Electron Transport Chain Pathways Research Articles

Single cell regulatory architecture of human pancreatic islets suggests sex differences in β cell function and the pathogenesis of type 2 diabetes.

Biological sex affects the pathogenesis of type 2 and type 1 diabetes (T2D, T1D) including the development of β cell failure observed more often in males. The mechanisms that drive sex differences in β cell failure is unknown. Studying sex differences in islet regulation and function represent a unique avenue to understand the sex-specific heterogeneity in β cell failure in diabetes. Here, we examined sex and race differences in human pancreatic islets from up to 52 donors with and without T2D (including 37 donors from the Human Pancreas Analysis Program [HPAP] dataset) using an orthogonal series of experiments including single cell RNA-seq (scRNA-seq), single nucleus assay for transposase-accessible chromatin sequencing (snATAC-seq), dynamic hormone secretion, and bioenergetics. In cultured islets from nondiabetic (ND) donors, in the absence of the in vivo hormonal environment, sex differences in islet cell type gene accessibility and expression predominantly involved sex chromosomes. Of particular interest were sex differences in the X-linked KDM6A and Y-linked KDM5D chromatin remodelers in female and male islet cells respectively. Islets from T2D donors exhibited similar sex differences in differentially expressed genes (DEGs) from sex chromosomes. However, in contrast to islets from ND donors, islets from T2D donors exhibited major sex differences in DEGs from autosomes. Comparing β cells from T2D and ND donors revealed that females had more DEGs from autosomes compared to male β cells. Gene set enrichment analysis of female β cell DEGs showed a suppression of oxidative phosphorylation and electron transport chain pathways, while male β cell had suppressed insulin secretion pathways. Thus, although sex-specific differences in gene accessibility and expression of cultured ND human islets predominantly affect sex chromosome genes, major differences in autosomal gene expression between sexes appear during the transition to T2D and which highlight mitochondrial failure in female β cells.

Sustained release of a novel bilayer packaging film loaded with binary microemulsion of melatonin/pummelo essential oil and its regulation of postharvest energy metabolism in Agaricus bisporus

Bio-based active soft packaging materials have excellent potential to alleviate existing environmental problems caused by conventional packaging products and delay the postharvest aging of fruit and vegetables. In this study, a novel bilayer packaging film (G/T-M-E) of glutenin/tamarind gum loaded with binary microemulsion of melatonin/pummelo essential oil was prepared. The results indicated that the G/T-M-E packaging had superior barrier and mechanical properties. Importantly, the melatonin (MT) and pomelo essential oil (PEO) loaded in soft packaging had good sustained-release properties, which could ensure release characteristics in different food simulants. Among them, MT had the highest release efficiency in 10% ethanol, while PEO is more easily released in distilled water. Meanwhile, MT and PEO migrated from the packaging film from the inside-out via different diffusion effects, which was beneficial for the synergistic effect of active ingredients. G/T-M-E maintained a higher level of ATP of white mushrooms, greater energy charge levels and higher activity of succinate dehydrogenase (SDH) and cytochrome c oxidase (CCO). The energy charge (EC) of mushrooms packaged in G/T-M-E was as high as 0.65, which was 1.43 times higher than that of the G/T treatment group on day 12. The SDH and CCO activity of the G/T-M-E treatment group were 2.62 and 2.19 times higher than those of the G/T group on day 10, respectively. Overall, the G/T-M-E film improved the electron transfer efficiency, reduced the excess active oxygen content, maintained the high energy state, regulated the tricarboxylic acid cycle and electron transport chain pathways, indicating that the film has the active potential to regulate the energy state of postharvest mushrooms.

Deciphering insights into the binding mechanism and plasticity of Telacebec with M. tuberculosis cytochrome bcc-aa3 supercomplex through an unbiased molecular dynamics simulation, free-energy analysis, and DFT study

The cytochrome bcc-aa3 supercomplex, a key component in the electron transport chain pathway involved in bacterial energy production and homeostasis, is a clinically validated target for tuberculosis (TB), leading to Telacebec (Q203). Telacebec is a potent candidate drug under Phase II clinical development for the treatment of drug-sensitive and drug-resistant TB. Recently, the cryo-electron microscopy structure of this supercomplex from Mycobacterium tuberculosis (Mtb) complexed with Q203 was resolved at 6.9 Å resolution (PDB ID: 7E1W). To understand the binding site (QP site) flexibility and Q203’s stability at the QP site of the Mtb cytochrome bcc complex, we conducted molecular dynamics (MD) simulation and free energy analysis on this complex in an explicit hydrated lipid bilayer environment for 500 ns. Through this study, the persistence of a range of direct and indirect interactions was observed over the course of the simulation. The significance of the interactions with His375, Tyr161, Ala178, Ala179, Ile183, His355, Leu356, and Thr313 is underlined. Electrostatic energy was the primary source of the net binding free energy, regardless of the important interacting residues. The overall binding free energy for Q203 was −112.84 ± 7.73 kcal/mol, of which the electrostatic and lipophilic energy contributions were −116.31 ± 1.14 and −21.32 ± 2.35 kcal/mol, respectively. Meanwhile, DFT calculations were utilized to elucidate Q203’s molecular properties. Overall, this study deciphers key insights into the cytochrome bcc-aa3 supercomplex with Q203 on the ground of molecular mechanics and quantum mechanics that may facilitate structure-based drug design and optimization for the discovery of the next-generation antitubercular drug(s). Communicated by Ramaswamy H. Sarma

