Modern electron microscopy permits scientists to study the fine detail of cells and tissues, using both two-dimensional and three-dimensional imaging modalities. However, achieving optimal preservation of ultrastructure for a variety of biological samples remains a challenge. Here, we describe practical methods to preserve the fine structure of mouse skeletal muscle and sciatic nerve and to obtain high-resolution images of mitochondria in cultured cells, flow-sorted T-cells, and mouse urothelium. We also propose an effective and economical workflow for three-dimensional electron microscopy in the context of a microscopy core facility.

The zygote is the first cell of a multicellular organism. In most angiosperms, the zygote divides asymmetrically to produce an embryo-precursor apical cell and a supporting basal cell. Zygotic division should properly segregate symbiotic organelles, because they cannot be synthesized de novo. In this study, we revealed the real-time dynamics of the principle source of ATP biogenesis, mitochondria, in Arabidopsis thaliana zygotes using live-cell observations and image quantifications. In the zygote, the mitochondria formed the extended structure associated with the longitudinal array of actin filaments (F-actins) and were polarly distributed along the apical–basal axis. The mitochondria were then temporally fragmented during zygotic division, and the resulting apical cells inherited mitochondria at higher concentration compared to the basal cells. Further observation of postembryonic organs showed that these mitochondrial behaviours are characteristic of the zygote. Overall, our results showed that the zygote has spatiotemporal regulation that unequally distributes the mitochondria.

Down syndrome (DS) is a genetically based disease caused by triplication of chromosome 21. DS is characterized by severe muscle weakness associated with motor deficits; however, understanding the DS-associated skeletal muscle condition is limited. In this study, we used a combined methodological approach involving light and electron microscopy, as well as nuclear magnetic resonance spectroscopy metabolomics, to investigate morphology and composition of the quadriceps muscles in the Ts65Dn mouse, a model of DS, to identify structural and/or functional trisomy-associated alterations. Morphometric analysis demonstrated a larger size of myofibers in trisomic versus euploid mice; however, myofibrils were thinner and contained higher amounts of mitochondria and lipid droplets. In trisomic mice, magnetic resonance spectroscopy showed a tendency to an overall increase in muscle metabolites involved in protein synthesis. These data strongly suggest that in DS, a sarcoplasmic hypertrophy associated with myofibril loss characterizes quadriceps myofibers. In addition, large-sized mitochondria suggestive of impaired fission/fusion events, as well as metabolites modifications suggestive of decreased mitochondrial function, were found in the trisomic muscle. Albeit preliminary, the results provided by this novel approach consistently indicate structural and compositional alterations of the DS skeletal muscle, which are typical of early aging.

Nonalcoholic fatty liver disease (NAFLD) represents a hepatic manifestation of metabolic syndrome. The aim of this study was to examine the effect of betaine on ultrastructural changes in the mouse liver with methionine- and choline-deficient (MCD) diet-induced NAFLD. Male C57BL/6 mice were divided into groups: Control—fed with standard chow, BET—standard chow supplemented with betaine (1.5% w/v drinking water), MCD—fed with MCD diet, and MCD + BET—MCD diet with betaine supplementation for 6 weeks. Liver samples were taken for pathohistology and transmission electron microscopy. The MCD diet-induced steatosis, inflammation, and balloon-altered hepatocytes were alleviated by betaine. MCD diet induced an increase in mitochondrial size versus the control group (p < 0.01), which was decreased in the betaine-treated group. In the MCD diet-fed group, the total mitochondrial count decreased versus the control group (p < 0.01), while it increased in the MCD + BET group versus MCD (p < 0.01). Electron microscopy showed an increase in the number of autophagosomes in the MCD and MCD + BET group versus control, and a significant difference in autophagosomes number was detected in the MCD + BET group by comparison with the MCD diet-treated group (p < 0.05). Betaine decreases the number of enlarged mitochondria, alleviates steatosis, and increases the number of autophagosomes in the liver of mice with NAFLD.

