environmental sources, has been reported with antibiotic resistance, raising concerns about food microbial safety. The antimicrobial resistance of S. saprophyticus requires the development of new strategies. Light- and photosensitizer-based antimicrobial photodynamic inactivation (PDI) is a promising approach to control microbial contamination, whereas there is limited information regarding the effectiveness of PDI on S. saprophyticus biofilm control. In this study, PDI mediated by natural bioactive compound (curcumin) associated with LED was evaluated for its potential to prevent and disrupt S. saprophyticus biofilms. Biofilms were treated with curcumin (50, 100, 200 µM) and LED fluence (4.32 J/cm2, 8.64 J/cm2, 17.28 J/cm2). Control groups included samples treated only with curcumin or light, and samples received neither curcumin nor light. The action was examined on biofilm mass, viability, cellular metabolic activity and cytoplasmic membrane integrity. PDI using curcumin associated with LED exhibited significant antibiofilm activities, inducing biofilm prevention and removal, metabolic inactivation, intracellular membrane damage and cell death. Likewise, scanning electronic microscopy observations demonstrated obvious structural injury and morphological alteration of S. saprophyticus biofilm after PDI application. In conclusion, curcumin is an effective photosensitizer for the photodynamic control of S. saprophyticus biofilm.

The Effects of Matriptase Inhibition on the Inflammatory and Redox Homeostasis of Chicken Hepatic Cell Culture Models

The function of the transmembrane serine protease matriptase is well described in mammals, but it has not been elucidated in avian species yet. Hence, the aim of the present study was to assess the effects of the 3-amidinophenylalanine (3-AphA)-type matriptase inhibitors MI432 and MI460 on the inflammatory and oxidative state of chicken primary hepatocyte mono-cultures and hepatocyte–nonparenchymal cell co-cultures, the latter serving as a proper model of hepatic inflammation in birds. Cell cultures were exposed to MI432 and MI460 for 4 and 24 h at 10, 25, and 50 µM concentrations, and thereafter the cellular metabolic activity, extracellular interleukin (IL-)6, IL-8, H2O2 and malondialdehyde concentrations were monitored. Both inhibitors caused a transient moderate reduction in the metabolic activity following 4 h exposure, which was restored after 24 h, reflecting the fast hepatic adaptation potential to matriptase inhibitor administration. Furthermore, MI432 triggered an intense elevation in the cellular proinflammatory IL-6 and IL-8 production after both incubation times in all concentrations, which was not coupled to enhanced oxidative stress and lipid peroxidation based on unchanged H2O2 production, malondialdehyde levels and glutathione peroxidase activity. These data suggest that physiological matriptase activities might have a key function in retaining the metabolic and inflammatory homeostasis of the liver in chicken, without being a major modulator of the hepatocellular redox state.

Dissecting drug-induced in vitro cytotoxicity and metabolic dysfunction in conditionally immortalized human proximal tubule cells

Acute kidney injury accounts for 20% of all hospitalized adults, and 14 to 26% is drug-induced, emphasizing the importance of proper nephrotoxicity assessment. The ‘gold standard’ MTT assay is widely used to measure cell viability, but depends on cellular metabolic activity. Consequently, MTT may not be most optimal to assess cytotoxicity, as nephrotoxicity often involves mitochondrial dysfunction. We compared MTT with a direct cell death assay based on a compromised plasma membrane permeability. Mature conditionally immortalized proximal tubule epithelial cells were dose- (0.1-1,000 µM) and time- (0.5, 1, 2, 4, 8 and 24 hours) dependently exposed to a selection of prototypic nephrotoxicants. Dose-dependent reductions in cellular metabolic activity were stronger compared to declines in fluorescence-based cell death, most prominently for cisplatin (1.6 ± 2.0% and 68 ± 4% (mean ± SEM), respectively) and chloroacetaldehyde (2.13 ± 0.05% and 61.0 ± 0.8%). Similar, but more pronounced time-dependent effects were observed, particularly for sanguinarine. We show that assessing cellular metabolic activity by MTT provides a composite readout of cellular metabolic activity and cell death. A nuclear staining approach is preferable when assessing nephrotoxicity of metabolically active compounds. We recommend both assays during drug development to discriminate between metabolically active versus non-active compounds.

IN VITRO ANALYSIS OF UNIVERSALLY UTILIZED IMPLANT RESTORATIVE DENTAL MATERIAL’S IMPACT ON THE ARCHITECTURAL STABILITY OF GINGIVAL FIBROBLASTS IN THE PRESENCE OF A COMMON ENDOTOXIN

Dental implants have been utilized in the last several decades to replace missing teeth. Various factors may result in the loss of teeth. The most common causes of tooth loss are often caries or periodontal disease. The use of a dental implant restored with a porcelain fused to metal crown is often the standard. The purpose of this study was to assess the architectural integrity of gingival fibroblasts at the cellular level when exposed to universally utilized restorative dental material; porcelain, in the presence of a periodontal pathogen, Porphyromonas gingivalis lipopolysaccharide (LPS-PG). Human gingival fibroblasts were exposed to Porcelain (.1 g) in combination with LPS-PG (10 μL), at 24, 48, and 72 hour durations. When assessing for cellular metabolic activity and viability, no significant differences were noted between the control and experimental groups. Contrastingly, when assessing for oxidative stress, the experimental groups were statistically significantly different from the control at the 48 and 72 hour phases (P<0.001). H&E staining of the experimental groups showed irregular shaped cells with loss of density, vacuolization, coarse cytoplasm, and hyperchromatic nuclei.

Flavin-Containing Monooxygenases Are Conserved Regulators of Stress Resistance and Metabolism

Flavin-Containing Monooxygenases are conserved xenobiotic-detoxifying enzymes. Recent studies have revealed endogenous functions of FMOs in regulating longevity in Caenorhabditis elegans and in regulating aspects of metabolism in mice. To explore the cellular mechanisms of FMO’s endogenous function, here we demonstrate that all five functional mammalian FMOs may play similar endogenous roles to improve resistance to a wide range of toxic stresses in both kidney and liver cells. We further find that stress-activated c-Jun N-terminal kinase activity is enhanced in FMO-overexpressing cells, which may lead to increased survival under stress. Furthermore, FMO expression modulates cellular metabolic activity as measured by mitochondrial respiration, glycolysis, and metabolomics analyses. FMO expression augments mitochondrial respiration and significantly changes central carbon metabolism, including amino acid and energy metabolism pathways. Together, our findings demonstrate an important endogenous role for the FMO family in regulation of cellular stress resistance and major cellular metabolic activities including central carbon metabolism.

Apoptosis, the only cell death pathway that can be measured in human diploid dermal fibroblasts following lethal UVB irradiation

Ultraviolet radiation (UVR) is a major environmental genotoxic agent. In skin, it can lead to the formation of mutagenic DNA damage. Several mechanisms are in place to prevent the conversion of these DNA damage into skin cancer-driver mutations. An important mutation prevention mechanism is the programmed cell death, which can safely dispose of the damaged cells. Apoptosis is the most studied and best characterised programmed cell death, but an increasing amount of new cell death pathways are emerging. Using different pharmacological cell death inhibitors and antioxidants, we have evaluated the implication of apoptosis, necroptosis, ferroptosis and parthanatos in UVB-induced cell death in human diploid dermal fibroblasts. Our results show that apoptosis is the only known cell death mechanism induced by UVB irradiation in fibroblasts. We also showed that lethal UVB irradiation induces a PARP-dependent drastic loss of cellular metabolic activity caused by an overused of NAD+.