Assessment of particulate matter (PM) in ambient air of different settings and its associated health risk in Haripur city, Pakistan / Avaliação de material particulado (MP) no ar ambiente de diferentes configurações e sua associação com o risco à saúde na cidade de Haripur, Paquistão

Particulate matter (PM) is a major air pollutant causing serious health problems. The aim of the present study was to find out concentration of PM in ambient air and its associated health risk in Haripur city, Pakistan. Twenty-three samples were taken at various educational institutes, hospitals, recreational areas and industries in Haripur city. Concentration of PM2.5 (µg/m3) and PM10 (µg/m3) was measured with Youngteng YT-HPC 3000A portable PM counter. The results revealed that values of both PM2.5 and PM10 were above the permissible limits (35 µg/m3 for PM2.5 and 150 µg/m3 for PM10) set by Environmental Protection Agency Pakistan (Pak-EPA) in all the educational institutes, hospitals, recreational areas and industries investigated. Furthermore, significant (p<0.05) variation was found in the concentration of both PM2.5 and PM10 in all the educational institutes, hospitals, recreational areas, and industries studied. The concentration of PM2.5 was positively correlated with the concentration of PM10 in all the sampling sites. Therefore, from 1-14 scale standard of health index, the values of PM2.5 and PM10 exhibited that the ambient air quality of Haripur city Pakistan is under high risk. If the regulatory authorities such as Environmental Protection Agency, Health Department and Local Government monitor PM pollution in different settings of Haripur city, then a decrease can be possible in the pollution level. The remedies that can be taken to overcome the problem of ambient air pollution such as PM are plantation of trees at the sites where there are higher levels of air pollutants and use of masks on personal protection basis along with implementation of pollution control system in industries of Hattar Industrial Estate Haripur city, Pakistan.

O material particulado (MP) é um importante poluente do ar que causa sérios problemas de saúde. O objetivo do presente estudo foi descobrir a concentração de MP no ar ambiente e sua associação com o risco à saúde na cidade de Haripur, Paquistão. Vinte e três amostras foram coletadas em várias instituições de ensino, hospitais, áreas recreativas e indústrias na cidade de Haripur. A concentração de MP2,5 (µg/m3) e MP10 (µg/m3) foi medida por meio do contador de MP portátil Youngteng YT-HPC 3000A. Os resultados revelaram que os valores de MP2,5 e MP10 estavam acima dos limites permitidos (35 µg/m3 para MP2,5 e 150 µg/m3 para MP10) estabelecidos pela Agência de Proteção Ambiental do Paquistão (Pak-EPA) em todas as instituições de ensino, hospitais, áreas recreativas e indústrias investigadas. Além disso, foi encontrada variação significativa (p < 0,05) na concentração de MP2,5 e MP10 em todos os locais estudados. A concentração de MP2,5 correlacionou-se positivamente com a concentração de MP10 em todos os locais de amostragem. Portanto, a partir da escala padrão 1-14 do índice de saúde, os valores de MP2,5 e MP10 mostraram que a qualidade do ar ambiente na cidade de Haripur, Paquistão, está sob alto risco. Se as autoridades reguladoras, como a Pak-EPA, o Departamento de Saúde e o governo local, monitorarem a poluição por MP em diferentes configurações da cidade de Haripur, pode ser que haja uma diminuição no nível de poluição. As medidas que podem ser tomadas para superar o problema da poluição do ar ambiente, como o MP, são o plantio de árvores nos locais onde há maiores níveis de poluentes atmosféricos, o uso de máscaras e a implantação de sistema de controle de poluição nas propriedades industriais de Hattar, na cidade Haripur, Paquistão.

Assessment of particulate matter (PM) in ambient air of different settings and its associated health risk in Haripur city, Pakistan

