Lactic Acid Release Research Articles

Vaginal microbiome: modern approaches for correction and/or restoration

Today, women’s health is a separate issue for doctors of many specialties, including obstetricians and gynecologists, family doctors, etc. There is a wide range of treatment options for managing diseases of the reproductive system in women. But despite this, many of these strategies do not work for our patients. Therefore, we need the latest personalized approaches to therapy. One of the newest trends in women’s health is the correction or restoration of the vaginal microbiome. In our previous publications, we have covered the issue of the normal composition of the vaginal microbiome and its changes in various pathological conditions. Modification of the vaginal microbiome can be a useful strategy in the treatment of sexually transmitted infections, bacterial vaginosis, candidiasis, benign, precancerous and even malignant diseases of the female reproductive system, miscarriage, preterm birth and other pregnancy complications.The concept of using exogenous microorganisms as a treatment has been known for centuries. Possible mechanisms by which exogenous strains of lactobacilli can affect vaginal microbiome include vaginal recolonization, increased production or release of lactic acid and other antimicrobial compounds, and modulation of the local mucosal immune response. From a microbiome perspective, “prebiotics” are nutraceutical compounds that induce bacterial growth or the activity of probiotics or beneficial endogenous microorganisms. One of the limitations of prebiotics is their dependence on the presence of lactobacilli, which are absent or almost absent in dysbiosis. Symbiotics are combinations of prebiotics and probiotics based on the concept that the first nutraceutical can improve the bacterial growth and function of the other. This review highlights the latest views on correction and/or restoration of the vaginal microbiome using not only probiotics, prebiotics, and symbiotic, but also phage-altering agents, phage therapy, vaginal microbiome transplantation, etc. Such methods of correction and/or restoration are currently relevant in the reproductive medicine, gynecology and obstetrics all over the world. Despite the fact that these are the newest methods of correction and/or restoration, they are developing every day and require more detailed coverage of this issue.

Antibacterial, Antifungal, and Cytotoxic Potential of PlumbaginLoaded pH-Responsive Vaginal Nanoformulations.

Plumbagin is a naphthoquinone from the roots of the Plumbago species and exhibits anticancer activity. Translational usage of plumbagin in biomedical sciences is restricted due to its poor solubility and bioavailability. Therefore, pH-responsive plumbagin-loaded vaginal nanoformulations with polylactic acid (PLA)-chitosan polymeric coat were fabricated by inotropic gelation technique. Among the four (F1, F2, F3, F4) nanoformulations prepared, F3 exhibited good interaction of polymers with plumbagin as evidenced by FTIR, XRD, and thermal analysis. The positive zeta potential (48.4 ± 5.57mV), optimal size (694 ± 65.76nm), low PDI (0.157), and good encapsulation efficiency (77.8 ± 3.62%) of F3 were significant. The indirect method of drug loading (58.35 ± 5.00%) confirmed the drug content of about 495.44 ± 5.00µg of plumbagin in 1mg of F3. The drug loading pattern was confirmed by TEM analysis, and the spherical morphology of the nanocomposite was confirmed by SEM analysis. F3 formulation showed 46% and 25.2% of drug release in 24h in simulated vaginal fluid at pH 4.5 and 7 respectively with sustained release and hydrolyses of lactic acid from PLA. Among all the nanoformulations evaluated, nanoformulation F3 with promising physicochemical properties showed good antifungal and antibacterial activity against various fungal and bacterial strains. F3 exhibited potent cytotoxicity with an IC50 of 3.6 ± 0.12µg/ml for HeLa and an IC50 of 0.81 ± 0.01µg/ml for SiHa cells. Altogether, the nanoformulation F3 exhibited potent antimicrobial activity against vaginal infections and cytotoxicity against cervical cancer cell lines.

Nrf2 Mediates Effect of Resveratrol in Ischemia-reperfusion Injury.

