Mitochondrial Metabolic Pathways Research Articles

Integrating the contributions of mitochondrial oxidative metabolism to lipotoxicity and inflammation in NAFLD pathogenesis

The prevalence of non-alcoholic fatty liver disease (NAFLD) is increasing globally. NAFLD includes non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH). NASH is the pathological form of the disease characterized by liver steatosis, inflammation, cell injury, and fibrosis. A fundamental contributor to NASH is the imbalance between lipid accretion and disposal. The accumulation of liver lipids precipitates lipotoxicity and the inflammatory contributions to disease progression. This review defines the role of dysregulated of lipid disposal in NAFLD pathophysiology. The characteristic changes in mitochondrial oxidative metabolism pathways and the factors promoting these changes across the spectrum of NAFLD severity are detailed. This includes pathway-specific and integrative perturbations in mitochondrial β-oxidation, citric acid cycle flux, oxidative phosphorylation, and ketogenesis. Moreover, well-recognized and emerging mechanisms through which dysregulated mitochondrial oxidative metabolism mediates inflammation, fibrosis, and disease progression are highlighted.

Identification of Genes Predicting Poor Response of Trastuzumab in Human Epidermal Growth Factor Receptor 2 Positive Breast Cancer.

Objective To identify trastuzumab-resistant genes predicting drug response and poor prognosis in human epidermal growth factor receptor 2 positive (HER2+) breast cancer. Methods Gene expression profiles from the GEO (Gene Expression Omnibus) database were obtained and analyzed. Differentially expressed genes (DEGs) between the pathological complete response (pCR) group and non-pCR group in a trastuzumab neoadjuvant therapy cohort and DEGs between Herceptin-resistant and wild-type cell lines were detected and evaluated. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analyses were performed to select the functional hub genes. The hub genes' prognostic power was validated by another trastuzumab adjuvant treatment cohort. Results Fifty upregulated overlapping DEGs were identified by analyzing two trastuzumab resistance-related GEO databases. Functional analysis picked out ten hub genes enriched in mitochondrial function and metabolism pathways: ASCL1, CPT2, DLD, ELVOL7, GAMT, NQO1, SLC23A1, SPR, UQCRB, and UQCRQ. These hub genes could distinguish patients with trastuzumab resistance from the sensitive ones. Further survival analysis of hub genes showed that DLD overexpression was significantly associated with an unfavorable prognosis in HER2+ breast cancer patients. Conclusion Ten novel trastuzumab resistance-related genes were discovered, of which DLD could be used for trastuzumab response prediction and prognostic prediction in HER2+ breast cancer.

Transcriptomic and proteomic retinal pigment epithelium signatures of age-related macular degeneration

There are currently no treatments for geographic atrophy, the advanced form of age-related macular degeneration. Hence, innovative studies are needed to model this condition and prevent or delay its progression. Induced pluripotent stem cells generated from patients with geographic atrophy and healthy individuals were differentiated to retinal pigment epithelium. Integrating transcriptional profiles of 127,659 retinal pigment epithelium cells generated from 43 individuals with geographic atrophy and 36 controls with genotype data, we identify 445 expression quantitative trait loci in cis that are asssociated with disease status and specific to retinal pigment epithelium subpopulations. Transcriptomics and proteomics approaches identify molecular pathways significantly upregulated in geographic atrophy, including in mitochondrial functions, metabolic pathways and extracellular cellular matrix reorganization. Five significant protein quantitative trait loci that regulate protein expression in the retinal pigment epithelium and in geographic atrophy are identified - two of which share variants with cis- expression quantitative trait loci, including proteins involved in mitochondrial biology and neurodegeneration. Investigation of mitochondrial metabolism confirms mitochondrial dysfunction as a core constitutive difference of the retinal pigment epithelium from patients with geographic atrophy. This study uncovers important differences in retinal pigment epithelium homeostasis associated with geographic atrophy.

Mitochondrial impairment and repair in the pathogenesis of systemic lupus erythematosus.