Antioxidants As a Novel Treatment to Revert Impaired Angiogenic Potential Driven By Metabolic Alterations Observed in Shwachman-Diamond Syndrome Derived Mesenchymal Stromal Cells

Introduction Shwachman-Diamond Syndrome (SDS) is a rare bone marrow (BM) failure disorder. SDS BM biopsies show tortuous and collapsed vessels, highlighting angiogenic abnormalities. Our group previously demonstrated that SDS BM mesenchymal stromal cells (MSCs) have in vitro and in vivo impaired angiogenic potential compared to healthy donors (HD)-MSCs. Interestingly, recent evidence underlines that pathological angiogenesis could be accompanied by altered metabolism and that antioxidants could represent a novel strategy to target angiogenic defects. Aims We aimed to characterize the metabolic status of SDS-MSCs and to investigate if the treatment with antioxidants may restore their impaired angiogenic potential. Methods BM derived HD- and SDS-MSCs were stimulated with DMEM ± N-Acetylcysteine (NAC, 1mM) or dimethylsulfoxide (DMSO, 0.05% v/v) for 48h. Oxidative phosphorylation (OxPhos) was assayed by oximetry and bioluminescent ATP synthesis assay. Spectrophotometric analyses were performed to evaluate ATP/AMP ratio, malondialdehyde (MDA) level, and Complex IV and lactate dehydrogenase (LDH) activity. The amount of reactive oxygen species (ROS) was quantified by flow cytometry. The angiogenic capability was evaluated by performing in vitro Matrigel angiogenesis assay. Results Firstly, we evaluated OxPhos oxygen consumption and ATP production (n=6). Concerning the I-III-IV mitochondrial complexes pathway, SDS-MSCs consumed 57% less oxygen (p=0.004) and produced 64% less ATP compared to HD-MSCs (p=0.002). Accordingly, the analysis of the II-III-IV complexes pathway demonstrated that the oxygen consumption was reduced by 62% (p=0.002) and the ATP synthesis was 67% lower than HD-MSCs (p=0.002). Furthermore, the P/O ratio, index of OxPhos efficiency, was significantly reduced in SDS-MSCs in both the electron transport chain pathways (p=0.002 for both). Therefore, we demonstrated that Complex IV activity was 61% lower in SDS- vs HD-MSCs (p=0.002), highlighting its role in the SDS OxPhos defect. As for the energetic status, SDS-MSCs showed a low intracellular ATP/AMP ratio (mean=1.1, range=0.8-1.6 vs mean=3.6, range=3.1-4.0, in HDs; p=0.002) that was accompanied by 30% increase of LDH activity in SDS-MSCs (p=0.002), an expression of increased anaerobic glycolysis to compensate the mitochondrial defect. The amount of ROS in SDS-MSCs was increased by 27% compared to HD-MSCs (n=5) and also the SDS-MSCs lipid peroxidation level was significantly higher over HDs (mean=12.7 µM of MDA/mg, range=10.5-14.1µM/mg vs mean=6.6µM/mg, range=5.9-7.4µM/mg; p=0.002). Then we treated SDS-MSCs for 48h with NAC, a broad-range antioxidant (n=5), or with DMSO, which acts at very low concentrations as scavenger, specifically on lipid peroxidation products (n=6). Interestingly, NAC and DMSO stimulated SDS-MSCs increased by 75% the oxygen consumption and by 70% the ATP synthesis in both the electron transport chain pathways, thus resulting in levels comparable to HD-MSCs. The SDS OxPhos restoration was respectively associated to a 53% and 60% increase of Complex IV activity after NAC and DMSO stimulation (p<0.0001 for both). Moreover, antioxidants corrected the SDS energetic defect by restoring ATP/AMP ratio and LDH activity to the levels of HDs. Importantly, SDS-MSCs lipid peroxidation level was drastically reduced after NAC and DMSO treatments (mean=12.7µM/mg, range=10.5-14.1µM/mg vs NAC mean=5.1µM/mg, range=4.3-5.4µM/mg and DMSO mean=6.2µM/mg, range=5.8-6.5µM/mg; p<0.0001 for both). Finally, we approached SDS-MSCs angiogenic defect and showed that SDS-MSCs defective capability to recreate a defined capillary-like network under angiogenic stimuli was completely restored after antioxidant stimulations (n≥5). As shown by ImageJ Angiogenesis Analyzer several angiogenic elements, including branches and segments, were significantly increased in NAC and DMSO stimulated SDS-MSCs that become comparable to HD-MSCs. Conclusions We demonstrated that the altered OxPhos metabolism of SDS-MSCs significantly contributes to their angiogenic defect and, importantly, that antioxidants restored the metabolic alterations and the angiogenic potential in SDS-MSCs, paving the way for new therapeutic strategies.

