Cytochrome Content Research Articles

Intra/extracellular electron transfer and metagenomic analysis elucidated the roles of magnetic iron powder (Fe3O4) on mixotrophic denitrification system

Elemental iron provides a viable strategy to improve the denitrification efficiency by expediting electron transport. However, the roles of magnetic iron powder (Fe3O4) on mixotrophic denitrification remains unknown. In this study, the intra/extracellular electron transfer (IET/EET) and microbial metabolism mechanisms were explored in a Fe3O4-mediated sulfide-autotrophic and heterotrophic denitrification system. The results showed that Fe3O4 promoted the formation of dense clump structure with filamentous cross-linking in activated sludge. Fe3O4 could increase the coenzyme Q activity in IET and the content of free riboflavin and cytochrome c in EET. Metagenomic analysis indicated that denitrification, sulfide oxidation and sulfate reduction were the main pathways of nitrogen and sulfur metabolism, and the enriched denitrifying bacteria (Halomonas and Hypobacterium) and sulfur-oxidizing bacteria (Marinicella) could stably support nitrate removal. This study expands our understanding of the IET/EET during Fe3O4-mediated mixotrophic denitrification process, providing a novel insight for nitrogen removal from marine recirculating aquaculture wastewater.

NLRP3 Inflammasome in the Pathogenesis of Miscarriages

Despite significant advances in prenatal medicine, spontaneous miscarriage remains one of the most common and serious pregnancy complications, affecting an increasing number of women. Since many aspects of the pathogenesis of spontaneous miscarriage remain unexplained, the aim of this study has been to assess the involvement of the NLRP3 inflammasome as a potential causative factor. The concentrations of NLRP3, IL-1β, IL-18, and cytochrome C in the serum of patients after miscarriage were measured by means of the immunoenzymatic method. In the placental tissue, the expression of NLRP3, IL-1β, IL-18, and Caspase-1 as well as that of the classical apoptosis biomarkers Fas, FasL, Bcl-2, and Ca was evaluated by means of immunohistochemistry techniques. Additionally, in whole blood, the concentrations of elements crucial for pregnancy progression, such as Ca, K, Mg, and Na, were examined by means of the ICP-OES method. Significantly higher concentrations of NLRP3 and IL-18 were demonstrated in the serum of patients with miscarriage as compared to the control group. In the placental tissue samples, a higher expression of IL-1β, IL-18, and Caspase-1 proteins was noted in women who had experienced miscarriage as compared to the control group. At the same time, a significantly lower expression of FasL and Bcl-2 proteins as well as Ca deposits was observed in women after miscarriage as compared to those with a normal pregnancy outcome. Significantly lower concentrations of Ca and K were recorded in the blood of patients with spontaneous miscarriage as compared to pregnant women. The analysis of the results x indicated a greater involvement of the inflammasome in women with spontaneous miscarriage associated with oxidative–antioxidative imbalance than in the case of miscarriage related to NET formation. Our research has provided evidence for the involvement of the inflammasome in the process of spontaneous miscarriage and identifies a new direction for diagnostics that includes NLRP3 as a preventive element in prenatal care, particularly in light of the steadily declining number of pregnancies and the increasing number of reproductive failures.

Electrodeposited zinc oxide nanoparticles: Synthesis, characterization, and anti-cervical cancer effects

In the present study, zinc oxide nanoparticles (ZnO NPs) were synthesized via the electrodeposition method and characterized. Then, the anticancer effects of ZnO NPs against cervical cancer HeLa cells were assessed by cell viability, oxidative stress, caspase activity, qRT-PCR, MMP, and ELISA assays. XRD analysis revealed the hexagonal wurtzite phase of ZnO. SEM image of ZnO NPs showed the homogeneous spherical/hexagonal-like structures of ZnO NPs, while TEM imaging revealed the successful synthesis of ZnO NPs with an average diameter of about 8 nm. The UV–vis absorption spectrum of ZnO NPs showed a characteristic absorption band around the wavelength of 368 nm with a band gap energy (Eg) of 3.36 eV. DLS study displayed that the obtained particle size of ZnO NPs has an average size of 29.24 nm and an average zeta potential value of −19 mV. Additionally, cellular findings indicated that the proliferation of cervical cancer HeLa cells was markedly mitigated after incubation with ZnO NPs at different concentrations of 10, 50 and 100 µg/ml, however, these concentrations were not able to trigger an apparent cytotoxic effect on HUVEC non-malignant cells. Also, it was detected that ZnO NPs led to overexpression of Bax/ Bcl-2, caspase-9/-3 genes, increased level of caspase-9/-3 activity, overproduction of MDA level, inhibition of SOD and CAT activity and reduction of GSH content, MMP collapse, and upregulation of cytoplasmic cytochrome c release. In general, these findings suggested that electrodeposited synthesized ZnO NPs can induce anticancer effects in cervical cancer HeLa cells through the mitochondrial-mediated apoptosis signaling pathway.