Nucleic acid autoantibodies, increase type I interferon (IFN-α) levels, and immune cell hyperactivation are hallmarks of systemic lupus erythematosus (SLE). Notably, immune cell activation requires high level of cellular energy that is predominately generated by the mitochondria. Mitochondrial reactive oxygen species (mROS), the byproduct of mitochondrial energy generation, serves as an essential mediator to control the activation and differentiation of cells and regulate the antigenicity of oxidized nucleoids within the mitochondria. Recently, clinical trials on normalization of mitochondrial redox imbalance by mROS scavengers and those investigating the recovery of defective mitophagy have provided novel insights into SLE prophylaxis and therapy. However, the precise mechanism underlying the role of oxidative stress-related mitochondrial molecules in skewing the cell fate at the molecular level remains unclear. This review outlines distinctive mitochondrial functions and pathways that are involved in immune responses and systematically delineates how mitochondrial dysfunction contributes to SLE pathogenesis. In addition, we provide a comprehensive overview of damaged mitochondrial function and impaired metabolic pathways in adaptive and innate immune cells and lupus-induced organ tissues. Furthermore, we summarize the potential of current mitochondria-targeting drugs for SLE treatment. Developing novel therapeutic approaches to regulate mitochondrial oxidative stress is a promising endeavor in the search for effective treatments for systemic autoimmune diseases, particularly SLE.

Differential In Vitro Effects of SGLT2 Inhibitors on Mitochondrial Oxidative Phosphorylation, Glucose Uptake and Cell Metabolism.

(1) The cardio-reno-metabolic benefits of the SGLT2 inhibitors canagliflozin (cana), dapagliflozin (dapa), ertugliflozin (ertu), and empagliflozin (empa) have been demonstrated, but it remains unclear whether they exert different off-target effects influencing clinical profiles. (2) We aimed to investigate the effects of SGLT2 inhibitors on mitochondrial function, cellular glucose-uptake (GU), and metabolic pathways in human-umbilical-vein endothelial cells (HUVECs). (3) At 100 µM (supra-pharmacological concentration), cana decreased ECAR by 45% and inhibited GU (IC5o: 14 µM). At 100 µM and 10 µM (pharmacological concentration), cana increased the ADP/ATP ratio, whereas dapa and ertu (3, 10 µM, about 10× the pharmacological concentration) showed no effect. Cana (100 µM) decreased the oxygen consumption rate (OCR) by 60%, while dapa decreased it by 7%, and ertu and empa (all 100 µM) had no significant effect. Cana (100 µM) inhibited GLUT1, but did not significantly affect GLUTs’ expression levels. Cana (100 µM) treatment reduced glycolysis, elevated the amino acids supplying the tricarboxylic-acid cycle, and significantly increased purine/pyrimidine-pathway metabolites, in contrast to dapa (3 µM) and ertu (10 µM). (4) The results confirmed cana´s inhibition of mitochondrial activity and GU at supra-pharmacological and pharmacological concentrations, whereas the dapa, ertu, and empa did not show effects even at supra-pharmacological concentrations. At supra-pharmacological concentrations, cana (but not dapa or ertu) affected multiple cellular pathways and inhibited GLUT1.

Transcriptional and metabolic remodeling in clear cell renal cell carcinoma caused by ATF4 activation and the integrated stress response (ISR).