Seed is a fertilized mature ovule, which possesses an embryonic plant. When the dry, mature seeds are subjected to imbibition, they release a wide range of organic substances, which include low molecular weight carbonyl compounds (gases and volatiles) and water-soluble organic substances (enzymes and polysaccharides). The volatile organic compounds (VOCs) are molecules of low molecular weight (300 g mol−1) and high vapour pressure (0.01 kPa at 20°C) and include diverse chemical compounds. The nature and emission kinetics of volatiles produced from seeds vary, depending on the moisture content of the seeds. Orthodox seeds stored at ‘low seed moisture content’ undergo seed deterioration, predominantly due to lipid peroxidation, initiated by autoxidation or enzymatic oxidation of unsaturated or polyunsaturated fatty acids. This peroxidation leads to emission of volatile compounds. The quantity of VOCs emitted is positively correlated with the advancement of seed deterioration. With respect to the seed germination process, exposure of seeds to ‘high moisture conditions’ leads to increased respiration, triggers glycolysis and mobilization of storage reserves, resulting in the emission of volatile metabolic products. The quantity of VOCs emitted on commencement of metabolic activity in germinating seeds depends on (1) vigour status and (2) amount of storage reserves. Since it has been established that there is a significant difference between high and low vigour seeds with respect to quantity and profile of VOCs emitted, there is great potential for utilizing the VOC profile to obtain a quick and reproducible test of vigour status of crop seeds. In order to harness the VOC profile for quick assessment of vigour status of seeds, research has to be taken up to develop standard protocols for fingerprinting of VOCs for the purpose of seed vigour assessment and to fix the standard volatile biomarker(s) specific to crop and vigour status of seeds.

Weaning is known to induce important nutritional and energetic stress in piglets. Low-birthweight (LBW) piglets, now frequently observed in swine production, are more likely to be affected. The weaning period is also associated with dysfunctional immune responses, uncontrolled inflammation and oxidative stress conditions that are recognized risk factors for infections and diseases. Mounting evidence indicates that mitochondria, the main cellular sources of energy in the form of adenosine 5′ triphosphate (ATP) and primary sites of reactive oxygen species production, are related to immunity, inflammation and bacterial pathogenesis. However, no information is currently available regarding the link between mitochondrial energy production and oxidative stress in weaned piglets. The objective of this study was to characterize markers of cellular and mitochondrial energy metabolism and oxidative status in both normal-birthweight (NBW) and LBW piglets throughout the peri-weaning period. To conduct the study, 30 multiparous sows were inseminated and litters were standardized to 12 piglets. All the piglets were weighted at day 1 and 120 piglets were selected and assigned to 1 of 2 experimental groups: NBW (n = 60, mean weight of 1.73 ± 0.01 kg) and LBW piglets weighing less than 1.2 kg (n = 60, 1.01 ± 0.01 kg). Then, 10 piglets from each group were selected at 14, 21 (weaning), 23, 25, 29 and 35 days of age to collect plasma and organ (liver, intestine and kidney) samples. Analysis revealed that ATP concentrations were lower in liver of piglets after weaning than during lactation (P < 0.05) thus suggesting a significant impact of weaning stress on mitochondrial energy production. Oxidative damage to DNA (8-hydroxy-2′-deoxyguanosine, 8-OHdG) and proteins (carbonyls) measured in plasma increased after weaning and this coincides with a rise in enzymatic antioxidant activity of glutathione peroxidase (GPx) and superoxide dismutase (SOD) (P < 0.05). Mitochondrial activities of both GPx and SOD are also significantly higher (P < 0.05) in kidney of piglets after weaning. Additionally, oxidative damage to macromolecules is more important in LBW piglets as measured concentrations of 8-OHdG and protein carbonyls are significantly higher (P < 0.05) in plasma and liver samples, respectively, than for NBW piglets. These results provide novel information about the nature, intensity and duration of weaning stress by revealing that weaning induces mitochondrial dysfunction and cellular oxidative stress conditions which last for at least 2 weeks and more severely impact smaller piglets.