Insights to the cooperation of double-working potential electroactive biofilm for performance of sulfamethoxazole removal: ARG fate and microorganism communities

Bioelectrochemical systems (BESs) have shown great potential in enhancing sulfamethoxazole (SMX) removal. However, electroactive biofilms (EBs) constructed with single potentials struggle due to limited biocatalytic activity, hindering deep SMX degradation. Here, we constructed a double-working potential BES (BES-D) to investigate its ability to eliminate SMX and reduce the levels of corresponding antibiotic resistance genes (ARGs). The preferable electrochemical activity of EB in BES-D was confirmed by electrochemical characterization, EPS analysis, physical structure, viability of the biofilm, and cytochrome content. BES-D exhibited a notably greater SMX removal efficiency (94.2 %) than did the single-working potential BES (BES-S) and the open-circuit group (OC). Degradation pathway analysis revealed that the cooperative EB could accelerate the in-depth removal of SMX. Moreover, EB interaction in BES-D decreased the relative abundance of ARGs in biofilms compared to that in BES-S, although the absolute number of ARG copies increased in BES-D effluents. Compared to those in BES-S and OC, more complex cross-niche microbial associations in the EB of BES-D were observed by network analysis of the bacterial community and ARG hosts, enhancing the degradation efficiency of SMX. In conclusion, BES-D has significant potential for SMX removal and the enhancement of EB activity. Nonetheless, the risk of ARG dissemination in effluent remains a concern.

Metabolism changes caused by glucose in normal and cancer human brain cell lines by Raman imaging and chemometric methods

Glucose is the main source of energy for the human brain. This paper presents a non-invasive technique to study metabolic changes caused by glucose in human brain cell lines. In this paper we present the spectroscopic characterization of human normal brain (NHA; astrocytes) and human cancer brain (CRL-1718; astrocytoma and U-87 MG; glioblastoma) control cell lines and cell lines upon supplementation with glucose. Based on Raman techniques we have identified biomarkers that can monitor metabolic changes in lipid droplets, mitochondria and nucleus caused by glucose. We have studied the vibrations at 750 cm−1, 1444 cm−1, 1584 cm−1 and 1656 cm−1 as a function of malignancy grade. We have compared the concentration of cytochrome, lipids and proteins in the grade of cancer aggressiveness in normal and cancer human brain cell lines. Chemometric analysis has shown that control normal, control cancer brain cell lines and normal and cancer cell lines after supplementation with glucose can be distinguished based on their unique vibrational properties. PLSDA (Partial Least Squares Discriminant Analysis) and ANOVA tests have confirmed the main role of cytochromes, proteins and lipids in differentiation of control human brain cells and cells upon supplementation with glucose. We have shown that Raman techniques combined with chemometric analysis provide additional insight to monitor the biology of astrocytes, astrocytoma and glioblastoma after glucose supplementation.

Biochar alleviates inhibition effects of humic acid on anaerobic digestion: Insights to performances and mechanisms

To recover methane from waste activated sludge through anaerobic digestion (AD) is one promising alternative to achieve carbon neutrality for wastewater treatment plants. However, humic acids (HAs) are one of the major compositions in waste activated sludge, and their accumulation performs inhibition effects on AD. This study investigated the potentials of biochar (BC) in alleviating inhibition effects of HAs on AD. Results showed that although the accumulated HAs reduced methane yield by 9.37% compared to control, the highest methane yield, 132.6 mL CH4/g VSS, was obtained after adding BC, which was 45.9% higher than that in HA group. Mechanism analysis showed that BC promoted the activities of hydrolase such as protease and α-glucosidase, which were 69.7% and 29.7% higher than those in HA group, respectively. The conversion of short-chain fatty acids was accelerated. In addition, the evolutions of electroactive microorganisms like Clostridium_sensu_stricto_13 and Methanosaeta were consistent with the activitiies of electron transfer and the contents of cytochrome c. Furthermore, parts of HAs rather than all of them were adsorbed by BC, and the remaining free HAs and BC formed synergistic effects on methanogenesis, then both CO2 reduction and acetoclastic methanogenesis pathways were improved. The findings may provide some solutions to alleviate inhibition effects of HAs on AD.