Research has shown extensive metabolic remodeling in clear cell renal cell carcinoma (ccRCC), with increased glutathione (GSH) levels. We hypothesized that activating transcription factor-4 (ATF4) and the integrated stress response (ISR) induce a metabolic shift, including increased GSH accumulation, and that Vitamin A deficiency (VAD), found in ccRCCs, can also activate ATF4 signaling in the kidney. To determine the role of ATF4, we used publicly available RNA sequencing (RNA-seq) data sets from The Cancer Genomics Atlas. Subsequently, we performed RNA-seq and liquid chromatography-mass spectrometry-based metabolomics analysis of the murine TRAnsgenic Cancer of the Kidney (TRACK) model for early-stage ccRCC. To validate our findings, we generated RCC4 cell lines with ATF4 gene edits (ATF4-knockout [KO]) and subjected these cells to metabolic isotope tracing. Analysis of variance, the two-sided Student's t test, and gene set enrichment analysis were used (p < 0.05) to determine statistical significance. Here we show that most human ccRCC tumors exhibit activation of the transcription factor ATF4. Activation of ATF4 is concomitant with enrichment of the ATF4 gene set and elevated expression of ATF4 target genes ASNS, ALDH1L2, MTHFD2, DDIT3 (CHOP), DDIT4, TRIB3, EIF4EBP1, SLC7A11, and PPP1R15A (GADD34). Transcript profiling and metabolomics analyses show that activated hypoxia-inducible factor-1α (HIF1α) signaling in our TRACK ccRCC murine model also induces an ATF4-mediated ISR. Notably, both normoxic HIF1α signaling in TRACK kidneys and VAD in wild-type kidneys diminish amino acid levels, increase ASNS, TRIB3, and MTHFD2 messenger RNA levels, and increase levels of lipids and GSH. By metabolic isotope tracing in human RCC4 kidney cancer parental and ATF4 gene-edited (ATF4-KO) cell lines, we show that ATF4 increases GSH accumulation in part via activation of the mitochondrial one-carbon metabolism pathway. Our results demonstrate for the first time that activation of ATF4 enhances GSH accumulation, increases purine and pyrimidine biosynthesis, and contributes to transcriptional and metabolic remodeling in ccRCC. Moreover, constitutive HIF1α expressed only in murine kidney proximal tubules activates ATF4, leading to the metabolic changes associated with the ISR. Our data indicate that HIF1α can promote ccRCC via ATF4 activation. Moreover, lack of Vitamin A in the kidney recapitulates aspects of the ISR.

Abstract 3781: Expression of ovarian cancer specific Drp1 splice variants regulate mitochondrial heterogeneity and cell plasticity during tumor progression

Abstract Mitochondrial shape is integral for its proper function and is maintained by a dynamic balance between the events of fission and fusion. Hence, a disruption in the balance is detrimental and has been associated with multiple pathologies including tumorigenesis. We noticed significant heterogeneity in mitochondrial morphology and function in ovarian cancer, which remains the deadliest gynecologic malignancy to date. We discovered that heterogenous mitochondrial dynamics in ovarian cancer cells were associated with specific transcript variant signatures of the fission protein Drp1 (encoded by the gene DNM1L), the primary GTPase responsible for mitochondrial fission. While several Drp1 splice variants have been reported, few studies have linked expression and potential interplay of splice variants of Drp1 on mitochondrial dynamics and function with pathophysiology especially in ovarian cancer. We used 3’RACE, western blotting and LC-MS/MS proteomics analysis to establish the identity of the major Drp1 splice variants expressed in ovarian cancer. We found ovarian cancer cell lines as well as patient-ascites derived cells, predominantly express two Drp1 variants: a transcript including both exons 16 and 17 (16/17) and a transcript lacking exon 16 (-/17). We also validated our findings in TCGA ovarian cancer specimens by analyzing Drp1 splice variant transcripts following annotation of TCGA raw RNAseq data and Salmon expression analysis. Our TCGA analysis of these variants highlighted significant difference in overall survival of ovarian cancer patients. Samples with high Drp1(-/17) expression were associated with poorer overall survival compared to those predominantly expressing Drp1(16/17). Furthermore, carrying out gene set enrichment analysis (GSEA) on TCGA specimens split by high expression of these two variants showed enrichment of distinct gene expression signatures. Overexpression and splice variant specific siRNA knockdown studies demonstrated that Drp1 variants have unique localization and effects on mitochondrial morphology and function. Furthermore, metabolic profiling and 13C metabolic flux analysis highlighted variant specific alterations in mitochondrial metabolic pathways and the TCA cycle. Drp1(-/17) expression enhanced mitochondrial respiratory function and as previously shown, Drp1(-/17) associated with both mitochondria and microtubules, potentially implying a more regulated fission activity as a consequence of controlled subcellular localization. Additionally, Drp1(-/17) was enriched and associated with quiescent phenotype compared to more proliferative phenotype of Drp1(16/17). Hence, expression of distinct Drp1 splice variants may be a novel mechanism to regulate mitochondrial fission, and integral to ovarian cancer cell plasticity under different selection pressures during tumor progression. Citation Format: Zaineb Javed, Dong-Hui Shin, Weihua Pan, Amal Taher Elhaw, Priscilla Tang, Rebecca Phaeton, Mohamed Trebak, Vonn Walter, Nadine Hempel. Expression of ovarian cancer specific Drp1 splice variants regulate mitochondrial heterogeneity and cell plasticity during tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3781.