Ischemia-Reperfusion Injury (IRI) is a paradoxical phenomenon where removing the source of injury can cause additional damage. Ischemia reduces ATP production and intracellular pH, reducing oxidative reactions, increasing lactic acid release, and activating anaerobic metabolism. Reperfusion restores aerobic respiration and increases ROS production, leading to malfunction of transmembrane transport, activation of proteases, DNA dissolution, and protein denaturation, leading to apoptotic cell death. Nrf2 is a transcription factor that regulates cellular inflammation and oxidative responses. It is activated by oxidants and electrophiles and enhances detoxifying enzyme expression, maintaining redox homeostasis. It also activates ARE, which activates several ARE-regulated genes that favor cell survival by exhibiting resistance to oxidants and electrophiles. Nrf2 regulates the antioxidant defense system by producing phase II and antioxidant defense enzymes, including HO-1, NQO-1, gglutamylcysteine synthetase, and rate-limiting enzymes for glutathione synthesis. Nrf2 protects mitochondria from damage and supports mitochondrial function in stress conditions. Resveratrol is a stilbene-based compound with a wide variety of health benefits for humans, including antioxidant, anticarcinogenic, antitumor, and estrogenic/antiestrogenic. Resveratrol protects against IRI through several signaling pathways, including the Nrf2/ARE pathway. Here, we review the studies that investigated the mechanisms of resveratrol protection against IRI through modulation of the Nrf2 signaling pathway.

Modulation of SEMA4D-modified titanium surface on M2 macrophage polarization via activation of Rho/ROCK-mediated lactate release of endothelial cells: In vitro and in vivo

SEMA4D-modified titanium surfaces can indirectly regulate macrophages through endothelial cells to achieve an anti-inflammatory effect, which is beneficial for healing soft tissues around the gingival abutment. However, the mechanism of surface-induced cellular phenotypic changes in SEMA4D-modified titanium has not yet been elucidated. SEMA4D activates the RhoA signaling pathway in endothelial cells, which coordinates metabolism and cytoskeletal remodeling. This study hypothesized that endothelial cells inoculated on SEMA4D-modified titanium surfaces can direct M2 polarization of macrophages via metabolites. An indirect co-culture model of endothelial cells and macrophages was constructed in vitro, and specific inhibitors were employed. Subsequently, endothelial cell adhesion and migration, metabolic changes, Rho/ROCK1 expression, and inflammatory expression of macrophages were assessed via immunofluorescence microscopy, specific kits, qRT-PCR, and Western blotting. Moreover, an in vivo rat bilateral maxillary implant model was constructed to evaluate the soft tissue healing effect. The in vitro experiments showed that the SEMA4D group had stronger endothelial cell adhesion and migration, increased Rho/ROCK1 expression, and enhanced release of lactate. Additionally, decreased macrophage inflammatory expression was observed. In contrast, the inhibitor group partially suppressed lactate metabolism and motility, whereas increased inflammatory expression. The in vivo analyses indicated that the SEMA4D group had faster and better angiogenic and anti-inflammatory effects, especially in the early stage. In conclusion, via the Rho/ROCK1 signaling pathway, the SEMA4D-modified titanium surface promotes endothelial cell adhesion and migration and lactic acid release, then the paracrine lactic acid promotes the polarization of macrophages to M2, thus obtaining the dual effects of angiogenesis and anti-inflammation.

NMAAP1 regulated macrophage polarizion into M1 type through glycolysis stimulated with BCG

Bacillus Calmette Guerin (BCG) perfusion is widely used as cancer adjuvant therapy, in which macrophages play an important role. Novel macrophage activated associated protein 1 (NMAAP1), upregulated after BCG's activation, was proved to promote macrophage polarization to the M1 type. We found that BCG could stimulate mice BMDM to the M1 type and kill tumor cells. After the deletion of NMAAP1, the tumor volume of mice became larger, and the number of M1 type macrophages in the tumor decreased significantly. When macrophages were induced into the M1 type, aerobic glycolysis, the Warburg effect manifested in the increased uptake of glucose and the conversion of pyruvate to lactic acid. NMAAP1 could bind with IP3R and regulate macrophage polarization to the M1 type. However, the specific mechanism of how NMAAP1 regulates macrophage polarization towards the M1 type and plays an antitumor role must be clarified. NMAAP1 could promote the release of lactic acid and pyruvate, enhance the glycolysis of macrophages, and affect the expression of HIF-1α. After inhibition of glycolysis by 2-DG and lactic acid generation by FX11, the effects of NMAAP1 promoting macrophage polarization to the antitumor M1 type were weakened. Furthermore, NMAAP1 upregulated the expression of HIF-1α, which is associated with glycolysis. Moreover, the Ca2+/NF-κB pathway regulated HIF-1α expression by NMAAP1 in the macrophages. NMAAP1 promotes the polarization of macrophages towards the M1 type by affecting the Warburg effect stimulated by BCG.