Maternal Obesity Programs the Premature Aging of Rat Offspring Liver Mitochondrial Electron Transport Chain Genes in a Sex-Dependent Manner.

We investigated whether maternal obesity affects the hepatic mitochondrial electron transport chain (ETC), sirtuins, and antioxidant enzymes in young (110 postnatal days (PND)) and old (650PND) male and female offspring in a sex- and age-related manner. Female Wistar rats ate a control (C) or high-fat (MO) diet from weaning, through pregnancy and lactation. After weaning, the offspring ate the C diet and were euthanized at 110 and 650PND. The livers were collected for RNA-seq and immunohistochemistry. Male offspring livers had more differentially expressed genes (DEGs) down-regulated by both MO and natural aging than females. C-650PND vs. C-110PND and MO-110PND vs. C-110PND comparisons revealed 1477 DEGs in common for males (premature aging by MO) and 35 DEGs for females. Analysis to identify KEGG pathways enriched from genes in common showed changes in 511 and 3 KEGG pathways in the male and female livers, respectively. Mitochondrial function pathways showed ETC-related gene down-regulation. All ETC complexes, sirtuin2, sirtuin3, sod-1, and catalase, exhibited gene down-regulation and decreased protein expression at young and old ages in MO males vs. C males; meanwhile, MO females down-regulated only at 650PND. Conclusions: MO accelerates the age-associated down-regulation of ETC pathway gene expression in male offspring livers, thereby causing sex-dependent oxidative stress, premature aging, and metabolic dysfunction.

Enhanced cyanide-resistant respiration as the predominant respiratory metabolic pathway in abnormal chilling injury behavior of postharvest papaya

An increasing number of studies have demonstrated that reactive oxygen species (ROS) play a crucial role in the chilling injury (CI) of postharvest fruit and vegetables. This is not only associated with the free radical scavenging system but is also intimately connected to the electron transport chain pathway. However, it remains unclear whether ROS is associated with the electron transport chain in the abnormal chilling behavior observed in postharvest papaya. As such, this study investigated the electron transport chain of papaya fruit stored at 1 ℃ and 6 ℃ and its connection to ROS. In comparison to storage at 6 ℃, storage at 1 ℃ suppressed the expression of CpCCO, and induced the upregulation of CpAOX expression in papaya fruit. Throughout the middle and late stages of storage, there was an increase in pyruvate content and ubiquinone reduction level. The increase had two effects: it inhibited cytochrome oxidase (CCO) activity and promoted alternative oxidase (AOX) activity, leading to a blocked cytochrome pathway and an increased proportion of cyanide-resistant respiratory pathways. Consequently, the cyanide-resistant respiratory pathway supplanted the cytochrome pathway as the primary electron transport pathway. This transition effectively curbed the accumulation of ROS, thereby diminishing the damage degree of fruit cell membrane, boosting chilling tolerance, and ultimately inhibiting the CI of papaya fruit, which led to the emergence of abnormal chilling injury.

Neurotoxicity of mycotoxin citrinin: Novel evidence in developing zebrafish and underlying mechanisms in human neuron cells.

Citrinin (CTN) is a mycotoxin that is found as a contaminant in various types of food/feed grains and fermented food supplements. Previous studies have already established the nephrotoxicity and hepatotoxicity of CTN, but the neurotoxicity of CTN has not been clearly examined. In this study, CTN at 2-20μM was first found to interfere with the neural ganglia formation and locomotive behavior of embryonic zebrafish, a vertebrate animal model, at 24 hpf and 6 dpf, respectively. Further exposure of human neuroblastoma SH-SY5Y cells to 10 and 20μM CTN for 72h indicated that pathways responsible for neuron differentiation and projection guidance were down-regulated while oxidative stress and electron transport chain pathways were up-regulated based on the enrichment results of GSEA in the transcriptomic profiling. PCR analysis verified that CTN significantly down-regulated the expression of marker genes involved in neuron differentiation and synaptic signaling. CTN at the doses impairing cellular neurite outgrowth did not trigger mitochondrial oxidative stress and dysfunction. The neurotoxic mechanisms of CTN provide new information that is valuable in the assessment of CTN-related health risk for the general public.

Study of Angiogenic Potential and Oxidative Metabolism in Mesenchymal Stromal Cells Derived from Shwachman-Diamond Syndrome Patients