Abstract Particulate matter (PM) is a major air pollutant causing serious health problems. The aim of the present study was to find out concentration of PM in ambient air and its associated health risk in Haripur city, Pakistan. Twenty-three samples were taken at various educational institutes, hospitals, recreational areas and industries in Haripur city. Concentration of PM2.5 (µg/m3) and PM10 (µg/m3) was measured with Youngteng YT-HPC 3000A portable PM counter. The results revealed that values of both PM2.5 and PM10 were above the permissible limits (35 µg/m3 for PM2.5 and 150 µg/m3 for PM10) set by Environmental Protection Agency Pakistan (Pak-EPA) in all the educational institutes, hospitals, recreational areas and industries investigated. Furthermore, significant (p 0.05) variation was found in the concentration of both PM2.5 and PM10 in all the educational institutes, hospitals, recreational areas, and industries studied. The concentration of PM2.5 was positively correlated with the concentration of PM10 in all the sampling sites. Therefore, from 1-14 scale standard of health index, the values of PM2.5 and PM10 exhibited that the ambient air quality of Haripur city Pakistan is under high risk. If the regulatory authorities such as Environmental Protection Agency, Health Department and Local Government monitor PM pollution in different settings of Haripur city, then a decrease can be possible in the pollution level. The remedies that can be taken to overcome the problem of ambient air pollution such as PM are plantation of trees at the sites where there are higher levels of air pollutants and use of masks on personal protection basis along with implementation of pollution control system in industries of Hattar Industrial Estate Haripur city, Pakistan.

Resumo O material particulado (MP) é um importante poluente do ar que causa sérios problemas de saúde. O objetivo do presente estudo foi descobrir a concentração de MP no ar ambiente e sua associação com o risco à saúde na cidade de Haripur, Paquistão. Vinte e três amostras foram coletadas em várias instituições de ensino, hospitais, áreas recreativas e indústrias na cidade de Haripur. A concentração de MP2,5 (µg/m3) e MP10 (µg/m3) foi medida por meio do contador de MP portátil Youngteng YT-HPC 3000A. Os resultados revelaram que os valores de MP2,5 e MP10 estavam acima dos limites permitidos (35 µg/m3 para MP2,5 e 150 µg/m3 para MP10) estabelecidos pela Agência de Proteção Ambiental do Paquistão (Pak-EPA) em todas as instituições de ensino, hospitais, áreas recreativas e indústrias investigadas. Além disso, foi encontrada variação significativa (p 0,05) na concentração de MP2,5 e MP10 em todos os locais estudados. A concentração de MP2,5 correlacionou-se positivamente com a concentração de MP10 em todos os locais de amostragem. Portanto, a partir da escala padrão 1-14 do índice de saúde, os valores de MP2,5 e MP10 mostraram que a qualidade do ar ambiente na cidade de Haripur, Paquistão, está sob alto risco. Se as autoridades reguladoras, como a Pak-EPA, o Departamento de Saúde e o governo local, monitorarem a poluição por MP em diferentes configurações da cidade de Haripur, pode ser que haja uma diminuição no nível de poluição. As medidas que podem ser tomadas para superar o problema da poluição do ar ambiente, como o MP, são o plantio de árvores nos locais onde há maiores níveis de poluentes atmosféricos, o uso de máscaras e a implantação de sistema de controle de poluição nas propriedades industriais de Hattar, na cidade Haripur, Paquistão.

Mitochondrial DNA - novel mechanisms of kidney damage and potential biomarker.

PURPOSE OF REVIEW: MtDNA copy number (CN), a putative noninvasive biomarker of mitochondrial dysfunction, is associated with renal disease. The purpose of this review is to describe studies which measured human blood mtDNA-CN in the context of chronic kidney disease (CKD), and to evaluate its potential as a clinical biomarker of kidney disease. RECENT FINDINGS: Following on from small scale cross-sectional studies implicating mtDNA-CN changes in diabetic kidney disease, recent large scale population studies provide compelling evidence of the association of mtDNA-CN and risk of renal disease in the general population and poor outcomes in CKD patients. SUMMARY: The kidney has high bioenergetic needs, renal cells are rich in mitochondrial content containing 100s to 1000s of mtDNA molecular per cell. MtDNA has emerged as both a potential mediator, and a putative biomarker of renal disease. Damage to mtDNA can result in bioenergetic deficit, and reduced MtDNA levels in the blood have been shown to correlate with CKD. Furthermore, leakage of mtDNA outside of mitochondria into the cytosol/periphery can directly cause inflammation and is implicated in acute kidney injury (AKI). Recent large-scale population studies show the association of mtDNA-CN and renal disease and provide a strong basis for the future evaluation of circulating DNA-CN in longitudinal studies to determine its utility as a clinical biomarker for monitoring renal function.

Stress-dependent macromolecular crowding in the mitochondrial matrix.