Several studies have proposed that cell-free DNA (cfDNA) is a potential biomarker present in follicular fluid (FF) for oocyte quality. Recently we reported that mitochondria-derived cfDNA (mt-cfDNA) closely reflects the amount of cfDNA in FFs. The present study investigated the mechanism regulating mt-cfDNA secretion from porcine granulosa cells. Oocytes and cumulus cell complexes or granulosa cells (GCs) were cultured in maturation medium for 24 or 48 h respectively. Then, nuclear-derived cell-free DNA (n-cfDNA) or mt-cfDNA contents in the spent medium were examined using real-time polymerase chain reaction. When 10 μM of MG132, a proteasome inhibitor, was added to the culture medium, cellular viability of both COCs and GCs decreased and n-cfDNA significantly increased in the culture medium, whereas mt-cfDNA significantly decreased. Supplementation of the culture medium with GW4869, an inhibitor of intracellular vesicle formation, significantly decreased the mt-cfDNA, whereas no effect was observed on n-cfDNA in the medium of both COCs and GCs. Furthermore, the addition of bafilomycin, an inhibitor of autophagy to the culture medium significantly increased mt-cfDNA in the culture medium. After filtration (0.22 μm) and centrifugation (23,000 g), the mt-cfDNA content of the medium decreased significantly. In conclusion, the proteasomal mitochondrial quality control system is upstream of mt-cfDNA secretion and autophagy plays a role in cellular digestion of mitochondrial DNA in the cytoplasm. It is further suggested that dsDNA is enclosed in certain vesicles or associated with small molecules and secreted into the medium.

Vitamin D receptor expression and associated function have been reported in various muscle models, including C2C12, L6 cell lines and primary human skeletal muscle cells. It is believed that 1,25-hydroxyvitamin D3 (1,25(OH)2D3), the active form of vitamin D, has a direct regulatory role in skeletal muscle function, where it participates in myogenesis, cell proliferation, differentiation, regulation of protein synthesis and mitochondrial metabolism through activation of various cellular signalling cascades, including the mitogen-activated protein kinase pathway(s). It has also been suggested that 1,25(OH)2D3 and its associated receptor have genomic targets, resulting in regulation of gene expression, as well as non-genomic functions that can alter cellular behaviour through binding and modification of targets not directly associated with transcriptional regulation. The molecular mechanisms of vitamin D signalling, however, have not been fully clarified. Vitamin D inadequacy or deficiency is associated with muscle fibre atrophy, increased risk of chronic musculoskeletal pain, sarcopenia and associated falls, and may also decrease RMR. The main purpose of the present review is to describe the molecular role of vitamin D in skeletal muscle tissue function and metabolism, specifically in relation to proliferation, differentiation and protein synthesis processes. In addition, the present review also includes discussion of possible genomic and non-genomic pathways of vitamin D action.

Colorectal cancer (CRC) is the third most common cancer globally. CRC risk is increased by obesity, and by its lifestyle determinants notably physical inactivity and poor nutrition. Obesity results in increased inflammation and oxidative stress which cause genomic damage and contribute to mitochondrial dysregulation and CRC risk. The mitochondrial dysfunction associated with obesity includes abnormal mitochondrial size, morphology and reduced autophagy, mitochondrial biogenesis and expression of key mitochondrial regulators. Although there is strong evidence that increased adiposity increases CRC risk, evidence for the effects of intentional weight loss on CRC risk is much more limited. In model systems, energy depletion leads to enhanced mitochondrial integrity, capacity, function and biogenesis but the effects of obesity and weight loss on mitochondria in the human colon are not known. We are using weight loss following bariatric surgery to investigate the effects of altered adiposity on mitochondrial structure and function in human colonocytes. In summary, there is strong and consistent evidence in model systems and more limited evidence in human subjects that over-feeding and/or obesity result in mitochondrial dysfunction and that weight loss might mitigate or reverse some of these effects.