Introduction: Shwachman-Diamond Syndrome (SDS) is a rare autosomal recessive bone marrow (BM) failure disorder. SDS BM biopsies showed tortuous vessels with collapsed lumens, which might promote progression of malignant clones. Mesenchymal stromal cells (MSCs) are crucial elements in the BM niche homeostasis. Our group previously demonstrated that the in vivo ability of semi-cartilaginous pellets derived from SDS-MSCs to generate complete heterotopic ossicles was severely impaired in comparison with healthy donors (HDs). In addition, after specific angiogenic stimuli, SDS-MSCs showed an in vitro defective capability to form a well-defined tubular network. The aim of this study was to deeply investigate the cellular and molecular mechanisms underlying the SDS-MSCs impaired angiogenic potential. Methods: The angiogenic capability of BM derived SDS- and HD-MSCs was evaluated by performing in vitro Matrigel angiogenesis assay, by analysing cell viability and the expression of key angiogenic molecules before and after the angiogenic assay (RT-PCRs and/or ELISA). Concerning energy metabolism, oxidative phosphorylation (OxPhos) has been assayed by oximetry and bioluminescent ATP synthesis assay, and ATP/AMP ratio, malondialdehyde (MDA) level, and complex IV and lactate dehydrogenase (LDH) activity have been evaluated by spectrophotometric analyses. Results: We confirmed the impaired angiogenic potential of SDS-MSCs, previously observed in a restricted cohort of patients (n=6), in 13 SDS- compared to 14 HD-MSCs. In detail, after 3h of angiogenic stimuli, SDS-MSCs showed a defective capability to recreate a defined capillary-like network compared to HD-MSCs in a Matrigel-based assay. Accordingly, ImageJ Angiogenesis Analyzer showed that several angiogenic elements, including branches and meshes, were significantly reduced in SDS-MSCs. Furthermore, we demonstrated that the angiogenic defect of SDS-MSCs was correlated neither to a lower cellular viability than HD-MSCs, nor to a different expression of the main angiogenic molecules. Different reports showed a close relationship between aberrant angiogenesis and oxidative stress. Based on previous studies that demonstrated a defective energy metabolism and an increased oxidative stress in SDS patient-derived lymphoblastoid cells, we performed metabolic analyses of 6 SDS- and 6 HD-MSCs from the cohort used for angiogenic assay. In detail, we assayed OxPhos metabolism, evaluating oxygen consumption and ATP production. Concerning the I, III, and IV complexes pathway, we demonstrated that SDS-MSCs consumed 54% less oxygen (p=0.009) and they produced 60% less ATP compared to HD-MSCs (p=0.004). Accordingly, the analysis of the II, III, and IV complexes pathway demonstrated that the oxygen consumption was reduced by 61% (p=0.002) and the ATP synthesis was 67% lower than HD-MSCs (p=0.002). In addition, the P/O ratio, an index of OxPhos efficiency, was significantly reduced in SDS-MSCs in both electron transport chain pathways (p=0.004 for both). Moreover, OxPhos defect of SDS-MSCs was associated to the decreased enzymatic activity of complex IV compared to HD-MSCs (mean=9.5mU of oxidated cytochrome c/mg protein, range=7.8-11.5mU/mg vs mean=25.6mU/mg, range=21.6-31.9mU/mg; p=0.002). Consequently, SDS-MSCs showed a low intracellular ATP/AMP ratio (mean=1.1, range=0.8-1.6 vs mean=3.6, range=3.1-4.0, in HDs; p=0.002). This decrease was accompanied by an increased LDH activity (mean=0.4mU/mg protein, range=0.4-0.6mU/mg vs mean=0.3mU/mg, range=0.27-0.32mU/mg, in HDs; p=0.0022) in SDS-MSCs, showing an attempt to compensate the mitochondrial defect by the anaerobic glycolysis enhancement. Finally, we demonstrated that the level of lipid peroxidation of SDS-MSCs was significantly increased compared to HDs (mean=11.8µM of MDA/mg protein, range=10.4-15.2µM/mg vs mean=6.5µM/mg, range=5.0-7.7µM/mg; p=0.0022), suggesting an oxidative stress production increment. Conclusions: We demonstrated that the angiogenic defect in SDS-MSCs coexists with metabolic alterations. In addition, we demonstrated for the first time to our knowledge, that SDS-MSCs showed a defective oxidative metabolism, characterized by a decreased energy production and a high level of oxidative damage. These results underline the key role of MSCs in SDS BM niche and provide new insights into the pathogenesis of this rare disease.

Identification of Global miRNA Targetomes within Human Hematopoietic Progenitors and Acute Myeloid Leukemia Uncovers Unique Molecular Networks Centered on Mitochondria Function in Leukemic Stem Cells