Topical application of Lactobacilli successfully eradicates Pseudomonas aeruginosa biofilms and promotes wound healing in chronic wounds

Chronic wounds are difficult to treat due to the presence of biofilm which prevents wound healing. Pseudomonas aeruginosa is one of the most common pathogens found in chronic wounds and conventional treatment strategies have been ineffective in the eradication of its biofilm, without harming the surrounding healthy tissue at the same time. Here, we introduced an innovative approach applying the probiotic product Bio-K+ (containing three lactobacilli) topically as an antimicrobial and antibiofilm agent. We identified lactic acid as the main active component. While antibiotics and antiseptics such as silver-ions only demonstrated limited efficacy, Bio-K+ was able to completely eradicate mature P. aeruginosa biofilms established in an in-vitro and ex-vivo human skin model. Furthermore, it demonstrated biocompatibility in the co-culture with human dermal fibroblasts and accelerated the migration of fibroblasts in a cell migration assay promoting wound healing. To enhance clinical practicability, we introduced Bio-K+ into the hydrocolloid dressing Aquacel, achieving sustained release of lactic acid and biofilm eradication. This new treatment approach applying probiotics could represent a major improvement in the management of chronic wounds and can be extended in treating other biofilm-associated infections.

BPA exposure enhances the metastatic aggression of ovarian cancer through the ERα/AKT/mTOR/HIF-1α signaling axis.

Long-term exposure to bisphenol A (BPA) in humans may promote ovarian cancer development. In present study, the mechanisms by which BPA mediates the aggression metastatic behavior of ovarian cancer were investigated in vitro/in vivo. The results showed that BPA (10μM) significantly promoted the proliferation, migration and invasion of human ovarian cancer cells (ES-2 and OVCAR-3cells); moreover, it promoted ES-2 and OVCAR-3cell glucose uptake, lactic acid release and intracellular ATP synthesis. After administration of 5μg/kg/day BPA, tumor volume was increased compared with that in control group. KEGG and GO enrichment analyses showed that the genes from ES-2cell in 10μM BPA-treated group were enriched mainly in central carbon metabolism and PI3K-AKT signaling pathway. Then, qRT‒PCR and western blotting results showed that BPA (10μM) increased the mRNA and protein expression levels of glycolysis-related genes and mTOR, p-AKT HIF-1α and ERα in vitro/vivo; whereas this effect was reduced after treatment with the ERα inhibitor methyl-piperidino-pyrazole. Furthermore, coimmunoprecipitation and mass spectrometry showed that BPA promoted the direct interaction of ERα with lactate dehydrogenase A. These results show that BPA directly promoted the proliferation, migration and invasion of ovarian cancer cells through the ERα/AKT/mTOR/HIF-1α signaling axis to enhance glycolysis.

ENO1 Promotes OSCC Migration and Invasion by Orchestrating IL-6 Secretion from Macrophages via a Positive Feedback Loop

Oral squamous cell carcinoma (OSCC) has a five-year survival rate of less than 50% due to its susceptibility to invasion and metastasis. Crosstalk between tumor cells and macrophages has been proven to play a critical role in tumor cell migration and invasion. However, the specific mechanisms by which tumor cells interact with macrophages have not been fully elucidated. This study sought to investigate the regulatory mechanism of tumor cell-derived alpha-enolase (ENO1) in the interaction between tumor cells and macrophages during OSCC progression. Small interfering RNA (siRNA) transfection and recombinant human ENO1 (rhENO1) stimulation were used to interfere with the interaction between tumor cells and macrophages. Our results showed that ENO1 was expressed higher in CAL27 cells than in HaCaT cells and regulated lactic acid release in CAL27 cells. Conditioned medium of macrophages (Macro-CM) significantly up-regulated the ENO1 mRNA expression and protein secretion in CAL27 cells. ENO1 promoted the migration and invasion of tumor cells by facilitating the epithelial-mesenchymal transition (EMT) through macrophages. ENO1 orchestrated the IL-6 secretion of macrophages via tumor cell-derived lactic acid and the paracrine ENO1/Toll-like receptor (TLR4) signaling pathway. In turn, IL-6 promoted the migration and invasion of tumor cells. Collectively, ENO1 promotes tumor cell migration and invasion by orchestrating IL-6 secretion of macrophages via a dual mechanism, thus forming a positive feedback loop to promote OSCC progression. ENO1 might be a promising therapeutic target which is expected to control OSCC progression.

