High Number Of Mitochondria Research Articles

Abstract A027: Targeting MYC-induced reprogramming of mitochondrial metabolism in breast cancer

Abstract Oncogenic MYC is frequently overexpressed in breast cancer, and its overexpression is associated with aggressiveness and unfavorable prognosis of breast cancer. MYC overexpression promotes tumor growth by stimulating cell cycle progression and rewiring metabolism – creating cancer vulnerabilities that can be therapeutically targeted via synthetic lethal strategies. A large part of our current understanding of the MYC overexpression biology and related synthetic lethal pathways is based on findings in various inducible MYC overexpression systems, which demonstrate the acute effects of MYC activation but may poorly represent long-term MYC overexpression status in real cancers. Here we classified 15 triple-negative breast cancer (TNBC) cell lines according to both MYC target gene set expression and MYC protein expression into MYC-high and MYC-low categories and explored the MYC-high status-coupled therapeutic responses and cancer-relevant metabolic changes. We show that mitochondrial respiration inhibiting biguanide drug metformin inhibits cell proliferation and generates senescence-like features in MYC-high TNBC cells. Furthermore, high-resolution respirometry experiments show that MYC-high TNBC cells have higher mitochondrial respiration rates than MYC-low TNBC cells, a phenotype not explained by a higher number of mitochondria. Also, targeting mitochondrial respiration with small molecule inhibitors specifically reduces proliferation in MYC-high TNBC cells and sensitizes them to apoptosis – indicating that MYC-high TNBC cells have higher dependency on mitochondrial respiration than the MYC-low cells. Targeted metabolomic flux experiments demonstrate that small molecule inhibitors of mitochondrial respiration rewire TCA cycle, resulting in suppression of MYC-driven glutamine anaplerosis, which offers a possible explanation for the apoptotic sensitization. In conclusion, our findings provide new insight into the long-term MYC overexpression-associated metabolic changes – highlighting mitochondrial respiration as a potential target for the development of MYC-dependent synthetic lethal strategies against breast cancer. Citation Format: Johanna M. Anttila, Mariel Savelius, Daniel Nicorici, Ryan Awadhpersad, Pauliina M. Munne, Linda Id, Topi A. Tervonen, Christopher Jackson, Anni I. Nieminen, Juha Klefström. Targeting MYC-induced reprogramming of mitochondrial metabolism in breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A027.

Role of circulating mitochondria in venous thrombosis in glioblastoma

BackgroundMany patients with glioblastoma multiforme (GBM) develop deep venous thrombosis or pulmonary emboli. Cell-free circulating mitochondria increase after brain injury and are associated with coagulopathy. ObjectivesThis study evaluated whether mitochondria play a role in the GBM-induced hypercoagulable state. MethodsWe examined the correlation between cell-free circulating mitochondria and venous thrombosis in patients with GBM and the impact of mitochondria on venous thrombosis in mice with inferior vena cava stenosis. ResultsUsing plasma samples of 82 patients with GBM, we found that patients with GBM had a higher number of mitochondria in their plasma (GBM with venous thromboembolism [VTE],: 2.8 × 107 mitochondria/mL; GBM without VTE, 1.9 × 107 mitochondria/mL) than that in healthy control subjects (n = 17) (0.3 × 107 mitochondria/mL). Interestingly, patients with GBM and VTE (n = 41) had a higher mitochondria concentration than patients with GBM without VTE (n = 41). In a murine model of inferior vena cava stenosis, intravenous delivery of mitochondria resulted in an increased rate of venous thrombosis compared with that in controls (70% and 28%, respectively). Mitochondria-induced venous thrombi were neutrophil-rich and contained more platelets than those in control thrombi. Furthermore, as mitochondria are the only source of cardiolipin in circulation, we compared the concentration of anticardiolipin immunoglobulin G in plasma samples of patients with GBM and found a higher concentration in patients with VTE (optical density, 0.69 ± 0.04) than in those without VTE (optical density, 0.51 ± 0.04). ConclusionWe concluded that mitochondria might play a role in the GBM-induced hypercoagulable state. We propose that quantifying circulating mitochondria or anticardiolipin antibody concentrations in patients with GBM might identify patients at increased risk of VTE.