Previous studies comparing transcriptome and proteome profiles of the cells that constitute both the normal and malignant hematopoietic hierarchies have revealed substantial uncoupling of transcript and protein expression levels. Despite the widely established role of miRNAs in regulating many cellular functions, the precise contribution of miRNA-mediated post-transcriptional regulation linking gene expression and proteome signatures of hematopoietic stem cells (HSC) and leukemic stem cells (LSC) remains largely unknown. Previous efforts to characterize miRNA targets have largely relied on computational prediction algorithms, which suffer from high rates of false-positive and negative target predictions and ultimately preclude discovery of non-canonical miRNA-target interactions. In this study, we perform Argonaute 2 (AGO2) crosslinking and immunoprecipitation (CLIP)-seq to experimentally map endogenous miRNA-target interactions in human CD34+ hematopoietic stem and progenitor cells (HSPC) from cord blood and six primary AML patient samples in an unbiased, transcriptome-wide manner (Manakov et al. 2022). Using this approach, we identify hundreds of miRNAs directly ligated to thousands of unique targets from miRNA-target chimeric sequencing reads. We find limited overlap (~30%) between miRNA targets captured by CLIP-seq and computationally predicted targets, underscoring the importance of functional validation of miRNA targets in specific cell types. Surprisingly, we find remarkable similarity between abundant miRNAs from HSPC and AML samples, including miR-142 and miRNAs encoded by the miR-17-92 cluster. However, the targets and the gene regulatory networks controlled by these highly abundant miRNAs are vastly different between HSPC and AML. In HSPC, we find that targets of abundant miRNAs are enriched in cell cycle regulation, histone modification and megakaryocyte/myeloid differentiation. In contrast, targets of the same miRNAs are enriched in protein localization/translation, neutrophil function, regulation of apoptosis and electron transport chain (ETC) pathways in AML. Collectively, we find that the composition of the abundant miRNAs is similar in normal HSPC and AML tumors; however they control divergent gene regulatory networks. To identify miRNA-target networks that play functional roles in LSC, we performed small RNA sequencing in 23 AML patient samples where each sorted fraction was assessed for LSC content by xenotransplantation. We derived an LSC-specific miRNA signature consisting of 5 miRNAs (LSC miR-5 score) predictive of overall survival and capable of stratifying AML patients independent of mRNA expression and other established indicators of outcome. Comparison of CLIP-seq targets of the LSC-specific miRNAs from HSPC and AML patient blasts revealed the existence of AML-specific gene regulatory networks centred on oxidative phosphorylation, ETC and cell-matrix adhesion. We further interrogated the functional significance of one miRNA from the LSC miR-5 score, miR-let-7c. We find that miR-let-7c is highly expressed in LSC-enriched populations of two AML in vitro models that recapitulate the leukemic hierarchy. Enforced expression of miR-let-7c in OCI-AML22 cells resulted in expansion of the LSC-enriched CD34+CD38- population and reduction in the CD34- terminally differentiated blast population. Comparison of miR-let-7c targets from HSPC and AML patient samples revealed the existence of distinct gene regulatory networks. In HSPC, miR-let-7c targets are enriched in cell cycle and regulation of transcription, whereas in AML, mitochondrial ETC, oxidative phosphorylation, and cell-matrix adhesion were the most enriched pathways. Moreover, analysis of miR-let-7c targets identified a high prevalence of transcripts encoded by the mitochondrial (mt) DNA (MT-ATP6, MT-ATP8, MT-ND1, -2, -4, -5, MT-CO1), pointing towards a largely uncharacterized function of miRNAs in regulating transcripts encoded by mtDNA in AML. Collectively, miRNA sequencing in LSC-enriched AML samples and identification of miRNA targetomes in HSPC and AML patient samples have revealed unexpected mechanisms of post-transcriptional regulation in normal and malignant hematopoietic stem cells.

Long-term effects of early postnatal stress on Sertoli cells.

Sertoli cells are somatic cells in testis essential for spermatogenesis, that support the development, maturation, and differentiation of germ cells. Sertoli cells are metabolically highly active and physiologically regulated by external signals, particularly factors in the blood stream. In disease conditions, circulating pathological signals may affect Sertoli cells and consequentially, alter germ cells and fertility. While the effects of stress on reproductive cells have been well studied, how Sertoli cells respond to stress remains poorly characterized. We used a mouse model of early postnatal stress to assess the effects of stress on Sertoli cells. We developed an improved strategy based on intracellular stainings and obtained enriched preparations of Sertoli cells from exposed males. We show that adult Sertoli cells have impaired electron transport chain (ETC) pathways and that several components of ETC complexes particularly complex I, III, and IV are persistently affected. We identify serum as potential mediator of the effects of stress on Sertoli cells by showing that it can recapitulate ETC alterations in primary cells. These results highlight Sertoli cells as cellular targets of stress in early life that can keep a trace of exposure until adulthood.

SLCO4A1 expression is associated with activated inflammatory pathways in high-grade serous ovarian cancer.

Patients with high-grade serous ovarian cancer (HGSOC) have a very poor overall survival. Current therapeutic approaches do not bring benefit to all patients. Although genetic alterations and molecular mechanisms are well characterized, the molecular pathological conditions are poorly investigated. Solute carrier organic anion transporter family member 4A1 (SLCO4A1) encodes OATP4A1, which is an uptake membrane transporter of metabolic products. Its expression may influence various signaling pathways associated with the molecular pathophysiological conditions of HGSOC and consequently tumor progression. RNA sequencing of 33 patient-derived HGSOC cell lines showed that SLCO4A1 expression was diverse by individual tumors, which was further confirmed by RT-qPCR, Western blotting and immunohistochemistry. Gene Set Enrichment Analysis revealed that higher SLCO4A1 level was associated with inflammation-associated pathways including NOD-like receptor, adipocytokine, TALL1, CD40, NF-κB, and TNF-receptor 2 signaling cascades, while low SLCO4A1 expression was associated with the mitochondrial electron transport chain pathway. The overall gene expression pattern in all cell lines was specific to each patient and remained largely unchanged during tumor progression. In addition, genes encoding ABCC3 along with SLCO4A1-antisense RNA 1, were associated with higher expression of the SLCO4A1, indicating their possible involvement in inflammation-associated pathways that are downstream to the prostaglandin E2/cAMP axis. Taken together, increased SLCO4A1/OATP4A1 expression is associated with the upregulation of specific inflammatory pathways, while the decreased level is associated with mitochondrial dysfunction. These molecular pathophysiological conditions are tumor specific and should be taken into consideration by the development of therapies against HGSOC.