Photoreceptors have high energy demands and densely packed mitochondria through which light passes before phototransduction. Old world primates including humans have three cone photoreceptor types mediating color vision with short (S blue), medium (M green), and long (L red) wavelength sensitivities. However, S-cones are enigmatic. They comprise <10% of the total cone population, their responses saturate early, and they are susceptible in aging and disease. Here, we show that primate S-cones actually have few mitochondria and are fueled by glycolysis, not by mitochondrial respiration. Glycolysis has a limited ability to sustain activity, potentially explaining early S-cone saturation. Mitochondria act as optical filters showing reduced light transmission at 400–450 nm where S-cones are most sensitive (420 nm). This absorbance is likely to arise in a mitochondrial porphyrin that absorbs strongly in the Soret band. Hence, reducing mitochondria will improve S-cone sensitivity but result in increased glycolysis as an alternative energy source, potentially increasing diabetic vulnerability due to restricted glucose access. Further, glycolysis carries a price resulting in premature functional decline as seen in aged S-cones. Soret band absorption may also impact on mitochondrial rich M and L cones by reducing sensitivity at the lower end of their spectral sensitivity range resulting in increased differentiation from S-cone responses. These data add to the list of unique characteristic of S-cones and may also explain aspects of their vulnerability.

Germ plasm-related structures (GPRS) are known to accompany meiotic cell differentiation but their dynamics are still poorly understood. In this study, we analyzed the ultrastructural mechanisms of GPRS transformation during oogenesis and spermatogenesis of the bivalve mollusc Ruditapes philippinarum (Manila clam), exploring patterns of GPRS activity occurring at meiosis onset, sex-specific difference/similarity of such patterns, and the involvement of mitochondria during GPRS-assigned events. In the two sexes, the zygotene–pachytene stage of meiosis is anticipated by three shared steps. First, the dispersion of germ plasm granules containing the germ line determinant VASA occurs. Second, the VASA protein deriving from germ plasm granules enters neighbouring mitochondria and appears to induce mitochondrial matter release, as supported by cytochrome B localization outside the mitochondria. Third, intranuclear VASA entrance occurs and the protein appears involved in chromatin reorganization, as supported by VASA localization in synaptonemal complexes. In spermatogenesis, these three steps are sufficient for the normal course of meiosis. In oogenesis, these are followed by the action of ‘germ plasm granule formation complex’, a novel type of structure that appears alternative to the Balbiani body. The possibility of germ plasm involvement in reproductive technologies is also suggested.

Endoperoxides kill malaria parasites via cleavage of their endoperoxide bridge by haem or iron, leading to generation of cytotoxic oxygen-centred radicals. In view of the Leishmania parasites having a relatively compromised anti-oxidant defense and high iron content, this study aims to establish the underlying mechanism(s) accounting for the apoptotic-like death of Leishmania promastigotes by artemisinin, an endoperoxide. The formation of reactive oxygen species was confirmed by flow cytometry and was accompanied by inhibition of mitochondrial complexes I–III and II–III. However, this did not translate into a generation of mitochondrial superoxide or decrease in oxygen consumption, indicating minimal impairment of the electron transport chain. Artemisinin caused depolarization of the mitochondrial membrane along with a substantial depletion of adenosine triphosphatase (ATP), but it was not accompanied by enhancement of ATP hydrolysis. Collectively, the endoperoxide-mediated radical formation by artemisinin in Leishmania promastigotes was the key step for triggering its antileishmanial activity, leading secondarily to mitochondrial dysfunction indicating that endoperoxides represent a promising therapeutic strategy against Leishmania worthy of pharmacological consideration.