Macromolecules of various sizes induce crowding of the cellular environment. This crowding impacts on biochemical reactions by increasing solvent viscosity, decreasing the water-accessible volume and altering protein shape, function, and interactions. Although mitochondria represent highly protein-rich organelles, most of these proteins are somehow immobilized. Therefore, whether the mitochondrial matrix solvent exhibits macromolecular crowding is still unclear. Here, we demonstrate that fluorescent protein fusion peptides (AcGFP1 concatemers) in the mitochondrial matrix of HeLa cells display an elongated molecular structure and that their diffusion constant decreases with increasing molecular weight in a manner typical of macromolecular crowding. Chloramphenicol (CAP) treatment impaired mitochondrial function and reduced the number of cristae without triggering mitochondrial orthodox-to-condensed transition or a mitochondrial unfolded protein response. CAP-treated cells displayed progressive concatemer immobilization with increasing molecular weight and an eightfold matrix viscosity increase, compatible with increased macromolecular crowding. These results establish that the matrix solvent exhibits macromolecular crowding in functional and dysfunctional mitochondria. Therefore, changes in matrix crowding likely affect matrix biochemical reactions in a manner depending on the molecular weight of the involved crowders and reactants.

Assessment of particulate matter (PM) in ambient air of different settings and its associated health risk in Haripur city, Pakistan.

Particulate matter (PM) is a major air pollutant causing serious health problems. The aim of the present study was to find out concentration of PM in ambient air and its associated health risk in Haripur city, Pakistan. Twenty-three samples were taken at various educational institutes, hospitals, recreational areas and industries in Haripur city. Concentration of PM2.5 (µg/m3) and PM10 (µg/m3) was measured with Youngteng YT-HPC 3000A portable PM counter. The results revealed that values of both PM2.5 and PM10 were above the permissible limits (35 µg/m3 for PM2.5 and 150 µg/m3 for PM10) set by Environmental Protection Agency Pakistan (Pak-EPA) in all the educational institutes, hospitals, recreational areas and industries investigated. Furthermore, significant (p<0.05) variation was found in the concentration of both PM2.5 and PM10 in all the educational institutes, hospitals, recreational areas, and industries studied. The concentration of PM2.5 was positively correlated with the concentration of PM10 in all the sampling sites. Therefore, from 1-14 scale standard of health index, the values of PM2.5 and PM10 exhibited that the ambient air quality of Haripur city Pakistan is under high risk. If the regulatory authorities such as Environmental Protection Agency, Health Department and Local Government monitor PM pollution in different settings of Haripur city, then a decrease can be possible in the pollution level. The remedies that can be taken to overcome the problem of ambient air pollution such as PM are plantation of trees at the sites where there are higher levels of air pollutants and use of masks on personal protection basis along with implementation of pollution control system in industries of Hattar Industrial Estate Haripur city, Pakistan.

Preclinical evidence for mitochondrial DNA as a potential blood biomarker for chemotherapy-induced peripheral neuropathy.

Chemotherapy-induced peripheral neuropathy (CIPN) is a serious dose-limiting side effect of several first-line chemotherapeutic agents including paclitaxel, oxaliplatin and bortezomib, for which no predictive marker is currently available. We have previously shown that mitochondrial dysfunction is associated with the development and maintenance of CIPN. The aim of this study was to evaluate the potential use of mitochondrial DNA (mtDNA) levels and complex I enzyme activity as blood biomarkers for CIPN. Real-time qPCR was used to measure mtDNA levels in whole blood collected from chemotherapy- and vehicle-treated rats at three key time-points of pain-like behaviour: prior to pain development, at the peak of mechanical hypersensitivity and at resolution of pain-like behaviour. Systemic oxaliplatin significantly increased mtDNA levels in whole blood prior to pain development. Furthermore, paclitaxel- and bortezomib-treated animals displayed significantly higher levels of mtDNA at the peak of mechanical hypersensitivity. Mitochondrial complex I activity in whole blood was assessed with an ELISA-based Complex I Enzyme Activity Dipstick Assay. Complex I activity was not altered by any of the three chemotherapeutic agents, either prior to or during pain-like behaviour. These data demonstrate that blood levels of mtDNA are altered after systemic administration of chemotherapy. Oxaliplatin, in particular, is associated with higher mtDNA levels before animals show any pain-like behaviour, thus suggesting a potential role for circulating mtDNA levels as non-invasive predictive biomarker for CIPN.