Enhanced Nitrate Nitrogen Removal from Constructed Wetland via Fe3O4/Granular Activated Carbon Anode Microbial Electrolysis Cell under Low C/N Ratio

In this study, a constructed wetland–Fe3O4/granular activated carbon anode microbial electrolysis cell (CW-FMEC) was constructed to enhance denitrification in low COD/N ratio wastewater. The introduction of Fe3O4 boosted the expression of functional genes involved in the denitrification pathway, and the abundance of narG, nirS, and nosZ increased by 99.29%, 70.54%, and 132.18%, respectively, compared to CW. In addition, the content of c-type cytochromes (c-Cyts) and EPS were also enhanced in the CW-FMEC. The microbial communities study displayed that Thauera, Dechloromonas, and Arenimonas became the main genera for denitrification. The denitrification performance at different COD/N ratios was investigated in depth. Under optimal working circumstances, the CW-FMEC had an excellent nitrate removal rate (88.9% ± 1.12%) while accumulating nearly no NO2−-N or NH4+-N in the effluent. This study provides a new direction for the development of CW-MEC and accelerates its implementation.

In-situ and label-free measurement of cytochrome C concentration with a Ti2C-MXene sensitized fiber-optic MZI sensor

BackgroundThe concentration of cytochrome C is demonstrated to be an effective indicator of the microbial corrosion strength of metals. Traditional cytochrome C sensor can detect cytochrome C with a low detection limit, but their use is limited by their high cost, cumbersome operation, and susceptibility to malignant environments. In addition, studies on the monitoring of cytochrome C in the field of microbial corrosion has still not been carried out. Therefore, there is a need for a highly sensitive, selective, low-cost, anti-interference, and stable cytochrome C sensor with online monitoring and remote sensing capabilities for in-situ measurement of microbial corrosion strength. ResultsThis paper proposed a highly sensitive label-free fiber-optic sensor based on Mach-Zehnder interferometer (MZI) for in-situ measurement of the microbial corrosion marker cytochrome C. Two-dimensional Ti2C-MXene material is uniformly immobilized onto the surface of the sensing area to improve the sensitivity, hydrophilicity, and specific surface area of the sensing area, as well as to facilitate the immobilization of specific sensitive materials. The cytochrome C antibody is modified on the surface of Ti2C-MXene to specifically recognize cytochrome C, whose concentration variation can be measured by monitoring the spectral shift of MZI sensor. Results demonstrate a measurement sensitivity of 1.428 nm/μM for cytochrome C concentrations ranging from 0 to 7.04 μM. The detection limit of the sensor is calculated to be 0.392 μM with remarkable performance, including selectivity, stability, and reliability. Besides, the measurement result of the proposed sensor in real microbial corrosive environment is consistent with that of the ideal environment. Significance and noveltyThis is the first instance of achieving in-situ and label-free measurement of cytochrome C by using a fiber-optic MZI sensor, which undoubtedly provides a feasible solution for the effective monitoring of microbial metal corrosion in the environment.

Inhibitory mechanism of Cr(VI) on sulfur-based denitrification: Bio-toxicity, bio-electron characteristics, and microbial evolution