Viability of mitochondria-labeled retinal ganglion cells in organotypic retinal explant cultures by two methods

Retinal explant cultures provide a valuable system to study retinal function in vitro. This study established a new retinal explant culture method to prolong the survival of retinal ganglion cells (RGCs). Explants were prepared in two different ways: with or without optic nerve. Retinas from newborn mice that had received an injection of MitoTracker Red into the contralateral superior colliculus to label axonal mitochondria were cultured as organotypic culture for 7 days in vitro. At several time points during the culture, viability of RGCs was assessed by multi-electrode array recording, and morphology by immunohistochemical methods. During the culture, the thickness of the retinal tissue in both groups gradually decreased, however, the structure of the layers of the retina could be identified. Massive apoptosis in the retinal ganglion cell layer (GCL) appeared on the first day of culture, thereafter the number of apoptotic cells decreased. Glial activation was observed throughout the culture, and there was no difference in morphology between the two groups. RGCs loss was exacerbated on 3rdday of culture, and RGCs loss in retinal explants with preserved optic nerve was significantly lower than in retinas that did not preserve the optic nerve. More and longer-lasting mitochondrial signals were observed in the injured area of the optic nerve-preserving explants. Retinal explants provide an invaluable tool for studying retinal function and developing treatments for ocular diseases. The optic nerve-preserving culture helps preserve the integrity of RGCs. The higher number of mitochondria in the nerve-preserving cultures may help maintain viability of RGCs.

CD177+ cells produce neutrophil extracellular traps that promote biliary atresia.

We have previously reported on the potential pathogenic role of neutrophils in biliary atresia (BA). Herein, we aimed to delineate the role of CD177+ neutrophils in the pathogenesis of BA. Immune cells from the livers of mice with rhesus rotavirus-induced BA were analysed. Single-cell RNA-sequencing was performed to specifically analyse Gr-1+ (Ly6C/Ly6G+) cells in the liver. Gene expression profiles of CD177+ cells were analysed using the Smart-Seq RNA-sequencing method, and the pathogenesis of BA was examined in Cd177-/- mice. Neutrophil extracellular trap (NET) inhibitors were used to determine the role of CD177+ cell-derived NETs in BA-associated bile duct damage, and a pilot clinical study evaluated the potential effects of N-acetylcysteine on NET release in BA. Increased levels of Gr-1+ cells were observed in the livers of mice with rhesus rotavirus-induced BA. RNA-sequencing analysis revealed that CD177+ cells were the main population of Gr-1+ cells and expressed elevated levels of both interferon-stimulated and neutrophil degranulation genes. Cd177-/- BALB/c mice exhibited delayed disease onset and reduced morbidity and mortality. High numbers of mitochondria were detected in CD177+ cells derived from mice with BA; these cells were associated with increased levels of reactive oxygen species and increased NET formation, which induced the apoptosis of biliary epithelial cells in cocultures. In a pilot clinical study, the administration of N-acetylcysteine to patients with BA reduced CD177+ cell numbers and reactive oxygen species levels, indicating a potential beneficial effect. Our data indicate that CD177+ cells play an important role in the initiation of BA pathogenesis via NET formation. The pilot study of N-acetylcysteine treatment in patients with BA was registered on the Chinese Clinical Trial Registry (ChiCTR2000040505). Neutrophils (a type of innate immune cell, i.e. an immune cell that doesn't target a specific antigen) are thought to play a role in the development of biliary atresia (a rare but potentially lethal condition of the bile ducts that occurs in infants). Herein, we found that neutrophils expressing a particular protein (CD177) played an important role in bile duct damage by releasing a special structure (NET) that can trap and kill pathogens but that can also cause severe tissue damage. A pilot study in patients with biliary atresia showed that inhibiting NETs could have a beneficial effect.