Copper Modulates Mitochondrial Oxidative Phosphorylation to Enhance Dermal Papilla Cells Proliferation in Rex Rabbits.

Copper (Cu) is an important coenzyme factor in cell signaling, such as cytochrome c oxidase (Complex IV). Metabolism plays an important role in regulating the fate of mammalian cells. The aim of this study is to experimentally investigate the effect of copper on cell metabolism in the dermal papilla cells of the Rex rabbit. In this study, Cu promoted proliferation of dermal papilla cells (p = 0.0008) while also increasing levels of cellular CIII, CIV, Complex IV and ATP. Moreover, fifty metabolites that were significantly different between Cu and controls were identified as potential biomarkers of Cu stimulation. Copper-stimulated cells had altered levels of arachidonic acid derivatives, S-glutamic acid, and citric acid, which were primarily linked to two different pathways: arachidonic acid metabolism (p < 0.0001) and alanine, aspartate and glutamate metabolism (p = 0.0003). The addition of Cu can increase the proliferation of Rex rabbit dermal papilla cells. Increased levels of ubiquinol-cytochrome c reductase complex core protein 2 (CIII) and cytochrome c oxidase subunit 1 (CIV) were associated with the increased levels of cellular cytochrome c oxidase (Complex IV) and adenosine triphosphate (ATP). In a word, copper promotes cell proliferation by maintaining the function of the cellular mitochondrial electron transport chain (ETC) pathway.

Abstract 13142: Combined Treatment With Dapagliflozin and Exenatide is More Cardioprotective Than Either Agent Alone in a Non-Diabetic Mouse Model of Ischemia-Reperfusion Injury

Introduction: Sodium-glucose cotransporter 2 inhibitors (SGLT2i) and glucagon-like peptide-1 receptor agonists (GLP-1RA) have individually shown cardiovascular benefits in patients with type 2 diabetes (T2D). SGLT2i also reduced events in patients with reduced ejection fraction heart failure, with or without T2D. However, it is not known whether the cardiovascular effects of SGLT2i and GLP-1RA are through shared or distinct mechanisms, and whether combining these agents would confer additive cardioprotection against ischemia-reperfusion injury (IRI) in the absence of diabetes. Methods &amp; Results: Non-diabetic male C56BL/6-J mice randomized to 7-d treatment with SGLT2i dapagliflozin (Dapa) and/or GLP-1RA exenatide (Ex-4) or vehicle-controls underwent IRI via 30-min left anterior descending (LAD) artery ligation. Analysis of late cardiac remodeling at d-28 post-IRI revealed preserved ejection fraction (EF) in mice treated with Dapa+Ex4 (50±2%; n=12, P&lt;0.05) vs. vehicle-treated controls (36±3; n=11, P&lt;0.05), whereas treatment with Dapa- and Ex-4-alone showed no such benefit. These improvements in LV function were associated with reduced infarct size (%LV) in mice treated with Dapa+Ex4 (17±3; n=8, P&lt;0.001) and Ex-4-alone (29±3; n=10, P&lt;0.05) vs. vehicle-control (44±5; n=10). Metabolome and merged proteome+phosphoproteome analyses revealed systemic and myocardial metabolic modulation following 7-d Dapa+Ex-4 treatment, with distinct enrichment of mitochondrial electron transport chain pathways. Cardiomyocytes isolated from mice treated in vivo for 7-d also showed enhanced aerobic respiration as measured by oxygen consumption rate (OCR;17.6±1.7 pmol/min/μg protein; n=4 mice) compared Dapa- (8.9±0.8; n=3, P&lt;0.001), Ex-4-alone (11.4±0.9; n=3, P&lt;0.01) and vehicle-treated controls (9.5±0.6; n=5, P&lt;0.001). Conclusions: Combined therapy with SGLT2i+GLP-1RA in non-diabetic mice better prevented late cardiac remodeling after IRI compared to either agent alone or vehicle controls. These findings provide rationale for the combined use of these agents in a condition (IRI) for which there remains no approved therapy, and implicate metabolic modulation as a cardioprotective strategy in the absence of diabetes.