Sulfur-based denitrification is a promising technology for efficient nitrogen removal in low-carbon wastewater, while it is easily affected by toxic substances. This study revealed the inhibitory mechanism of Cr(VI) on thiosulfate-based denitrification, including bio-toxicity and bio-electron characteristics response. The activity of nitrite reductase (NIR) was more sensitive to Cr(VI) than that of nitrate reductase (NAR), and NIR was inhibited by 21.32 % and 19.86 % under 5 and 10 mg/L Cr(VI), resulting in 10.12 and 15.62 mg/L of NO2--N accumulation. The biofilm intercepted 36.57 % of chromium extracellularly by increasing 25.78 % of extracellular polymeric substances, thereby protecting microbes from bio-toxicity under 5 mg/L Cr(VI). However, it was unable to resist 20–30 mg/L of Cr(VI) bio-toxicity as 19.95 and 14.29 mg Cr/(g volatile suspended solids) invaded intracellularly, inducing the accumulation of reactive oxygen species by 165.98 % and 169.12 %, which triggered microbial oxidative-stress and damaged the cells. In terms of electron transfer, S2O32- oxidation was inhibited, and parts of electrons were redirected intracellularly to maintain microbial activity, resulting in insufficient electron donors. Meanwhile, the contents of flavin adenine dinucleotide and cytochrome c decreased under 5–30 mg/L Cr(VI), reducing the electron acquisition rate of denitrification. Thermomonas (the dominant genus) possessed denitrification and Cr(VI) resistance abilities, playing an important role in antioxidant stress and biofilm formation. Environmental ImplicationSulfur-based denitrification (SBD) is a promising method for nitrate removal in low-carbon wastewater, while toxic heavy metals such as Cr(VI) negatively impair denitrification. This study elucidated Cr(VI) inhibitory mechanisms on SBD, including bio-toxicity response, bio-electron characteristics, and microbial community structure. Higher concentrations Cr(VI) led to intracellular invasion and oxidative stress, evidenced by ROS accumulation. Moreover, Cr(VI) disrupted electron flow by inhibiting thiosulfate oxidation and affecting electron acquisition by denitrifying enzymes. This study provided valuable insights into Cr(VI) toxicity, which is of great significance for improving wastewater treatment technologies and maintaining efficient and stable operation of SBD in the face of complex environmental challenges.

Enhanced Sb(V) removal of sulfate-rich wastewater by anaerobic granular sludge assisted with Fe/C amendment

Antimony (Sb) and sulfate are two common pollutants in Sb mine drainage and Sb-containing textile wastewater. In this paper, it was found that iron‑carbon (Fe/C) enhanced Sb(V) removal from sulfate-rich wastewater by anaerobic granular sludge (AnGS). Sulfate inhibited Sb(V) removal (S + Sb, k = 0.101), while Fe/C alleviated the inhibition and increased Sb(V) removal rate by 2.3 times (Fe/C + S + Sb, k = 0.236). Fe/C could promote the removal of Sb(III), and Sb(III) content decreased significantly after 8 h. Meanwhile, Fe/C enhanced the removal of sulfate. The 3D-EEM spectrum of supernatant in Fe/C + S + Sb group (at 24 h) showed that Fe/C stimulated the production of soluble microbial products (SMP) in wastewater. SMP alleviated the inhibition of sulfate, promoting AnGS to reduce Sb(V). Sb(V) could be reduced to Sb(III) both by AnGS and sulfides produced from sulfate reduction. Further analysis of extracellular polymeric substances (EPS) and AnGS showed that Fe/C increased the adsorbed Sb(V) in EPS and the c-type cytochrome content in AnGS, which may be beneficial for Sb(V) removal. Sb(V) reduction in Fe/C + S + Sb group may be related to the genus Acinetobacter, while in Sb group, several bacteria may be involved in Sb(V) reduction, such as Acinetobacter, Pseudomonas and Corynebacterium. This study provided insights into Fe/C-enhanced Sb(V) removal from sulfate-rich wastewater.

Effects of exogenous riboflavin or cytochrome addition on the cathodic reduction of Cr(VI) in microbial fuel cell with Shewanella putrefaciens.

Bioreduction of Cr(VI) is recognized as a cost-effective and environmentally friendly method, attracting widespread interest. However, the slow rate of Cr(VI) bioreduction remains a practical challenge. Additionally, the direct removal efficiency of microbes for high concentrations of Cr(VI) is not ideal due to the toxicity. Therefore, this study investigated the effects of exogenous riboflavin or cytochrome on the cathodic reduction of Cr(VI) in microbial fuel cells. The results demonstrated that the exogenous riboflavin or cytochrome effectively improved the voltage output of the cells, with riboflavin increasing the voltage by 52.08%. Within the first 24h, the Cr(VI) removal ratio in the normal, cytochrome, and riboflavin groups was 14.3%, 29.3%, and 53.8%, respectively. And the final removal ratio was 55.1%, 69.1%, and 98.0%, respectively. These results showed different enhancement effects of riboflavin and cytochrome on Cr(VI) removal. The analysis of riboflavin and cytochrome contents revealed that the additions did not have a significant impact on the autocrine riboflavin of S. putrefaciens, but affected the autocrine cytochrome. SEM, XPS, and FTIR results confirmed the presence of reduced Cr(III) on the cathode, which formed precipitate and adhered to the cathode surface. The EDS analysis showed that the amount of Cr on the cathode in normal, cytochrome, and riboflavin groups was 4.71%, 6.37%, 7.56%, respectively, which was consistent with the voltage and Cr(VI) removal data. These findings demonstrated the significant enhancement of exogenous riboflavin or cytochrome on Cr(VI) reduction, thereby providing data reference for the future bio-assisted remediation of Cr(VI) pollution.