Plant Growth-Promoting Attributes of Zinc Solubilizing Dietzia maris Isolated from Polyhouse Rhizospheric Soil of Punjab.

Zinc solubilizing rhizobacteria (ZSR) enhance the phyto-availability of Zn by converting its insoluble forms into usable forms that are essential for the growth and nutritional quality of crops. In the present study, a potential ZSR, hereafter referred to as strain N14, was isolated from the polyhouse rhizospheric soil of Punjab, India. The isolated rhizobacteria was found to be Gram-positive, aerobic, rod-shaped, and demonstrated a solubilization index of 63.75 on the Bunt Rovira (BR) medium. The 16S rRNA gene sequence analysis revealed that isolated strain N14 matches substantially with type strain Dietzia maris DSM 43672T. In its ZnO broth assay, a significant amount of soluble Zn was detected along with a simultaneous decrease in pH of the broth. Ultra-performance liquid chromatography analysis revealed the release of organic acids, specifically, lactic acid and acetic acid by D. maris strain N14 which could be the reason for the decrease in broth pH. The production of indole acetic acid (29.91µg/ml), gibberellic acid (4.72µg/ml), ammonia (38.87µg/ml), siderophore (0.89%), along with the release of HCN and appearance of phosphate solubilization zone (14.4mm) with this strain suggested its possible plant growth-promoting (PGP) characteristics. Therefore, this strain was employed in the formulation of pellets which were applied for in vivo PGPstudies using tomato plants. The developed bioformulated pellets showed a significant enhancement in plant growth as compared to control and vermicompost treated plants. To the best of our knowledge, this is the first report describing the Zn solubilizing and PGP characteristics of D. maris.

Investigation of the Interaction of Adipose-Derived Mesenchymal Stem Cells with ε-Polycaprolactone and Egg White Scaffolds

The development of three-dimensional (3D) cell culture models is becoming increasingly important due to their numerous advantages over conventional monolayer culture. This study aimed to examine the interaction of adipose tissue-derived mesenchymal stem cells (AD-MSCs) with scaffolds composed of ε-polycaprolactone (ε-PCL) and egg white. In our study, ε-PCL and egg white scaffolds were produced from their monomers by tin octoate catalyzed and heat polymerization, respectively. Characterization of ε-PCL was carried out by Gel Permeation Chromatography (GPC), Fourier Transform Infrared Spectrophotometry (FTIR), Proton Nuclear Magnetic Resonance (H-NMR), Differential Scanning Calorimetry (DSC) and Scanning Electron Microscopy (SEM). AD-MSCs labeled with red fluorescent CellTracker CM-DiI were cultured on egg white and ε-PCL scaffolds for 12 days. Cell viability was determined using 3-(4.5-Dimethylthiazol-2yl)-2.5-diphenyltetrazolium bromide (MTT) and nitric oxide (NO) level was evaluated for toxicity. The results showed that the number of AD-MSCs in the egg white scaffold increased periodically for 12 days compared to the other groups. Although the number of AD-MSCs in the ε-PCL scaffold increased until day 6 of the culture, the number of cells started to decrease after day 6. These results were associated with the toxic effect of lactic acid release on cells resulting from the decomposition of ε-PCL scaffolds through catabolic reactions. Therefore, these results indicated that the egg white scaffold enhanced and maintained cell adhesion and cell viability more than the ε-Polycaprolactone scaffold and could be used as a scaffold in tissue engineering studies involving stem cells.