Reactive oxygen species (ROS)-mediated regulation of muscle texture in grass carp fed with dietary oxidants

Natural oxidant faba beans (FB) improve the muscle textural quality of grass carp (Ctenopharyngodon idellus). This study further explored the role and spatiotemporal dynamics of ROS during the 120-day process of texture quality change in grass carp fed with four feeds including commercial feed, FB only, FB and antioxidants (vitamin C and vitamin E), and commercial feed and oxidant (vitamin K3). The results showed that ROS (total ROS, H2O2, ·O2− and ·OH) time-dependently accumulated from intestine to blood, liver and muscle, improving the muscle texture by day 90 in grass carp fed solely with FB. Dietary antioxidants supplementation almost completely abolished the effect. It is notable that the textural quality and level of total ROS in the commercial feed and oxidant group supplemented vitamin K3 were higher than those of other three groups by day 60 (p < 0.05), suggesting that oxidant supplementation effectively improved the muscle textural quality of grass carp. Also, there was a positive feedback between ROS accumulation and production mainly in the mitochondria of tested organs as demonstrated by measuring the activity of the elevated electron transport chain (ETC) pathway, openness degree of mitochondrial membrane permeability transition pore (MPTP), and mitochondrial membrane potential (Δψ). These results validated the regulatory role of ROS in the improvement of muscle texture in grass carp fed with the natural oxidants and also demonstrated that oxidant supplementations (vitamin K3, or/and FB) have the potential to be applied to aquafeed for the improvement of muscle textural quality in freshwater fishes.

Structural and Biochemical Features of Eimeria tenella Dihydroorotate Dehydrogenase, a Potential Drug Target.

Dihydroorotate dehydrogenase (DHODH) is a mitochondrial monotopic membrane protein that plays an essential role in the pyrimidine de novo biosynthesis and electron transport chain pathways. In Eimeria tenella, an intracellular apicomplexan parasite that causes the most severe form of chicken coccidiosis, the activity of pyrimidine salvage pathway at the intracellular stage is negligible and it relies on the pyrimidine de novo biosynthesis pathway. Therefore, the enzymes of the de novo pathway are considered potential drug target candidates for the design of compounds with activity against this parasite. Although, DHODHs from E. tenella (EtDHODH), Plasmodium falciparum (PfDHODH), and human (HsDHODH) show distinct sensitivities to classical DHODH inhibitors, in this paper, we identify ferulenol as a potent inhibitor of both EtDHODH and HsDHODH. Additionally, we report the crystal structures of EtDHODH and HsDHODH in the absence and presence of ferulenol. Comparison of these enzymes showed that despite similar overall structures, the EtDHODH has a long insertion in the N-terminal helix region that assumes a disordered configuration. In addition, the crystal structures revealed that the ferulenol binding pocket of EtDHODH is larger than that of HsDHODH. These differences can be explored to accelerate structure-based design of inhibitors specifically targeting EtDHODH.

Ca2+ ionophore A23187 inhibits ATP generation reducing mouse sperm motility and PKA-dependent phosphorylation

Male infertility is a global problem in modern society of which capacitating defects are a major cause. Previous studies have demonstrated that Ca2+ ionophore A23187 can make mouse sperm capable of fertilizing in vitro, which may aid in clinical treatment of capacitating defects. However, the detailed role and mechanism of Ca2+ in the capacitating process are still unclear especially how A23187 quickly renders sperm immotile and inhibits cAMP/PKA-mediated phosphorylation. We report that A23187 induces a Ca2+ flux in the mitochondria enriched sperm tail and excess Ca2+ inhibits key metabolic enzymes involved in acetyl-CoA biosynthesis, TCA cycle and electron transport chain pathways resulting in reduced ATP and overall energy production, however this flux does not destroy the structure of the sperm tail. Due to the decrease in ATP production, which is the main phosphate group donator and the power of sperm, the sperm is rendered immobile and PKA-mediated phosphorylation is inhibited. Our study proposed a possible mechanism through which A23187 reduces sperm motility and PKA-mediated phosphorylation from ATP generation, thus providing basic data for exploring the functional roles of Ca2+ in sperm in the future.

Age-associated changes in human CD4+ T cells point to mitochondrial dysfunction consequent to impaired autophagy.

To gain understanding on the mechanisms that drive immunosenescence in humans, we examined CD4+ T cells obtained from younger (20-39 years-old) and older (70+ years-old) healthy participants of the Baltimore Longitudinal Study on Aging (BLSA). We found that mitochondrial proteins involved in the electron transport chain were overrepresented in cells from older participants, with prevalent dysregulation of oxidative phosphorylation and energy metabolism molecular pathways. Surprisingly, gene transcripts coding for mitochondrial proteins pertaining to oxidative phosphorylation and electron transport chain pathways were underrepresented in older individuals. Paralleling the observed decrease in gene expression, mitochondrial respiration was impaired in CD4+ T cells from older subjects. Though mitochondrial number in both naïve and memory cells visualized with electron microcopy was similar in older versus younger participants, there were a significantly higher number of autophagosomes, many of them containing undegraded mitochondria, in older individuals. The presence of mitochondria inside the accumulated autophagic compartments in CD4+ T cells from older individuals was confirmed by immunofluorescence. These findings suggest that older age is associated with persistence of dysfunctional mitochondria in CD4+ T lymphocytes caused by defective mitochondrial turnover by autophagy, which may trigger chronic inflammation and contribute to the impairment of immune defense in older persons.