Exploration of mechanism of total triterpenoids from fruits of Chaenomeles speciosa against senescent GES-1 cells induced by D-galactose based on Gln/GLS1/α-KG metabolic axis and mitochondrial apoptosis signaling pathway

Total triterpenoids from the fruits of Chaenomeles speciosa(TCS) are active components in the prevention and treatment of gastric mucosal damage, which have potential anti-aging effects. However, it is still unclear whether TCS can improve gastric aging, especially its molecular mechanism against gastric aging. On this basis, this study explored the effect and mechanism of TCS on senescent GES-1 cells induced by D-galactose(D-gal) to provide scientific data for the clinical use of TCS to prevent gastric aging. GES-1 cells cultured in vitro and those transfected with overexpression GLS1(GLS1-OE) plasmid of glutaminase 1(GLS1) were induced to aging by D-gal, and then TCS and or GLS1 inhibitor bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl) ethyl sulfide(BPTES) were given. Cell survival rate, positive rate of β-galactosidase(SA-β-gal) staining, mitochondrial membrane potential(MMP), and apoptosis were investigated. GLS1 activity, levels of glutamine(Gln), glutamate(Glu), α-ketoglutarate(α-KG), urea, and ammonia in supernatant and cells were detected by enzyme-linked immunosorbent assay(ELISA) and colorimetric methods. The mRNA and protein expressions of GLS1 and the related genes of the mitochondrial apoptosis signaling pathway were measured by real-time fluorescence quantitative PCR and Western blot. The results manifested that compared with the D-gal model group and GLS1-OE D-gal model group, TCS significantly decreased the SA-β-gal staining positive cell rate and MMP of D-gal-induced senescent GES-1 cells and GLS1-OE senescent GES-1 cells, inhibited the survival of senescent cells, and promoted their apoptosis(P<0.01). It decreased the activity of GLS1 and the content of Gln, Glu, α-KG, urea, and ammonia in supernatant and cell(P<0.01), reduced the concentration of cytochrome C(Cyto C) in mitochondria and the mRNA and protein expressions of GLS1 and proliferating nuclear antigen in cells(P<0.01). The mRNA expression of Bcl-2 and Bcl-xl, the protein expression of pro-caspase-9 and pro-caspase-3, and the ratio of Bcl-2/Bax and Bcl-xl/Bad in cells were decreased(P<0.01). Cyto C concentration in the cytoplasm, the mRNA expressions of Bax, Bad, apoptosis protease activating factor 1(Apaf-1), and protein expressions of cleaved-caspase-9, cleaved-caspase-3, cleaved-PARP-1 were increased(P<0.01). The aforementioned results indicate that TCS can counteract the senescent GES-1 cells induced by D-gal, and its mechanism may be closely related to suppressing the Gln/GLS1/α-KG metabolic axis, activating the mitochondrial apoptosis pathway, and thereby accelerating the apoptosis of the senescent cells and eliminating senescent cells.

Electron Transfer of Cellobiose Dehydrogenase in Polyethyleneimine Films

AbstractCellobiose dehydrogenase (CDH) is applied as a bioelectrocatalyst in biosensors because its mobile cytochrome domain is capable of direct electron transfer. This study investigates the electron transfer mechanism of CDH molecules embedded in the polycation polyethyleneimine (PEI), which has been reported as a current‐boosting component of CDH‐based biosensors. By immobilizing different concentrations of CDH and its isolated cytochrome domain in PEI films, we found that increasing concentrations of cytochrome enhanced the film conductivity (up to 251±8 mS cm−1) through improved electron transfer between the protein redox centers. The increased electrical conductivity of the film contacts CDH molecules at a greater distance from the electrode. The cross‐linker poly(ethylene glycol) diglycidyl ether improves the packing and contacting of the cytochrome domains, whereas glutaraldehyde reduces the current obtained. Deglycosylation of CDH enhances the conductivity of enzyme‐polymer films by up to 34 %, implying a higher number of productive electron‐hopping events between cytochrome domains due to enhanced mobility or reduced shielding. By balancing negative charges on the CDH surface at neutral and alkaline pH, PEI increases the interdomain electron transfer and the electrical film conductivity. The resulting increased current output is relevant for in vivo bioanalytical applications.