Mitochondria, oxidative stress and nonalcoholic fatty liver disease: A complex relationship.

According to the 'multiple-hit' hypothesis, several factors can act simultaneously in nonalcoholic fatty liver disease (NAFLD) progression. Increased nitro-oxidative (nitroso-oxidative) stress may be considered one of the main contributors involved in the development and risk of NAFLD progression to nonalcoholic steatohepatitis (NASH) characterized by inflammation and fibrosis. Moreover, it has been repeatedly postulated that mitochondrial abnormalities are closely related to the development and progression of liver steatosis and NAFLD pathogenesis. However, it is difficult to determine with certainty whether mitochondrial dysfunction or oxidative stress are primary events or a simple consequence of NAFLD development. On the one hand, increasing lipid accumulation in hepatocytes could cause a wide range of effects from mild to severe mitochondrial damage with a negative impact on cell fate. This can start the cascade of events, including an increase of cellular reactive nitrogen species (RNS) and reactive oxygen species (ROS) production that promotes disease progression from simple steatosis to more severe NAFLD stages. On the other hand, progressing mitochondrial bioenergetic catastrophe and oxidative stress manifestation could be considered accompanying events in the vast spectrum of abnormalities observed during the transition from NAFL to NASH and cirrhosis. This review updates our current understanding of NAFLD pathogenesis and clarifies whether mitochondrial dysfunction and ROS/RNS are culprits or bystanders of NAFLD progression.

Accurate Measurement of Cellular and Cell-Free Circulating Mitochondrial DNA Content from Human Blood Samples Using Real-Time Quantitative PCR.

Changes in circulating mitochondrial DNA (mtDNA) are widely used to indicate mitochondrial dysfunction in common non-genetic diseases where mitochondrial dysfunction may play a role. However, the methodology being used is not always specific and reproducible, and most studies use whole blood rather than evaluating cellular and cell-free mtDNA separately. Cellular mtDNA is contained within the mitochondrion and encodes vital subunits of the OXPHOS machinery. Conversely, cell-free mtDNA can have harmful effects, triggering inflammatory responses and potentially contributing to pathogenic processes. In this chapter, we describe a protocol to accurately measure the amount of cellular and cell-free human mtDNA in peripheral blood. Absolute quantification is carried out using real-time quantitative PCR (qPCR) to quantify cellular mtDNA, measured as the mitochondrial genome to nuclear genome ratio (designated the Mt/N ratio) in whole blood and peripheral blood mononuclear cells (PBMCs) and the number of mtDNA copies per µL in plasma and serum. We describe how to (1) separate whole blood into PBMCs, plasma, and serum fractions, (2) prepare DNA from each of these fractions, (3) prepare dilution standards for absolute quantification, (4) carry out qPCR for either relative or absolute quantification from test samples, (5) analyze qPCR data, and (6) calculate the sample size to adequately power studies. The protocol presented here is suitable for high-throughput use and can be modified to quantify mtDNA from other body fluids, human cells, and tissues.

Supplementation with a prebiotic (polydextrose) in obese mouse pregnancy improves maternal glucose homeostasis and protects against offspring obesity.