Effects of polylactic acid (PLA) and polybutylene adipate-co-terephthalate (PBAT) biodegradable microplastics on the abundance and diversity of denitrifying and anammox bacteria in freshwater sediment

Microplastics (MPs) have been widely distributed on Earth and have drawn global concern for freshwater and marine ecosystems. Biodegradable plastics have risen in popularity to replace nonbiodegradable plastics all over the world. The effects of biodegradable plastics on denitrifying and anammox bacteria in freshwater sediment remain largely unknown. In this study, water column reactors containing polylactic acid (PLA) or polybutylene adipate-co-terephthalate (PBAT) MPs in sediment were established to simulate lake ecosystems and analyze the effects of biodegradable MPs on sedimentary nitrogen transformation microorganisms. The total organic carbon (TOC) concentrations in the PLA and PBAT groups were slightly higher than those in the control group, which might be related to the slow degradation of these two plastics. Denitrifying and anammox bacterial diversities decreased after adding MPs to sediments for 30 days, and the dominant OTUs of these two bacteria were differentiated from the control. The abundance levels of nirS denitrifying and anammox bacteria on the PLA MP surface were significantly higher than those in the other groups (P < 0.05), but they were lower in the PBAT groups than in the other groups. As an excellent electron donor for the denitrification process, lactic acid release from PLA degradation resulted in the enrichment of denitrifying and anammox bacteria on the MP surfaces. However, PBAT led to various responses of bacteria in an anaerobic environment. In addition, the redundancy analysis results indicated that total phosphorus, TOC and nitrate were strongly negatively correlated with the abundance levels of denitrifying and anammox bacteria. Our findings provided insight into the effects of MPs, especially the biodegradable ones, on sedimentary nitrogen-transformation bacteria.

Hydrolytic degradation mechanism of modified polylactic acid in different food simulants

Hydrolytic degradation of non-compatibilized and compatibilized toughened polylactic acid (PLA) blends produced with polyolefin elastomer (POE) at 5 and 15 wt percent were examined in distilled water and both 50 % and 95 % ethanol solutions at 40 °C for up to 180 days and were compared with pure PLA. The change in swelling degree, molecular weight, crystallinity, thermal properties, and lactic acid (LA) release were monitored. All samples demonstrated higher hydrolytic degradation in contact with 50 % ethanol in comparison to 95 % ethanol and distilled water, which were in conformity with the results of the LA release during the exposure time. During the exposure period, the crystallinity degree of samples increased specifically in contact with 50 % ethanol solution. that could be due to amorphous regions degradation and solvent-induced crystallization. Blending PLA with POE and compatibilizing with polyolefin elastomer-graft-maleic anhydride (POE-g-MA) resulted in lower hydrolytic degradation and subsequently restricted polymer chain mobility as highlighted by glass transition temperature and the crystallization change.

Effects of Drug Physicochemical Properties on In-Situ Forming Implant Polymer Degradation and Drug Release Kinetics.

In-situ forming implants (ISFIs) represent a simple, tunable, and biodegradable polymer-based platform for long-acting drug delivery. However, drugs with different physicochemical properties and physical states in the polymer-solvent system exhibit different drug release kinetics. Although a few limited studies have been performed attempting to elucidate these effects, a large, systematic study has not been performed until now. The purpose of this study was to characterize the in vitro drug release of 12 different small molecule drugs with differing logP and pKa values from ISFIs. Drug release was compared with polymer degradation as measured by lactic acid (LA) release and change in poly(DL-lactide-co-glycolide) (PLGA) molecular weight (MW) measured by size exclusion chromatography with multi-angle laser light scattering (SEC-MALS). Drug physical state and morphology were also measured using differential scanning calorimetry (DSC) and scanning electron microscopy (SEM). Together, these results demonstrated that hydrophilic drugs have higher burst release at 24 h (22.8–68.4%) and complete drug release within 60 days, while hydrophobic drugs have lower burst release at 24 h (1.8–18.9%) and can sustain drug release over 60–285 days. Overall, drug logP and drug physical state in the polymer–solvent system are the most important factors when predicting the drug release rate in an ISFI for small-molecule drugs. Hydrophilic drugs exhibit high initial burst and less sustained release due to their miscibility with the aqueous phase, while hydrophobic drugs have lower initial burst and more sustained release due to their affinity for the hydrophobic PLGA. Additionally, while hydrophilic drugs seem to accelerate the degradation of PLGA, hydrophobic drugs on the other hand seem to slow down the PLGA degradation process compared with placebo ISFIs. Furthermore, drugs that were in a crystalline state within the ISFI drugs exhibited more sustained release compared with amorphous drugs.