Candidatus Midichloria mitochondrii is a maternally inherited bacterium of ticks with a unique intra-mitochondrial lifestyle. Here, we investigate on the evolutionary history of these associations and the degree of Midichloria–tick specificity. While previous surveys used the 16S rRNA gene as an exclusive molecular marker, we rather developed a multi-locus typing method based on four more variable housekeeping genes (groEL, rpoB, dnaK and ftsZ) and on one flagellum gene (fliC) present in Midichloria genomes. Using this method, multi-locus phylogenetic analyses revealed the structuring of a wide Midichloria genetic diversity into three distinct lineages associated with ticks. Overall, two distinct evolutionary strategies are obvious depending on lineage: two Midichloria lineages are generalists with infections acquired through horizontal transfers between distantly related tick species but one other Midichloria lineage rather show a high specificity degree to the Ixodes tick genus. This pattern suggests a capacity of certain Midichloria strains to maintain infections in only limited range of related tick species. These different infection strategies of Midichloria highlight an unexpected variability in their dependency to their tick hosts. We further conjecture that this pattern is also likely to indicate variability in their effects on ticks.

Mosquitoes’ importance as vectors of pathogens that drive disease underscores the importance of precise and comparable methods of taxa identification among their species. While several molecular targets have been used to study mosquitoes since the initiation of PCR in the 1980s, its application to mosquito identification took off in the early 1990s. This review follows the research's recent journey into the use of mitochondrial DNA (mtDNA) cytochrome oxidase 1 (COI or COX1) as a DNA barcode target for mosquito species identification – a target whose utility for discriminating mosquitoes is now escalating. The pros and cons of using a mitochondrial genome target are discussed with a broad sweep of the mosquito literature suggesting that nuclear introgressions of mtDNA sequences appear to be uncommon and that the COI works well for distantly related taxa and shows encouraging utility in discriminating more closely related species such as cryptic/sibling species groups. However, the utility of COI in discriminating some closely related groups can be problematic and investigators are advised to proceed with caution as problems with incomplete lineage sorting and introgression events can result in indistinguishable COI sequences appearing in reproductively independent populations. In these – if not all – cases, it is advisable to run a nuclear marker alongside the mtDNA and thus the utility of the ribosomal DNA – and in particular the internal transcribed spacer 2 – is also briefly discussed as a useful counterpoint to the COI.

The treatment of leishmaniasis relies primarily on highly toxic, parenterally administered drugs. Therefore, the search for more effective and safer drugs is considered a priority for leishmaniasis’ control. The antileishmanial activity of the oestrogen receptor modulator tamoxifen was previously described in experimental models of leishmaniasis. However, the mechanisms responsible for the antileishmanial activity of tamoxifen remain unknown. Since tamoxifen has been shown to affect plasma and intracellular membranes in tumour cells, the aim of this study was to investigate the activity of the drug on Leishmania amazonensis membranes. Through morphological analysis and labelling with propidium iodide and DiSBAC2(3), we demonstrated that tamoxifen led to plasma membrane depolarization without general membrane disruption or permeabilization. Tamoxifen also caused mitochondrial damage, with loss of membrane potential, as shown by Rhodamine 123 accumulation. Mitochondrial swelling followed, signalling the mitochondrial dysfunction. Therefore, the effect of tamoxifen on Leishmania is mediated, at least in part, by disorder in parasite's membranes. These alterations are sufficient to trigger a series of lethal events.

Parthenotes are characterized by poor in vitro developmental potential either due to the ploidy status or the absence of paternal factors. In the present study, we demonstrate the beneficial role of sperm-derived factors (SDF) on the in vitro development of mouse parthenotes. Mature (MII) oocytes collected from superovulated Swiss albino mice were activated using strontium chloride (SrCl2) in the presence or absence of various concentrations of SDF in M16 medium. The presence of SDF in activation medium did not have any significant influence on the activation rate. However, a significant increase in the developmental potential of the embryos and increased blastocyst rate (P < 0.01) was observed at 50 µg/ml concentration. Furthermore, the activated oocytes from this group exhibited early cleavage and cortical distribution of cortical granules that was similar to that of normally fertilized zygotes. Culturing 2-cell stage parthenotes in the presence of SDF significantly improved the developmental potential (P < 0.05) indicating that they also play a significant role in embryo development. In conclusion, artificial activation of oocytes with SDF can improve the developmental potential of parthenotes in vitro.