Sodium sulfite-driven Helicobacter pylori eradication: Unraveling oxygen dynamics through multi-omics investigation

Due to the emergence and spread of multidrug resistance in Helicobacter pylori (H. pylori), its eradication has become difficult. Sodium sulfite (SS), a widely used food additive for ensuring food safety and storage, has been recognized as an effective nonbactericidal agent for H. pylori eradication. However, the mechanism by which H. pylori adapts and eventually succumbs under low- or no-oxygen conditions remains unknown. In this study, we aimed to evaluate the anti-H. pylori effect of SS and investigated the multiomics mechanism by which SS kills H. pylori. The results demonstrated that SS effectively eradicated H. pylori both in vitro and in vivo. H. pylori responds to the oxygen changes regulated by SS, downregulates the HcpE gene, which is responsible for redox homeostasis in bacteria, decreases the activities of enzymes related to oxidative stress, and disrupts the outer membrane structure, increasing susceptibility to oxidative stress. Furthermore, SS downregulates the content of cytochrome C in the microaerobic respiratory chain, leading to a sharp decrease in ATP synthesis. Consequently, the accumulation of triglycerides (TGs) in bacteria due to oxidative stress supports anaerobic respiration, meeting their energy requirements. The multifaceted death of H. pylori caused by SS does not result in drug resistance. Thus, screening of the redox homeostasis of HcpE as a new target for H. pylori infection treatment could lead to the development of a novel approach for H. pylori eradication therapy.

Magnetite-equipped algal-rich sediments for microbial fuel cells: Remediation of sediment organic matter pollution and mechanisms of remote electron transfer

Using the bio-electrochemical methods for the restoration of high algae sediments is full of potential and challenges. How to promote extracellular electron transfer (EET) process in microbial fuel cells (MFC) is the key bottleneck. The study had explored the potential application of magnetite on accelerating electron transfer for improving the output of MFC and sediment pollution remediation. The results indicated that the organic matter degradation rate showed a remarkable increase of 27.45 %, and the voltage output was approximately 1.68 times higher compared to the MFC configured with regular sediment. Abundant electroactive bacteria (EABs), such as Geobacter and Burkholderiaceae, and fermentative bacteria were responsible for these results, accompanied by the enhanced fluorescence of humic substances (HS), increased concentration and activity of cytochrome C (25.05 % and 21.12 %), as well as elevated extracellular polymeric substance content. Moreover, the intrinsic EET mechanisms among Fe-oxides, HS, and EABs were explored. According to the electrochemical analysis and substance transformation, the EET process involved four stages: magnetite-enhanced direct electron transfer via strong conductivity, iron respiration mediating electron transfer to the electrode, the model quinone substance acting as an electron shuttle facilitating EET and iron reduction, and iron cycling mediating electron transfer. This study provides an effective strategy for pollution remediation in algal-rich sediment, which was beneficial for the harmless treatment and resource utilization of both algae and sediment, simultaneously.

The ratio of cytochrome c oxidase subunit 4 isoform 4I1 and 4I2 mRNA is changed in permanent atrial fibrillation.