Synthesis of paramylon ester-graft-PLA copolymers and its two-step enzymatic degradation by proteinase K and β-1,3-glucanase

Paramylon ester-graft-poly(lactic acid) (PLA) copolymers were synthesized by the ring opening polymerization of l-lactide with paramylon acetate (PaAc) samples having an acetate degree of substitution (DS) from 0.5 to 2.2 (PaAc-g-PLA) or paramylon propionate (PaPr) samples with a propionate DS of 1.6 or 2.0 (PaPr-g-PLA). These copolymers exhibited thermoplasticity and some formed self-supporting melt-pressed films. Analyses of annealed films using differential scanning calorimetry showed that their thermal properties varied with the PLA DS. The enzymatic degradation behaviors of the neat paramylon as well as the PaAc and PaPr were investigated by treating these materials with β-1,3-glucanase followed by spectrometric analysis of the glucose levels in the buffer solutions, prior to the investigation of the copolymers. The data showed that 9 wt% glucose was obtained from the neat paramylon after 7 d and that the PaAc and PaPr were also degraded by β-1,3-glucanase. The concentration of liberated glucose was also found to decrease as the DS increased. Two-step enzymatic degradation tests were subsequently carried out with PaAc-g-PLA and PaPr-g-PLA specimens. In the first step, proteinase K was applied to degrade the PLA side-chains and high performance liquid chromatography (HPLC) was used to assess the release of lactic acid. Both HPLC and nuclear magnetic resonance (NMR) analyses established that the PLA side-chains of both the PaAc-g-PLA and PaPr-g-PLA specimens were successfully degraded. In the second step, β-1,3-glucanase was employed to decompose the remaining paramylon ester backbones. Monitoring the release of glucose showed that these backbones were successfully degraded in trials using the PaAc0.5-g-PLA and PaAc1.0-g-PLAs after 7 d These results confirmed that both copolymers could be broken down via this two-step enzymatic treatment, and that degradation by β-1,3-glucanase was dependent on the DS of the paramylon ester backbone. The results of this work show that PaAc1.0-g-PLA is a plastic material capable of thermal processing and is also completely biodegradable.

Extracellular ATP is increased by release of ATP-loaded microparticles triggered by nutrient deprivation.

Rationale: Caloric restriction improves the efficacy of anti-cancer therapy. This effect is largely dependent on the increase of the extracellular ATP concentration in the tumor microenvironment (TME). Pathways for ATP release triggered by nutrient deprivation are largely unknown.Methods: The extracellular ATP (eATP) concentration was in vivo measured in the tumor microenvironment of B16F10-inoculated C57Bl/6 mice with the pmeLuc probe. Alternatively, the pmeLuc-TG-mouse was used. Caloric restriction was in vivo induced with hydroxycitrate (HC). B16F10 melanoma cells or CT26 colon carcinoma cells were in vitro exposed to serum starvation to mimic nutrient deprivation. Energy metabolism was monitored by Seahorse. Microparticle release was measured by ultracentrifugation and by Nanosight.Results: Nutrient deprivation increases eATP release despite the dramatic inhibition of intracellular energy synthesis. Under these conditions oxidative phosphorylation was dramatically impaired, mitochondria fragmented and glycolysis and lactic acid release were enhanced. Nutrient deprivation stimulated a P2X7-dependent release of ATP-loaded, mitochondria-containing, microparticles as well as of naked mitochondria.Conclusions: Nutrient deprivation promotes a striking accumulation of eATP paralleled by a large release of ATP-laden microparticles and of naked mitochondria. This is likely to be a main mechanism driving the accumulation of eATP into the TME.

Development of Non-Invasive Impedance Sensors for Real Time Monitoring of Bacteria Growth