Differential network analysis and protein-protein interaction study reveals active protein modules in glucocorticoid resistance for infant acute lymphoblastic leukemia

BackgroundAcute lymphoblastic leukemia (ALL) is the most common type of cancer diagnosed in children and Glucocorticoids (GCs) form an essential component of the standard chemotherapy in most treatment regimens. The category of infant ALL patients carrying a translocation involving the mixed lineage leukemia (MLL) gene (gene KMT2A) is characterized by resistance to GCs and poor clinical outcome. Although some studies examined GC-resistance in infant ALL patients, the understanding of this phenomenon remains limited and impede the efforts to improve prognosis.MethodsThis study integrates differential co-expression (DC) and protein-protein interaction (PPI) networks to find active protein modules associated with GC-resistance in MLL-rearranged infant ALL patients. A network was constructed by linking differentially co-expressed gene pairs between GC-resistance and GC-sensitive samples and later integrated with PPI networks by keeping the links that are also present in the PPI network. The resulting network was decomposed into two sub-networks, specific to each phenotype. Finally, both sub-networks were clustered into modules using weighted gene co-expression network analysis (WGCNA) and further analyzed with functional enrichment analysis.ResultsThrough the integration of DC analysis and PPI network, four protein modules were found active under the GC-resistance phenotype but not under the GC-sensitive. Functional enrichment analysis revealed that these modules are related to proteasome, electron transport chain, tRNA-aminoacyl biosynthesis, and peroxisome signaling pathways. These findings are in accordance with previous findings related to GC-resistance in other hematological malignancies such as pediatric ALL.ConclusionsDifferential co-expression analysis is a promising approach to incorporate the dynamic context of gene expression profiles into the well-documented protein interaction networks. The approach allows the detection of relevant protein modules that are highly enriched with DC gene pairs. Functional enrichment analysis of detected protein modules generates new biological hypotheses and may help in explaining the GC-resistance in MLL-rearranged infant ALL patients.

294-LB: Transcriptome-Wide Signals Converge Upon Mitochondrial Function in Developmental Stages of Type 2 Diabetes in Human Skeletal Muscle

Skeletal muscle is a major contributor to overall glucose homeostasis in the body and together with the liver is chiefly responsible for the whole-body insulin sensitivity. However, few studies have investigated the transcriptional signature of insulin resistance in skeletal muscles, at different stages of T2D development. Thus, the aim of this study was to identify the transcriptional signature of dysregulated glucose metabolism in skeletal muscle in humans by profiling the transcriptome with next-generation sequencing (NGS). We selected 40 sedentary males, aged 40-65 years, with BMI 26-33 kg/m2, diagnosed into three groups: 18 normoglycemics, 14 prediabetics and 8 subjects with type 2 diabetes. mRNA was extracted from biopsied Vastus lateralis muscles and sequenced with Illumina HiSeq 4000.Comparing normoglycemic and diabetic subjects revealed 4404 significantly differentially expressed (DE) genes, which represents around 30% of all mapped genes in studied samples. For the comparison between type 2 diabetics and prediabetics, the number of DE genes was 4055 genes. Interestingly we found no significantly DE genes between normoglycemics and prediabetics. KEGG enrichment analysis revealed that DE pathways between diabetics vs. nondiabetics were enriched for pathways related to oxidative phosphorylation, thermogenesis, nonalcoholic fatty liver disease and neurological diseases, which share the same underlying mechanism: mitochondrial dysfunction.These results indicate that skeletal muscle contributes most to overt T2D, with a possible indication that the liver contributes most to early stages the disease development. Differences observed between type 2 diabetics and the two remaining groups, occurring primarily in oxidative phosphorylation and electron transport chain pathways suggests that mitochondrial dysfunction plays the predominant role in the pathogenesis of T2D. Disclosure L. Szczerbinski: None. M. Niemira: None. K. Szczerbinski: None. U. Puchta: None. E. Siewiec: None. A. Citko: None. J. Zapolska: None. M. Taylor: None. S. Larsen: None. M. Gorska: None. A. Kretowski: None.

Enniatin B induces expression changes in the electron transport chain pathway related genes in lymphoblastic T-cell line

Enniatin B is a ionophoric and lipophilic mycotoxin which reaches the bloodstream and has the ability to penetrate into cellular membranes. The purpose of this study was to reveal changes in the gene expression profile caused by enniatin B in human Jurkat lymphoblastic T-cells after 24 h of exposure at 1.5, 3 and 5 μM by next generation sequencing. It was found that up to 27% of human genome expression levels were significantly altered (5750 genes for both down-regulation and up-regulation). In the three enniatin B concentrations studied 245 differentially expressed genes were found to be overlapped, 83 were down and 162 up-regulated. ConsensusPathDB analysis of over-representation of differentially expressed genes provided a list of gene ontology terms in which several biological processes related to nucleoside monophosphate metabolic process, respiratory chain complex, electron transport chain, oxidative phosphorylation and cellular respiration were the most altered. Also, an interesting correlation was found between enniatin B toxicity and the up-regulation of the UCP protein complex. In summary, the transcriptomic analysis revealed that mitochondria are the organelles showing more related differentially expressed genes. Consequently, differentially expressed genes involved in biological processes, molecular functions and pathways related to mitochondrial metabolism and respiration were significantly changed.