The conditions of hypoxia are suggested to induce permanent atrial fibrillation (AF). The regulation of COX4I2 and COX4I1 depends on oxygen availability in tissues. A role of COX4I2 in the myocardium of AF patients is supposed for pathogenesis of AF and subsequent alterations in the electron transfer chain (ETC) under hypoxia. In vitro, influence of hypoxia on HeLa 53 cells was studied and elevated parts of COX 4I2 were confirmed. Myocardial biopsies were taken ex vivo from the patients' Right Atria with SR (n=31) and AF (n=11), respectively. RT- PCR for mRNA expresson, mitochondrial respiration by polarography and the protein content of cytochrome c oxidase (CytOx) subunit 4I1 and CytOx subunit 4I2 by ELISA were studied. Clinical data were correlated to the findings of gene expressions in parallel. Patients with permanent AF had a change in isoform 4I2/4I1 expression along with a decrease of isoform COX 4I1 expression. The 4I2/4I1 ratio of mRNA expression was increased from 0.630 to 1.058 in comparison. However, the protein content of CytOx subunit 4 was much lower in the AF group, whereas the respiration/units enzyme activity in both groups remained the same. This study describes a possible molecular correlate for the development of AF. Due to the known functional significance of COX 4I2, mitochondrial dysfunction can be assumed as a part of the pathogenesis of AF.

Validation of mitochondrial function measurements in permeabilized myocardial tissue from porcine left ventricular specimens with ischemia/reperfusion

Abstract Background Mitochondrial function is critical to myocardial ischemia/reperfusion injury and cardioprotection. The measurement of mitochondrial function in isolated mitochondria requires cardiac specimens of about 300 mg and is therefore only possible at the end of an animal experiment or during cardiosurgical interventions in humans. As an alternative, permeabilized myocardial tissue can be used for quantification of mitochondrial respiration in small myocardial specimens of about only 2-5 mg, which can be retrieved by sequential biopsies in animal experiments and during cardiac catheterization in humans. Whether or not mitochondrial dysfunction during ischemia/reperfusion is reflected to the same extent in saponin-permeabilized myocardial tissue specimens (PMT) and in isolated mitochondria is still unclear. Purpose We attempted to validate measurements of mitochondrial respiration from PMT by comparison to those from isolated mitochondria in left ventricular myocardium from anesthetized pigs undergoing coronary occlusion/reperfusion. Methods Göttingen open-chest, anesthetized minipigs (n=10) were subjected to 60 min distal left anterior descending artery occlusion and 180 min reperfusion. At the end of reperfusion, PMT and isolated mitochondria were prepared from remote and ischemic/reperfused myocardium. Baseline, adenosine diphosphate-stimulated complex I (without/with cytochrome c, to account for absence or presence of the outer mitochondrial membrane integrity) and complex IV respiration was measured at 37°C during magnetic stirring using a high-resolution respirometer. Mitochondrial respiration in PMT and isolated mitochondria was normalized to protein content and the content of the mitochondrial marker protein cytochrome c oxidase 4. Results Mitochondrial respiration measurements in PMT and isolated mitochondria correlated well (Figure 1A) and agreed well in Bland-Altman plot (Figure 1B). While baseline respiration in PMT was somewhat higher than that in isolated mitochondria (Figure 2A), mitochondrial dysfunction during ischemia/reperfusion was equally reflected in PMT and isolated mitochondria (Figure 2B & 2C). Complex IV respiration was not different between remote myocardium and ischemic/reperfused myocardium, independently of the preparation, indicating a comparable amount of viable mitochondria (Figure 2D). Conclusion Mitochondrial function measurements in PMT can substitute for that in isolated mitochondria to reflect their dysfunction in ischemia/reperfusion. Thus, mitoprotective strategies targeting ischemia/reperfusion injury in small tissue samples and also in human myocardial biopsies can be followed in PMT.Figure 1Figure 2

Muscone inhibits opening of mPTP to alleviate OGD/R-induced injury of HT22 cells

This study aims to investigate the mechanism of muscone in inhibiting the opening of mitochondrial permeability transition pore(mPTP) to alleviate the oxygen and glucose deprivation/reoxygenation(OGD/R)-induced injury of mouse hippocampal neurons(HT22). An in vitro model of HT22 cells injured by OGD/R was established. CCK-8 assay was employed to examine the viability of HT22 cells, fluorescence microscopy to measure the mitochondrial membrane potential, the content of reactive oxygen species(ROS), and the opening of mPTP in HT22 cells. Enzyme-linked immunosorbent assay was employed to determine the level of ATP and the content of cytochrome C(Cyt C) in mitochondria of HT22 cells. Flow cytometry was employed to determine the Ca~(2+) content and apoptosis of HT22 cells. The expression of Bcl-2(B-cell lymphoma-2) and Bcl-2-associated X protein(Bax) was measured by Western blot. Molecular docking and Western blot were employed to examine the binding between muscone and methyl ethyl ketone(MEK) after pronase hydrolysis of HT22 cell proteins. After the HT22 cells were treated with U0126, an inhibitor of MEK, the expression levels of MEK, p-ERK, and CypD were measured by Western blot. The results showed that compared with the OGD/R model group, muscone significantly increased the viability, mitochondrial ATP activity, and mitochondrial membrane potential, lowered the levels of ROS, Cyt C, and Ca~(2+), and reduced mPTP opening to inhibit the apoptosis of HT22 cells. In addition, muscone up-regulated the expression of MEK, p-ERK, and down-regulated that of CypD. Molecular docking showed strong binding activity between muscone and MEK. In conclusion, muscone inhibits the opening of mPTP to inhibit apoptosis, thus exerting a protective effect on OGD/R-injured HT22 cells, which is associated with the activation of MEK/ERK/CypD signaling pathway.