Ultrastructure of Terpene and Polyphenol Synthesis in the Bark of Cupressus sempervirens After Seiridium cardinale Infection.

Cypress Canker Disease (CCD) pandemic caused by Seiridium cardinale is the major constraint of many Cupressaceae worldwide. One of the main symptoms of the disease is the flow of resin from the cankered barks. While inducible phloem axial resin duct-like structures (PARDs) have recently been characterized from an anatomical point of view, their actual resin production is still being debated and has never been demonstrated. Although the involvement of polyphenolic parenchyma cells (PP cells) in the bark of Cupressus sempervirens after S. cardinale infection was revealed in one of our previous studies using light microscopy, their evolution from the phloem parenchyma cells is yet to be clarified. This study investigated functional and ultrastructural aspects of both PARD-like structures and PP cells by means of more in-depth light (LM) and fluorescence microscopy (FM) combined with histochemical staining (using Sudan red, Fluorol Yellow, NADI Aniline blue black, and Toluidine blue staining), in addition to Transmission Electron Microscope (TEM). Two-year-old stem sections of a C. sempervirens canker-resistant clone (var. “Bolgheri”), artificially inoculated with S. cardinale, were sampled 5, 7, 14, 21, and 45 days after inoculation, for time-course observations. FM observation using Fluorol yellow dye clearly showed the presence of lipid material in PARD-like structures lining cells of the cavity and during their secretion into the duct space/cavity. The same tissues were also positive for NADI staining, revealing the presence of terpenoids. The cytoplasm of the ducts' lining cells was also positive for Sudan red. TEM observation highlighted the involvement of plastids and endoplasmic reticulum in the production of terpenoids and the consequent secretion of terpenoids directly through the plasma membrane, without exhibiting vesicle formation. The presence of a high number of mitochondria around the area of terpenoid production suggests that this process is active and consumes ATP. The LM observations showed that PP cells originated from the phloem parenchyma cells (and possibly albuminous cells) through the accumulation of phenolic substances in the vacuole. Here, plastids were again involved in their production. Thus, the findings of this work suggest that the PARD-like structures can actually be considered PARDs or even bark traumatic resin ducts (BTRD).

Renal mitochondrial injury in the pathogenesis of CKD: mtDNA and mitomiRs.

Chronic kidney disease (CKD) is a public health concern that affects over 200 million people worldwide and is associated with a tremendous economic burden. Therefore, deciphering the mechanisms underpinning CKD is crucial to decelerate its progression towards end-stage renal disease (ESRD). Renal tubular cells are populated with a high number of mitochondria, which produce cellular energy and modulate several important cellular processes, including generation of reactive oxygen species (ROS), calcium homeostasis, proliferation, and apoptosis. Over the past few years, increasing evidence has implicated renal mitochondrial damage in the pathogenesis of common etiologies of CKD, such as diabetes, hypertension, metabolic syndrome (MetS), chronic renal ischemia, and polycystic kidney disease (PKD). However, most compelling evidence is based on preclinical studies because renal biopsies are not routinely performed in many patients with CKD. Previous studies have shown that urinary mitochondrial DNA (mtDNA) copy numbers may serve as non-invasive biomarkers of renal mitochondrial dysfunction. Emerging data also suggest that CKD is associated with altered expression of mitochondria-related microRNAs (mitomiRs), which localize in mitochondria and regulate the expression of mtDNA and nucleus-encoded mitochondrial genes. This review summarizes relevant evidence regarding the involvement of renal mitochondrial injury and dysfunction in frequent forms of CKD. We further provide an overview of non-invasive biomarkers and potential mechanisms of renal mitochondrial damage, especially focusing on mtDNA and mitomiRs.