Cross-species metabolomic analysis of tau- and DDT-related toxicity.

Exposure to the pesticide dichlorodiphenyltrichloroethane (DDT) has been associated with increased risk of Alzheimer's disease (AD), a disease also associated with hyperphosphorylated tau (p-tau) protein aggregation. We investigated whether exposure to DDT can exacerbate tau protein toxicity in Caenorhabditiselegans using a transgenic strain that expresses human tau protein prone to aggregation by measuring changes in size, swim behavior, respiration, lifespan, learning, and metabolism. In addition, we examined the association between cerebrospinal fluid (CSF) p-tau protein—as a marker of postmortem tau burden—and global metabolism in both a human population study and in C. elegans, using the same p-tau transgenic strain. From the human population study, plasma and CSF-derived metabolic features associated with p-tau levels were related to drug, amino acid, fatty acid, and mitochondrial metabolism pathways. A total of five metabolites overlapped between plasma and C. elegans, and four between CSF and C. elegans. DDT exacerbated the inhibitory effect of p-tau protein on growth and basal respiration. In the presence of p-tau protein, DDT induced more curling and was associated with reduced levels of amino acids but increased levels of uric acid and adenosylselenohomocysteine. Our findings in C. elegans indicate that DDT exposure and p-tau aggregation both inhibit mitochondrial function and DDT exposure can exacerbate the mitochondrial inhibitory effects of p-tau aggregation. Further, biological pathways associated with exposure to DDT and p-tau protein appear to be conserved between species.

Mitochondrial adaptations throughout the Trypanosoma brucei lifecycle.

The unicellular parasite Trypanosoma brucei has a digenetic life cycle that alternates between a mammalian host and an insect vector. During programmed development, this extracellular parasite encounters strikingly different environments that determine its energy metabolism. Functioning as a bioenergetic, biosynthetic, and signaling center, the single mitochondrion of T.brucei is drastically remodeled to support the dynamic cellular demands of the parasite. This manuscript will provide an up-to-date overview of how the distinct T.brucei developmental stages differ in their mitochondrial metabolic and bioenergetic pathways, with a focus on the electron transport chain, proline oxidation, TCA cycle, acetate production, and ATP generation. Although mitochondrial metabolic rewiring has always been simply viewed as a consequence of the differentiation process, the possibility that certain mitochondrial activities reinforce parasite differentiation will be explored.

Chronic Stress Induces Hippocampal Mitochondrial Damage in APPPS1 Model Mice and Wildtype Littermates.

Alzheimer's disease (AD) is a neurodegenerative disorder and the most common cause of dementia worldwide. Despite decades of investigation, the etiology of AD is not fully understood, although emerging evidence suggest that chronic environmental and psychological stress plays a role in the mechanisms and contributes to the risk of developing AD. Thus, dissecting the impact of stress on the brain could improve our understanding of the pathological mechanisms. We aimed to study the effect of chronic stress on the hippocampal proteome in male APPPS1 transgenic mice and wildtype (WT) littermates. APPPS1 and WT mice were subjected to 4 weeks of chronic stress followed by 3 weeks of continued diurnal disruption. Hippocampal tissue was used for proteomics analysis using label-free quantitative DIA based LC-MS/MS analysis. We identified significantly up- and downregulated proteins in both APPPS1 and WT mice exposed to chronic stress compared to the control groups. Via interaction network mapping, significant proteins could be annotated to specific pathways of mitochondrial function (oxidative phosphorylation and TCA cycle), metabolic pathways, AD pathway and synaptic functions (long term potentiation). In WT mice, chronic stress showed the highest impact on complex I of the oxidative phosphorylation pathway, while in APPPS1 mice this pathway was compromised broadly by chronic stress. Our data shows that chronic stress and amyloidosis additively contribute to mitochondrial damage in hippocampus. Although these results do not explain all effects of chronic stress in AD, they add to the scientific knowledge on the topic.

A human coronavirus OC43-derived polypeptide causes neuropathic pain.