OBJECTIVES: We hypothesised that maternal diet-induced-obesity has adverse consequences for offspring energy expenditure and susceptibility to obesity in adulthood, and that the prebiotic polydextrose (PDX) would prevent the consequences of programming by maternal obesity. METHODS: Female mice were fed a control (Con) or obesogenic diet (Ob) for 6 weeks prior to mating and throughout pregnancy and lactation. Half the obese dams were supplemented with 5% PDX (ObPDX) in drinking water throughout pregnancy and lactation. Offspring were weaned onto standard chow. At 3 and 6 months, offspring energy intake (EI) and energy expenditure (EE by indirect calorimetry) were measured, and a glucose-tolerance test performed. Offspring of control (OffCon), obese (OffOb) and PDX supplemented (OffObP) dams were subsequently challenged for 3 weeks with Ob, and energy balanced reassessed. Potential modifiers of offspring energy balance including gut microbiota and biomarkers of mitochondrial activity were also evaluated. RESULTS: Six-month-old male OffOb demonstrated increased bodyweight (BW, P < 0.001) and white adipose tissue mass (P < 0.05), decreased brown adipose tissue mass (BAT, P < 0.01), lower night-time EE (P < 0.001) versus OffCon, which were prevented in OffObP. Both male and female OffOb showed abnormal glucose-tolerance test (peak [Glucose] P < 0.001; AUC, P < 0.05) which was prevented by PDX. The Ob challenge resulted in greater BW gain in both male and female OffOb versus OffCon (P < 0.05), also associated with increased EI (P < 0.05) and reduced EE in females (P < 0.01). OffObP were protected from accelerated BW gain on the OB diet compared with controls, associated with increased night-time EE in both male (P < 0.05) and female OffObP (P < 0.001). PDX also prevented an increase in skeletal muscle mtDNA copy number in OffOb versus OffCon (P < 0.01) and increased the percentage of Bacteroides cells in faecal samples from male OffObP relative to controls. CONCLUSIONS: Maternal obesity adversely influences adult offspring energy balance and propensity for obesity, which is ameliorated by maternal PDX treatment with associated changes in gut microbiota composition and skeletal muscle mitochondrial function.

A case for measuring both cellular and cell-free mitochondrial DNA as a disease biomarker in human blood.

Circulating mitochondrial DNA (mtDNA), widely studied as a disease biomarker, comprises of mtDNA located within mitochondria, indicative of mitochondrial function, and cell-free (cf) mtDNA linked to inflammation. The purpose of this study was to determine the ranges of, and relationship between, cellular and cf mtDNA in human blood. Whole blood from 23 controls (HC) and 20 patients with diabetes was separated into peripheral blood mononuclear cells (PBMCs), plasma, and serum. Total DNA was isolated and mtDNA copy numbers were determined using absolute quantification. Cellular mtDNA content in PBMCs was higher than in peripheral blood and a surprisingly high level of cf mtDNA was present in serum and plasma of HC, with no direct relationship between cellular and cf mtDNA content within individuals. Diabetes patients had similar levels of cellular mtDNA compared to healthy participants but a significantly higher cf mtDNA content. Furthermore, only in patients with diabetes, we observed a correlation between whole blood and plasma mtDNA levels, indicating that the relationship between cellular and cf mtDNA content is affected by disease status. In conclusion, when evaluating mtDNA in human blood as a biomarker of mitochondrial dysfunction, it is important to measure both cellular and cf mtDNA.

Optimizing beta cell function through mesenchymal stromal cell-mediated mitochondria transfer.

Pretransplant islet culture is associated with the loss of islet cell mass and insulin secretory function. Insulin secretion from islet ß-cells is primarily controlled by mitochondrial ATP generation in response to elevations in extracellular glucose. Coculture of islets with mesenchymal stromal cells (MSCs) improves islet insulin secretory function in vitro, which correlates with superior islet graft function in vivo. This study aimed to determine whether the improved islet function is associated with mitochondrial transfer from MSCs to cocultured islets. We have demonstrated mitochondrial transfer from human adipose MSCs to human islet ß-cells in coculture. Fluorescence imaging showed that mitochondrial transfer occurs, at least partially, through tunneling nanotube (TNT)-like structures. The extent of mitochondrial transfer to clinically relevant human islets was greater than that to experimental mouse islets. Human islets are subjected to more extreme cellular stressors than mouse islets, which may induce "danger signals" for MSCs, initiating the donation of MSC-derived mitochondria to human islet ß-cells. Our observations of increased MSC-mediated mitochondria transfer to hypoxia-exposed mouse islets are consistent with this and suggest that MSCs are most effective in supporting the secretory function of compromised ß-cells. Ensuring optimal MSC-derived mitochondria transfer in preculture and/or cotransplantation strategies could be used to maximize the therapeutic efficacy of MSCs, thus enabling the more widespread application of clinical islet transplantation.

The Detection and Partial Localisation of Heteroplasmic Mutations in the Mitochondrial Genome of Patients with Diabetic Retinopathy.