Mucosal surface in the gastrointestinal track and oral cavity are colonized by more than 700 types of microorganism. The bacteria interaction is depended on the location of its formation and dynamics. Streptococcus mutans, is known pathogenic bacteria present in oral microbiome, which metabolizes sucrose and other carbohydrates to produce lactic acid and lowers the pH 5.5 or less, which causes caries and periodontal diseases. Therefore, study of growth and dynamics of Sm bacteria is critical to understand oral cavity formation. Fluorescent in-situ hybridization probe (FISH) with confocal laser scanning microscopy (CLSM) is widely used for monitoring of biofilm with spatial resolution. However, these sophisticated instruments are not economically viable for multiple replication or high throughput applications. Here, we have used single frequency impedance spectroscopy as fast and non-invasive technique to monitor real time growth of Sm biofilm and kinetics of its formation.Real time monitoring of Sm biofilm was performed by the continuous recording of impedance while Sm bacteria was grown on glass substrate in BMM with 30 mM sucrose feeding. The cell-sensors impedance has been reported in the arbitrary unit as normalized impedance called cell-index (CI). Sensors were first tested in phosphate buffer solution (PBS) and then changed to BMM as background signal. After stabilization of sensors, streptococcus mutane was injected. There is increase of CI after Sm injection from zero to 0.3 at 10kHz frequency then decreased ~ 0.2. The decrease of CI at 10 kHz is lactic acid release by the Sm bacteria which causes decrease in solution resistance. The CI value at 1 Hz is increased from zero to ~ 1 after bacteria inoculation. The increase in CI at lower frequency is due to growth of Sm bacteria on the electrodes surface which causes increase of capacitance. After 56 hrs of growth of Sm, surfactant sodium dodecyl sulphate (SDS, CMC) was injected for investigation of sensitivity of impedancemetry sensors on destruction of bacteria biofilm. There is decrease in CI value at 1 Hz suggests that Sm biofilm matrix was partially collapsed. So, this study suggests that, high frequency impedance-based sensor can be used as solution resistance sensors and low frequency impedance based sensor can be used as capacitive based sensor for monitoring of biofilm growth.

Untargeted metabolomics and lipidomics analysis identified the role of FOXA1 in remodeling the metabolic pattern of BaP-transformed 16HBE cells

Benzo[a]pyrene (BaP) is a strong carcinogen for lung cancer, and forkhead-box A1 (FOXA1) plays an oncogenic role in BaP-transformed cell THBEc1. To explore the remodeling of metabolic pattern caused by BaP-induced transformation and the possible role FOXA1 might play in it, we compared metabolic patterns between THBEc1 cells and control using untargeted metabolomics and lipidomics analysis, and determined the effects of FOXA1 knockout on the metabolic pattern of THBEc1 cells. Metabolomics and lipidomics identified a total of 15 and 46 differential metabolites and lipids between THBEc1 and 16HBE cells, respectively, and a total of 4 and 1 differential metabolites and lipids between FOXA1 knockout cell THBEc1-ΔFOXA1-c34 and control cell THBEc1-ctrl, respectively. Analysis results of metabolites and metabolic pathways indicated the metabolic pattern remodeling may be related to the alteration in glucose metabolism during BaP-induced transformation. Western blotting revealed the up-regulation of enolase-2 (ENO2), pyruvate carboxylase (PCB), aconitase-2 (ACO2) and phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) (Thr202/Tyr204), the down-regulation of succinate dehydrogenase complex subunit A (SDHA) and phosphoenolpyruvate carboxykinase 2 (PCK2) in THBEc1 cells. The detection results of metabolites related to glucose metabolism demonstrated the decreasing of lactic acid content in cells, lactic acid production in culture medium and citric acid content in mitochondria, and the increasing of ATP production of THBEc1 cells. FOXA1 knockout partially reversed the changes of ENO2, SDHA, PCK2 and p-ERK1/2 (Thr202/Tyr204) levels, lactic acid release, citric acid content in mitochondria of THBEc1 cells. In conclusion, FOXA1 knockout partially reversed the remodeling of glucose metabolism caused by BaP-induced malignant transformation. Our findings provide a clue for the possible role of FOXA1 in glucose metabolism regulation.

Aquaporin family lactic acid channel NIP2;1 promotes plant survival under low oxygen stress in Arabidopsis.