Human coronaviruses have been recently implicated in neurological sequelae by insufficiently understood mechanisms. We here identify an amino acid sequence within the HCoV‐OC43 p65‐like protein homologous to the evolutionarily conserved motif of myelin basic protein (MBP). Because MBP‐derived peptide exposure in the sciatic nerve produces pronociceptive activity in female rodents, we examined whether a synthetic peptide derived from the homologous region of HCoV‐OC43 (OC43p) acts by molecular mimicry to promote neuropathic pain. OC43p, but not scrambled peptides, induces mechanical hypersensitivity in rats following intrasciatic injections. Transcriptome analyses of the corresponding spinal cords reveal upregulation of genes and signaling pathways with known nociception‐, immune‐, and cellular energy‐related activities. Affinity capture shows the association of OC43p with an Na+/K+‐transporting ATPase, providing a potential direct target and mechanistic insight into virus‐induced effects on energy homeostasis and the sensory neuraxis. We propose that HCoV‐OC43 polypeptides released during infection dysregulate normal nervous system functions through molecular mimicry of MBP, leading to mechanical hypersensitivity. Our findings might provide a new paradigm for virus‐induced neuropathic pain.

Deletion of Aldh4a1 Leads to Impaired Sperm Maturation in Mice

ALDH4A1, a member of the aldehyde dehydrogenase superfamily, is a key enzyme in the mitochondrial proline metabolism pathway. Recent studies have shown that mutations in aldh4a1 lead to reduced fertility and reproductive premature aging of male nematodes. However, the effect of ALDH4A1 on fertility of male mice has not been studied. In this study, we used CRISPR-Cas9 technology to construct a knockout mouse model of Aldh4a1 for the first time to explore the effect of this gene on the reproduction of male mice. The results showed that compared with WT male mice, Aldh4a1^(-/-) male mice were fertile, had normal spermatogenesis but defect in sperm maturation in the epididymis documented by impaired motility, increased morphological abnormalities and increased spontaneous acrosome reaction. In addition, transmission electron microscopy showed vacuoles in the sperm mitochondria, and fracture in the neck of sperms and vacuoles in these mice. These results revealed that ALDH4A1 plays a vital role in the structure of sperm flagellum and the process of sperm maturation in mice.

Total flavonoids of Selaginella tamariscina (P.Beauv.) Spring ameliorates doxorubicin-induced cardiotoxicity by modulating mitochondrial dysfunction and endoplasmic reticulum stress via activating MFN2/PERK

BackgroundDoxorubicin (DOX) is a highly effective chemotherapeutic that is effective for various tumours. However, the clinical application of DOX has been limited by adverse reactions such as cardiotoxicity and heart failure. Since DOX-induced cardiotoxicity is irreversible, drugs to prevent DOX-induced cardiotoxicity are needed. PurposeThis study aimed to investigate the effect of total flavonoids of Selaginella tamariscina (P.Beauv.) Spring (TFST) on doxorubicin-induced cardiotoxicity. MethodsThe present study established DOX-induced cardiotoxicity models in C57BL/6 mice treated with DOX (cumulative dose: 20 mg/kg body weight) and H9c2 cells incubated with DOX (1 μM/l) to explore the intervention effect and potential mechanism of TFST. Echocardiography was performed to evaluate left ventricular functions. Heart tissue samples were collected for histological evaluation. Myocardial injury markers and oxidative stress markers were examined. Mitochondrial energy metabolism pathway associated proteins PPARα/PGC-1α/Sirt3 were detected. We also explored the effects of TFST on endoplasmic reticulum (ER) stress and apoptosis. To further investigate the protective mechanism of TFST, we used the specific small interfering RNA MFN2 (siMFN2) to explore the effect of MFN2 on TFST against DOX-induced cardiotoxicity in vitro. Flow cytometry detected reactive oxygen species, mitochondrial membrane potential and apoptosis. Cell mitochondrial stress was measured by Seahorse XF analyser. ResultsBoth in vivo and in vitro studies verified that TFST observably alleviated DOX-induced mitochondrial dysfunction and ER stress. However, these effects were reversed after transfected siMFN2. ConclusionOur results indicated that TFST ameliorates DOX-induced cardiotoxicity by alleviating mitochondrial dysfunction and ER stress by activating MFN2/PERK. MFN2/PERK pathway activation may be a novel mechanism to protect against DOX-induced cardiotoxicity.