Diabetic retinopathy (DR) is a common complication of diabetes and a major cause of acquired blindness in adults. Mitochondria are cellular organelles involved in energy production which contain mitochondrial DNA (mtDNA). We previously showed that levels of circulating mtDNA were dysregulated in DR patients, and there was some evidence of mtDNA damage. In the current project, our aim was to confirm the presence of, and determine the location and prevalence of, mtDNA mutation in DR. DNA isolated from peripheral blood from diabetes patients (n = 59) with and without DR was used to amplify specific mtDNA regions which were digested with surveyor nuclease S1 to determine the presence and location of heteroplasmic mtDNA mutations were present. An initial screen of the entire mtDNA genome of 6 DR patients detected a higher prevalence of mutations in amplicon P, covering nucleotides 14,443 to 1066 and spanning the control region. Further analysis of 42 subjects showed the presence of putative mutations in amplicon P in 36% (14/39) of DR subjects and in 10% (2/20) non-DR subjects. The prevalence of mutations in DR was not related to the severity of the disease. The detection of a high-prevalence of putative mtDNA mutations within a specific region of the mitochondrial genome supports the view that mtDNA damage contributes to DR. The exact location and functional impact of these mutations remains to be determined.

Psychopharmacology of Pediatric Anxiety Disorders: A Narrative Review.

Anxiety disorders are common among children and adolescents; almost one-third of this population has an anxiety disorder. The most common anxiety disorders in this population are specific phobia (19.3%), social anxiety disorder/ social phobia (9.1 %), and separation anxiety disorder (7.6 %). Pediatric anxiety disorders are often associated with poor psychosocial functioning, academic underachievement, learning difficulties, substance abuse, relationship problems, and suicide behaviors. Psychotherapy, particularly cognitive behavioral therapy as a stand-alone treatment or in combination with medication, is found to be efficacious in the treatment of various anxiety disorders. The early recognition and treatment of anxiety disorders result in better long-term outcomes in children and adolescents. This article summarizes the evidence-based pharmacologic treatments for anxiety disorders in youth, including social anxiety disorder generalized anxiety disorder, separation anxiety disorder, and panic disorder.

A Diet Induced Maladaptive Increase in Hepatic Mitochondrial DNA Precedes OXPHOS Defects and May Contribute to Non-Alcoholic Fatty Liver Disease.

Non-alcoholic fatty liver disease (NAFLD), an increasingly prevalent and underdiagnosed disease, is postulated to be caused by hepatic fat mediated pathological mechanisms. Mitochondrial dysfunction is proposed to be involved, but it is not known whether this is a pathological driver or a consequence of NAFLD. We postulate that changes to liver mitochondrial DNA (mtDNA) are an early event that precedes mitochondrial dysfunction and irreversible liver damage. To test this hypothesis, we evaluated the impact of diet on liver steatosis, hepatic mtDNA content, and levels of key mitochondrial proteins. Liver tissues from C57BL/6 mice fed with high fat (HF) diet (HFD) and Western diet (WD, high fat and high sugar) for 16 weeks were used. Steatosis/fibrosis were assessed using haematoxylin and eosin (H&E) Oil Red and Masson's trichome staining and collagen content. Total DNA was isolated, and mtDNA content was determined by quantifying absolute mtDNA copy number/cell using quantitative PCR. Selected mitochondrial proteins were analysed from a proteomics screen. As expected, both HFD and WD resulted in steatosis. Mouse liver contained a high mtDNA content (3617 ± 233 copies per cell), which significantly increased in HFD diet, but this increase was not functional, as indicated by changes in mitochondrial proteins. In the WD fed mice, liver dysfunction was accelerated alongside downregulation of mitochondrial oxidative phosphorylation (OXPHOS) and mtDNA replication machinery as well as upregulation of mtDNA-induced inflammatory pathways. These results demonstrate that diet induced changes in liver mtDNA can occur in a relatively short time; whether these contribute directly or indirectly to subsequent mitochondrial dysfunction and the development of NAFLD remains to be determined. If this hypothesis can be substantiated, then strategies to prevent mtDNA damage in the liver may be needed to prevent development and progression of NAFLD.

Regional mitochondrial DNA and cell-type changes in post-mortem brains of non-diabetic Alzheimer's disease are not present in diabetic Alzheimer's disease.