Under anaerobic stress, Arabidopsis thaliana induces the expression of a collection of core hypoxia genes that encode proteins for an adaptive response. Among these genes is NIP2;1, which encodes a member of the "Nodulin 26-like Intrinsic Protein" (NIP) subgroup of the aquaporin superfamily of membrane channel proteins. NIP2;1 expression is limited to the "anoxia core" region of the root stele under normal growth conditions, but shows substantial induction (up to 1,000-fold by 2-4 h of hypoxia) by low oxygen stress, and accumulation in all root tissues. During hypoxia, NIP2;1-GFP accumulates predominantly on the plasma membrane by 2 h, is distributed between the plasma and internal membranes during sustained hypoxia, and remains elevated in root tissues through 4 h of reoxygenation recovery. In response to hypoxia challenge, T-DNA insertion mutant nip2;1 plants exhibit elevated lactic acid within root tissues, reduced efflux of lactic acid, and reduced acidification of the external medium compared to wild-type plants. Previous biochemical evidence demonstrates that NIP2;1 has lactic acid channel activity, and our work supports the hypothesis that NIP2;1 prevents lactic acid toxicity by facilitating release of cellular lactic acid from the cytosol to the apoplast, supporting eventual efflux to the rhizosphere. In evidence, nip2;1 plants demonstrate poorer survival during argon-induced hypoxia stress. Expressions of the ethanolic fermentation transcript Alcohol Dehydrogenase1 and the core hypoxia-induced transcript Alanine Aminotransferase1 are elevated in nip2;1, and expression of the Glycolate Oxidase3 transcript is reduced, suggesting NIP2;1 lactic acid efflux regulates other pyruvate and lactate metabolism pathways.

Roxarsone Promotes Glycolysis and Angiogenesis by Inducing Hypoxia-Inducible Factor-1α In Vitro and In Vivo.

Roxarsone (Rox) is an organic arsenic compound used as a feed additive to promote animal growth. The release of Rox into the environment poses risks to human health. Rox demonstrated tumor-promoting and proangiogenic effects in xenograft models. Increasing studies revealed the tight relationship among angiogenesis, carcinogenesis, tumorigenesis, and glycolysis. Glycolysis, via hypoxia-inducible factor-1α (HIF-1α), controls vascular endothelial cell (VEC) growth. To date, there has been no literature report on the effect of Rox on HIF-1α-dependent glycolysis. Herein, we report that Rox promoted glycolysis in rat VECs, as shown by the increased adenosine triphosphate production, the lactic acid release, the activity and content of aldolase (ALD), and the expression levels of ALD A and glucose transporter 1 (GLUT1). Rox also increased the cellular levels of HIF-1α. Treatment with the HIF-1α inhibitor YC-1 reversed Rox-increased ALD A and GLUT1 levels and attenuated Rox-induced VEC viability, suggesting that Rox-induced HIF-1α contributes to the glycolytic and angiogenic effects of Rox. Rox also promoted tumor growth and angiogenesis and increased the levels of ALD A, GLUT1, and HIF-1α in the tumor tissue of a mouse xenograft model, whereas these effects were abolished using YC-1. Our findings indicated that Rox induces HIF-1α in VECs to promote glycolysis and angiogenesis thus enhancing the tumor growth.

Glycolysis-derived acidic microenvironment as a driver of endothelial dysfunction in systemic sclerosis.

SSc is an autoimmune disease characterized by peripheral vasculopathy and skin and internal organ fibrosis. Accumulating evidence underlines a close association between a metabolic reprogramming of activated fibroblasts and fibrosis. This prompted us to determine the metabolism of SSc dermal fibroblasts and the effect on the vasculopathy characterizing the disease. A Seahorse XF96 Extracellular Flux Analyzer was used to evaluate SSc fibroblast metabolism. In vitro invasion and capillary morphogenesis assays were used to determine the angiogenic ability of endothelial cells (ECs). Immunofluorescence, flow cytometry and real-time PCR techniques provided evidence of the molecular mechanism behind the impaired vascularization that characterizes SSc patients. SSc fibroblasts, compared with controls, showed a boosted glycolytic metabolism with increased lactic acid release and subsequent extracellular acidification that in turn was found to impair EC invasion and organization in capillary-like networks without altering cell viability. A molecular link between extracellular acidosis and endothelial dysfunction was identified as acidic ECs upregulated MMP-12, which cleaves and inactivates urokinase-type plasminogen activator receptor, impairing angiogenesis in SSc. Moreover, the acidic environment was found to induce the loss of endothelial markers and the acquisition of mesenchymal-like features in ECs, thus promoting the endothelial-to-mesenchymal transition process that contributes to both capillary rarefaction and tissue fibrosis in SSc. This study showed the relationship of the metabolic reprogramming of SSc dermal fibroblasts, extracellular acidosis and endothelial dysfunction that may contribute to the impairment and loss of peripheral capillary networks in SSc disease.