Effect of COVID-19 mRNA Vaccine on Human Lung Carcinoma Cells In Vitro by Means of Raman Spectroscopy and Imaging.

The effect of COVID-19 mRNA vaccine on human lung epithelial carcinoma cells (A549) in vitro as a convenient preclinical model was studied by means of Raman spectroscopy and imaging. The article focuses on Raman imaging as a tool to explore apoptosis and oxidative phosphorylation in mitochondrial dysfunctions. The Raman results demonstrate alterations in the oxidation-reduction pathways associated with cytochrome c. We found that the COVID-19 mRNA vaccine downregulates the concentration of cytochrome c upon incubation with tumorous lung cells. The concentration of the oxidized form of cytochrome c in the mitochondria of lung cells decreases upon incubation with the COVID-19 mRNA vaccine. A lower concentration of oxidized cytochrome c in mitochondria illustrates lower effectiveness of oxidative phosphorylation (respiration), reduced apoptosis, and lessened ATP production. Moreover, mRNA vaccine significantly increases de novo lipids synthesis in lipid droplets up to 96 h and alterations in biochemical composition. It seems that the lipid composition of cells returns to the normal level for a longer incubation time (14 days). In the cell nucleus, the mRNA vaccine does not produce statistically significant changes.

Yak-Derived CXCL14 Activates the Pro-Inflammatory Response of Macrophages and Inhibits the Proliferation and Migration of HepG2.

CXCL14 (C-X-C motif chemokine ligand 14) is an important chemokine involved in infection and immunity and plays an important role in a variety of immune-related diseases. The 446 bp cDNA sequence of the CXCL14 gene in yaks was obtained. Additionally, the prokaryotic expression vector of the CXCL14 protein with a molecular weight of 27 kDa was successfully constructed and expressed. The proliferation activities and migration abilities of spleen macrophages were significantly inhibited after treatment with the CXCL14 protein at different concentrations (1, 10 and 20 μg/mL) (p < 0.05). Furthermore, the expressions of pro-inflammatory cytokines interleukin 1 beta (IL-1β), interleukin 6 (IL6), interleukin 8 (IL8) and interferon-α (TNF-α) were significantly increased (p < 0.05), but the expression of anti-inflammatory factor interleukin 10 (IL10) was significantly decreased (p < 0.05). The contents of inflammatory factors in the supernatant of cells were detected using ELISA, and it was also found that the contents of TNF-α, IL6 and cytochrome c oxidase subunit II (COX2) were significantly increased under different CXCL14 protein concentrations (p < 0.05). Finally, the exogenous addition of CXCL14 inhibited the activity, clonal formation and migration of hepatoma cells (HepG2). Additionally, after HepG2 cells were treated with 20 μg/mL CXCL14 protein for 12 h, 24 h and 36 h, the expression levels of BCL2 homologous antagonist/killer (BAK) and the BCL2-associated X apoptosis regulator (BAX) were increased to varying degrees, while the expression levels of hypoxia-inducible factor 1 subunit alpha (HIF1A), the mechanistic target of rapamycin kinase (mTOR) and cyclin-dependent kinase 1 (CDK1) genes decreased compared to the control group. In conclusion, the CXCL14 protein can inhibit the proliferation and migration of HepG2 cells by inducing the expression of macrophage pro-inflammatory factors and activating apoptosis-related genes to exert innate immunity. These results are helpful to further study the function of the CXCL14 protein and provide research data for the innate immune mechanism of yaks under harsh plateau environments.