Diabetes increases the risk of Alzheimer's disease (AD), and mitochondrial dysfunction is implicated in both diseases, however the impact of both diabetes and AD on brain mitochondria is not known. We measured mitochondrial DNA (mtDNA), an indicator of mitochondrial function, in frontal, parietal, and cerebellar regions of post-mortem human brains (n = 74) from non-cognitively impaired controls (NCI), mild-cognitively impaired (MCI) and AD cases. In a subset of parietal cortices, we measured mRNAs corresponding to cell types and mitochondrial function and semi-automated stereological assessment was performed on immune-staining of parietal cortex sections. mtDNA showed significant regional variation, highest in parietal cortex, and lowest in cerebellum. Irrespective of cognitive status, all brain regions had significantly higher mtDNA in diabetic cases. In the absence of diabetes, AD parietal cortices had decreased mtDNA, reduced MAP2 (neuronal) and increased GFAP (astrocyte) mRNA, relative to NCI. However, in the presence of diabetes, we did not observe these AD-related changes, suggesting that the pathology observed in diabetic AD may be different to that seen in non-diabetic AD. The lack of clear functional changes in mitochondrial parameters in diabetic AD suggest different cellular mechanisms contributing to cognitive impairment in diabetes which remain to be fully understood.

Reduced mitochondrial lipid oxidation leads to fat accumulation in myosteatosis.

Myosteatosis is the pathologic accumulation of lipid that can occur in conjunction with atrophy and fibrosis following skeletal muscle injury. Little is known about the mechanisms by which lipid accumulates in myosteatosis, but many clinical studies have demonstrated that the degree of lipid infiltration negatively correlates with muscle function and regeneration. Our objective was to determine the pathologic changes that result in lipid accumulation in injured muscle fibers. We used a rat model of rotator cuff injury in this study because the rotator cuff muscle group is particularly prone to the development of myosteatosis after injury. Muscles were collected from uninjured controls or 10, 30, or 60 d after injury and analyzed using a combination of muscle fiber contractility assessments, RNA sequencing, and undirected metabolomics, lipidomics, and proteomics, along with bioinformatics techniques to identify potential pathways and cellular processes that are dysregulated after rotator cuff tear. Bioinformatics analyses indicated that mitochondrial function was likely disrupted after injury. Based on these findings and given the role that mitochondria play in lipid metabolism, we then performed targeted biochemical and imaging studies and determined that mitochondrial dysfunction and reduced fatty acid oxidation likely leads to the accumulation of lipid in myosteatosis.-Gumucio, J. P., Qasawa, A. H., Ferrara, P. J., Malik, A. N., Funai, K., McDonagh, B., Mendias, C. L. Reduced mitochondrial lipid oxidation leads to fat accumulation in myosteatosis.

Mitochondrial genes are altered in blood early in Alzheimer's disease.

Although mitochondrial dysfunction is a consistent feature of Alzheimer's disease in the brain and blood, the molecular mechanisms behind these phenomena are unknown. Here we have replicated our previous findings demonstrating reduced expression of nuclear-encoded oxidative phosphorylation (OXPHOS) subunits and subunits required for the translation of mitochondrial-encoded OXPHOS genes in blood from people with Alzheimer's disease and mild cognitive impairment. Interestingly this was accompanied by increased expression of some mitochondrial-encoded OXPHOS genes, namely those residing closest to the transcription start site of the polycistronic heavy chain mitochondrial transcript (MT-ND1, MT-ND2, MT-ATP6, MT-CO1, MT-CO2, MT-C03) and MT-ND6 transcribed from the light chain. Further we show that mitochondrial DNA copy number was unchanged suggesting no change in steady-state numbers of mitochondria. We suggest that an imbalance in nuclear and mitochondrial genome-encoded OXPHOS transcripts may drive a negative feedback loop reducing mitochondrial translation and compromising OXPHOS efficiency, which is likely to generate damaging reactive oxygen species.

Hyperglycemia induced damage to mitochondrial respiration in renal mesangial and tubular cells: Implications for diabetic nephropathy.