A Link Between Mitochondrial Dysfunction and the Immune Microenvironment of Salivary Glands in Primary Sjogren's Syndrome.

BackgroundPrimary Sjogren’s syndrome (pSS) is a slowly progressive, inflammatory autoimmune disease characterized by lymphocytic infiltration into salivary and lacrimal glands. It becomes more recognized that morphology alterations of epithelial mitochondria are involved in altered cellular bioenergetics in pSS patients. The integrated analysis of the mitochondrial role in the pathogenesis and aberrant immune microenvironment in pSS remains unknown.MethodsThe mitochondria-related genes and gene expression data were downloaded from the MitoMiner, MitoCarta, and NCBI GEO databases. We performed novel transcriptomic analysis and constructed a network between the mitochondrial function and immune microenvironment in pSS-salivary glands by computer-aided algorithms. Subsequently, real-time PCR was performed in clinical samples in order to validate the bioinformatics results. Histological staining and transmission electron microscopy (TEM) were further studied on labial salivary gland samples of non-pSS and pSS patients characterized for mitochondria-related phenotypic observation in the different stages of the disease.ResultsThe bioinformatic analysis revealed that the expression of several mitochondria-related genes was altered in pSS. Quantitative real-time PCR showed that four hub genes, CD38, CMPK2, TBC1D9, and PYCR1, were differentially expressed in the pSS clinical samples. These hub genes were associated with the degree of immune cell infiltration in salivary glands, the mitochondrial respiratory chain complexes, mitochondrial metabolic pathway in gluconeogenesis, TCA cycle, and pyruvate/ketone/lipid/amino acid metabolism in pSS. Clinical data revealed that the gene expression of fission (Fis1, DRP1, and MFF) and fusion (MFN1, MFN2, and OPA1) was downregulated in pSS samples, consistent with the results from the public validation database. As the disease progressed, cytochrome c and Bcl-2 proteins were regionally distributed in salivary glands from pSS patients. TEM revealed cytoplasmic lipid droplets and progressively swollen mitochondria in salivary epithelial cells.ConclusionOur study revealed cross talk between mitochondrial dysfunction and the immune microenvironment in salivary glands of pSS patients, which may provide important insights into SS clinical management based on modulation of mitochondrial function.

Mechanism of Longevity Extension of Caenorhabditis elegans Induced by Schizophyllum commune Fermented Supernatant With Added Radix Puerariae.

Schizophyllum commune (S. commune) fermented supernatant with added Radix Puerariae (SC-RP) showed significant antioxidant activity in our previous work. However, the possible lifespan and healthspan extending the capacity of Caenorhabditis elegans (C. elegans) and the underlying mechanism were not illuminated. In this study, the effect of SC-RP on extending the lifespan and improving stress resistance of C. elegans were examined. Additionally, the underlying lifespan extending molecular mechanisms of SC-RP were explored. Treated with SC-RP at 10 μg/mL, the lifespan of C. elegans increased by 24.89% (P < 0.01). Also, SC-RP prolonged the healthspan of the nematode, including reducing lipofuscin levels, improving mobility and enhancing resistance to oxidative stress and heat shock. Moreover, superoxide dismutase and catalase activities were increased for SC-RP treated C. elegans. Meantime the intracellular levels of thiobarbituric acid reactive substances (TBARS) and reactive oxygen species (ROS) were attenuated. Express levels of eight genes including daf-2, daf-16, sod-3, skn-1, gst-4, clk-1, age-1 and mev-1 were analyzed by RT-PCR method for possible C. elegan anti-aging mechanisms of SC-RP. Expression levels of key genes daf-2, gst-4 and sod-3 were up-regulated, while that of daf-16, skn-1, and clk-1 were down-regulated. The results suggest that SC-RP could extend the lifespan and healthspan of C. elegans significantly, and the IIS pathway, SKN-1/Nrf2 pathway and mitochondrial metabolism pathway were primarily considered associated. Thus, SC-RP is a potential component to improve aging and aging-related symptoms as new functional materials.

Dissecting Plasmodium yoelii Pathobiology: Proteomic Approaches for Decoding Novel Translational and Post-Translational Modifications.