Damage to renal tubular and mesangial cells is central to the development of diabetic nephropathy (DN), a complication of diabetes which can lead to renal failure. Mitochondria are the site of cellular respiration and produce energy in the form of ATP via oxidative phosphorylation, and mitochondrial dysfunction has been implicated in DN. Since the kidney is an organ with high bioenergetic needs, we postulated that hyperglycemia causes damage to renal mitochondria resulting in bioenergetic deficit. The bioenergetic profiles and the effect of hyperglycemia on cellular respiration of human primary mesangial (HMCs) and proximal tubular cells (HK-2) were compared in normoglycemic and hyperglycemic conditions using the seahorse bio-analyzer. In normoglycemia, HK-2 had significantly lower basal, ATP-linked and maximal respiration rates, and lower reserve capacity compared to HMCs. Hyperglycemia caused a down-regulation of all respiratory parameters within 4 days in HK-2 but not in HMCs. After 8 days of hyperglycemia, down-regulation of respiratory parameters persisted in tubular cells with compensatory up-regulated glycolysis. HMCs had reduced maximal respiration and reserve capacity at 8 days, and by 12 days had compromised mitochondrial respiration despite which they did not enhance glycolysis. These data suggest that diabetes is likely to lead to a cellular deficit in ATP production in both cell types, although with different sensitivities, and this mechanism could significantly contribute to the cellular damage seen in the diabetic kidney. Prevention of diabetes induced damage to renal mitochondrial respiration may be a novel therapeutic approach for the prevention/treatment of DN.

Accurate quantification of mouse mitochondrial DNA without co-amplification of nuclear mitochondrial insertion sequences.

BACKGROUND: Mitochondria contain an extra-nuclear genome in the form of mitochondrial DNA (MtDNA), damage to which can lead to inflammation and bioenergetic deficit. Changes in MtDNA levels are increasingly used as a biomarker of mitochondrial dysfunction. We previously reported that in humans, fragments in the nuclear genome known as nuclear mitochondrial insertion sequences (NumtS) affect accurate quantification of MtDNA. In the current paper our aim was to determine whether mouse NumtS affect the quantification of MtDNA and to establish a method designed to avoid this. METHODS: The existence of NumtS in the mouse genome was confirmed using blast N, unique MtDNA regions were identified using FASTA, and MtDNA primers which do not co-amplify NumtS were designed and tested. MtDNA copy numbers were determined in a range of mouse tissues as the ratio of the mitochondrial and nuclear genome using real time qPCR and absolute quantification. RESULTS: Approximately 95% of mouse MtDNA was duplicated in the nuclear genome as NumtS which were located in 15 out of 21 chromosomes. A unique region was identified and primers flanking this region were used. MtDNA levels differed significantly in mouse tissues being the highest in the heart, with levels in descending order (highest to lowest) in kidney, liver, blood, brain, islets and lung. CONCLUSION: The presence of NumtS in the nuclear genome of mouse could lead to erroneous data when studying MtDNA content or mutation. The unique primers described here will allow accurate quantification of MtDNA content in mouse models without co-amplification of NumtS.

Altered circulating mitochondrial DNA and increased inflammation in patients with diabetic retinopathy.

AIMS: We previously showed that circulating mitochondrial DNA (MtDNA) levels are altered in diabetic nephropathy. The aim of the current study was to determine if circulating MtDNA levels are altered in patients with diabetic retinopathy. METHODS: Patients with diabetes (n=220) were studied in a clinical setting using a cross-sectional study design as the following groups: DR-0 (no retinopathy, n=53), DR-m (mild non-proliferative diabetic retinopathy NPDR, n=98) and DR-s (severe proliferative diabetic retinopathy, n=69). MtDNA content in peripheral blood DNA was measured as the mitochondrial to nuclear genome ratio using real time qPCR. Circulating cytokines were measured using the luminex assay and MtDNA damage was assessed using PCR. Differences were considered significant at P<0.05. RESULTS: Circulating MtDNA values were higher in DR-m compared to DR-0 (P=0.02) and decreased in DR-s compared to DR-m (P=0.001). These changes remained significant after adjusting for associated parameters. In parallel there were increased levels of circulating cytokines IL-4 (P=0.005) and TNF-α (P=0.02) in the DR-s group and increased MtDNA damage in DR-m patients compared to DR-0 (P=0.03). CONCLUSIONS: Our data show that circulating MtDNA levels are independently associated with diabetic retinopathy, showing an increase in DR-m and decrease in DR-s with a parallel increase in MtDNA damage and inflammation. Hyperglycemia-induced changes in MtDNA in early diabetes may contribute to inflammation and progression of diabetic retinopathy. Longitudinal studies should be carried out to determine a potential causality of MtDNA in diabetic retinopathy.