Malaria is a vector-borne disease. It is caused by Plasmodium parasites. Plasmodium yoelii is a rodent model parasite, primarily used for studying parasite development in liver cells and vectors. To better understand parasite biology, we carried out a high-throughput-based proteomic analysis of P. yoelii. From the same mass spectrometry (MS)/MS data set, we also captured several post-translational modified peptides by following a bioinformatics analysis without any prior enrichment. Further, we carried out a proteogenomic analysis, which resulted in improvements to some of the existing gene models along with the identification of several novel genes. Analysis of proteome and post-translational modifications (PTMs) together resulted in the identification of 3124 proteins. The identified PTMs were found to be enriched in mitochondrial metabolic pathways. Subsequent bioinformatics analysis provided an insight into proteins associated with metabolic regulatory mechanisms. Among these, the tricarboxylic acid (TCA) cycle and the isoprenoid synthesis pathway are found to be essential for parasite survival and drug resistance. The proteogenomic analysis discovered 43 novel protein-coding genes. The availability of an in-depth proteomic landscape of a malaria pathogen model will likely facilitate further molecular-level investigations on pre-erythrocytic stages of malaria.

Low and high doses of ionizing radiation evoke discrete global (phospho)proteome responses

BackgroundAlthough cancer risk is assumed to be linear with ionizing radiation (IR) dose, it is unclear to what extent low doses (LD) of IR from medical and occupational exposures pose a cancer risk for humans. Improved mechanistic understanding of the signaling responses to LD may help to clarify this uncertainty. Here, we performed quantitative mass spectrometry-based proteomics and phosphoproteomics experiments, using mouse embryonic stem cells, at 0.5 h and 4 h after exposure to LD (0.1 Gy) and high doses (HD; 1 Gy) of IR. ResultsThe proteome remained relatively stable (29; 0.5% proteins responded), whereas the phosphoproteome changed dynamically (819; 7% phosphosites changed) upon irradiation. Dose-dependent alterations of 25 IR-responsive proteins were identified, with only four in common between LD and HD. Mitochondrial metabolic proteins and pathways responded to LD, whereas transporter proteins and mitochondrial uncoupling pathways responded to HD. Congruently, mitochondrial respiration increased after LD exposure but decreased after HD exposure. While the bulk of the phosphoproteome response to LD (76%) occurred already at 0.5 h, an equivalent proportion of the phosphosites responded to HD at both time points. Motif, kinome/phosphatome, kinase-substrate, and pathway analyses revealed a robust DNA damage response (DDR) activation after HD exposure but not after LD exposure. Instead, LD-irradiation induced (de)phosphorylation of kinases, kinase-substrates and phosphatases that predominantly respond to reactive oxygen species (ROS) production. ConclusionOur analyses identify discrete global proteome and phosphoproteome responses after LD and HD, uncovering novel proteins and protein (de)phosphorylation events involved in the dose-dependent ionizing radiation responses.

The Epidemiology and Phenotypes of Ocular Manifestations in Childhood and Juvenile Myasthenia Gravis: A Review.

Myasthenia gravis (MG) appears to have a similar incidence among adult populations worldwide. However, epidemiological and phenotypic differences have been noted among children and juveniles with MG. We reviewed the literature on childhood- and juvenile-onset MG among different populations, with the focus on ocular involvement, antibody profiles, the genetic susceptibility to juvenile MG phenotypes, the use of immune treatments, and the reported responses of extraocular muscles to therapies. Although epidemiological studies used different methodologies, reports from Asia, compared to Europe, showed more than two-fold higher proportions of prepubertal onset (before 12 years) vs. postpubertal-onset juveniles with MG. Compared to European children, ocular MG was 4-fold more frequent among Asian children, and 2–3-fold more frequent among children with African ancestry both in prepubertal and postpubertal ages at onset. These results suggest genetic influences. In Asia, HLA-B*46 and DRB1*09 appeared overrepresented in children with ocular MG. In Europe, children with MG had a significantly higher rate of transforming from ocular to generalized disease and with an overrepresentation of HLADRB1*04. Although treatment regimens vary widely and the responses to immune therapies of the ocular muscles involved in MG were generally poorly described, there were indications that earlier use of steroid therapy may have better outcomes. Reports of treatment-resistant ophthalmoplegia may be more frequent in African and Asian juvenile MG cohorts compared to Europeans. Genetic and muscle gene expression studies point to dysregulated muscle atrophy signaling and mitochondrial metabolism pathways as pathogenetic mechanisms underpinning treatment-resistant ophthalmoplegia in susceptible individuals. In conclusion, phenotypic differences in juveniles with ocular manifestations of MG were evident in different populations suggesting pathogenetic influences. Treatment responses in MG-associated ocular disease should attract more careful descriptive reports. In MG, extraocular muscles may be vulnerable to critical periods of poor force generation and certain individuals may be particularly susceptible to developing treatment-resistant ophthalmoplegia. The development of prognostic biomarkers to identify these susceptible individuals is an unmet need.

Acrolein inhalation acutely affects the regulation of mitochondrial metabolism in rat lung

Exposure of the airways to cigarette smoke (CS) is the primary risk factor for developing several lung diseases such as Chronic Obstructive Pulmonary Disease (COPD). CS consists of a complex mixture of over 6000 chemicals including the highly reactive α,β-unsaturated aldehyde acrolein. Acrolein is thought to be responsible for a large proportion of the non-cancer disease risk associated with smoking. Emerging evidence suggest a key role for CS-induced abnormalities in mitochondrial morphology and function in airway epithelial cells in COPD pathogenesis. Although in vitro studies suggest acrolein-induced mitochondrial dysfunction in airway epithelial cells, it is unknown if in vivo inhalation of acrolein affects mitochondrial content or the pathways controlling this. In this study, rats were acutely exposed to acrolein by inhalation (nose-only; 0−4 ppm), 4 h/day for 1 or 2 consecutive days (n = 6/group). Subsequently, the activity and abundance of key constituents of mitochondrial metabolic pathways as well as expression of critical proteins and genes controlling mitochondrial biogenesis and mitophagy were investigated in lung homogenates. A transient decreasing response in protein and transcript abundance of subunits of the electron transport chain complexes was observed following acrolein inhalation. Moreover, acrolein inhalation caused a decreased abundance of key regulators associated with mitochondrial biogenesis, respectively a differential response on day 1 versus day 2. Abundance of components of the mitophagy machinery was in general unaltered in response to acrolein exposure in rat lung. Collectively, this study demonstrates that acrolein inhalation acutely and dose-dependently disrupts the molecular regulation of mitochondrial metabolism in rat lung. Hence, understanding the effect of acrolein on mitochondrial function will provide a scientifically supported reasoning to shortlist aldehydes regulation in tobacco smoke.

Short-Term Adaptations in Skeletal Muscle Mitochondrial Oxidative Capacity and Metabolic Pathways to Breaking up Sedentary Behaviors in Overweight or Obese Adults.

Breaking up sedentary behavior with short-frequent bouts of physical activity (PA) differentially influences metabolic health compared with the performance of a single-continuous bout of PA matched for total active time. However, the underlying mechanisms are unknown. We compared skeletal muscle mitochondrial respiration (high-resolution respirometry) and molecular adaptations (RNA sequencing) following 4-day exposure to breaks vs. energy-matched single-continuous PA bout in inactive adults with overweight/obesity. Participants (9M/10F, 32.2 ± 6.4 years, 30.3 ± 3.0 kg/m2) completed three 4-day interventions of a randomized cross-over study: SED, sedentary control; MICRO, 5 min brisk walking each hour for 9 h; ONE: 45 min/d continuous brisk walking bout. Fasted muscle biopsies were collected on day 5. Mitochondrial coupling in the presence of lipid-associated substrates was higher after ONE (4.8 ± 2.5) compared to MICRO (3.1 ± 1.1, p = 0.02) and SED (2.3 ± 1.0, p = 0.001). Respiratory rates did not differ across groups with carbohydrate-associated substrates. In pathways associated with muscle contraction transcription signaling, ONE and MICRO similarly enhanced Oxidative Phosphorylation and Sirtuin Signaling expression (p < 0.0001, for both). However, ONE (p < 0.001, for all), but not MICRO, had greater pathway enrichment, including Ca++, mTOR, AMPK, and HIF1α signaling, than SED. Although breaking up sedentary behavior triggered skeletal muscle molecular adaptations favoring oxidative capacity, it did not improve mitochondrial function over the short term.