Ultrastructural investigation of the pneumocytes in piglets that live in a trashed environment

The present study provides the ultrastructure of the pneumocytes types II and I in piglets living in the trash environment. Samples of the lungs of twelve piglets two months old were used. By light microscopy, the pneumocytes type I were squamous and somewhat flattened cells, while pneumocytes type II was cuboidal to spherical projected within the alveolar lumen and characterized by a spherical nucleus and foamy cytoplasm, it contained vacuolated bodies that were polygonal with variable size manly at the poles of the cell. The electron microscopy investigation showed blood air barrier between the endothelial lining of pneumocytes type I and therefore the endothelial lining of blood capillary and their nucleus were irregular in shape varied from nearly irregular triangular to polygon rough endoplasmic reticulum represented at their cytoplasm. The pneumocytes type II were frequently covered by pneumocytes type I extensions and united to them by a tight junction. It had been characterized by a high number of mitochondria within the cytoplasm and vacuolated bodies encircled the nucleus and at the two extremities of the cell. The lamellar vacuolated bodies were connected to the endoplasmic reticulum membranes and therefore the intravascular macrophages were attached to the endothelial cells within the pulmonary capillaries until two months old piglets. The occurrence of the intravascular macrophages could be attributed to the higher resistance to the respiratory diseases of the piglets.

N-butylidenephthalide ameliorates high-fat diet-induced obesity in mice and promotes browning through adrenergic response/AMPK activation in mouse beige adipocytes

Thermogenesis (non-exercise activity) in brown adipose tissue (BAT) promotes energy expenditure because of its higher number of mitochondria than white adipose tissue (WAT). The main function of thermogenesis in BAT can counteract obesity through the dissipation of calories as heat. N-butylidenephthalide (BP) is a natural derivative from Angelica sinensis, a Chinese herb that has been used for thousands of years. In this report, we demonstrated that BP improved the metabolic profiles of mice with high fat diet-induced obesity (DIO) by preventing weight gain, improving serum blood parameters, enhancing energy expenditure, stimulating white fat browning, and reversing hepatic steatosis. Further investigations demonstrated that BP administration upregulated the mRNA expression of beige (CD137, TMEM26) and brown fat selected genes (UCP1, PRDM16, PGC-1α, PPARγ) in white adipose tissues. In vitro studies, BP treatment increased multilocular lipid droplet levels, induced β-adrenergic receptor (cAMP/PKA) and AMP-activated protein kinase (AMPK) signaling (AMPK/acetyl-CoA carboxylase/SIRT1), and increased oxygen consumption in murine differentiated beige adipocytes, and the effects of BP were blocked by an AMPK inhibitor. BP promoted the interaction of AMPK with PGC-1α in beige adipocytes. Our findings provide novel insights into the application of BP in regulating energy metabolism and suggest its utility for clinical use in the treatment of obesity and related diseases.

Abstract 100: Mitochondrial Dynamics Directs Macrophage Polarization During Atherosclerosis: Implications For Disease Regression

Coronary heart disease is caused by atherosclerosis, a cholesterol-driven inflammatory disease of the vessel wall. A hallmark of atherogenesis is the accumulation of pro-inflammatory (M1) macrophages within the vessel wall, which sustains the inflammatory environment of the atherosclerotic plaque. In contrast, during disease regression, macrophages adopt a pro-resolving, anti-inflammatory phenotype (M2). Macrophage subtypes also exhibit different metabolic programs, where M1 macrophages' preferentiality use glycolytic metabolism and are associated with high levels of reactive oxygen species (ROS), while M2 macrophages primarily use oxidative metabolism. Macrophages adapt to these different metabolic needs and protect from mitochondrial DNA damage by ROS by undergoing cycles of fission (fragmentation) & fusion (elongation), which have been demonstrated to direct differentiation and function in other cell types, including T-cells. We hypothesize that the balance of mitochondrial fusion & fission directs macrophage polarization and may influence macrophage plasticity during atherosclerosis progression and regression. We found that mouse bone marrow-derived macrophages polarized to an M1 phenotype have a more elongated mitochondrial network, whereas both resting (M0) and M2 macrophages have a more fragmented mitochondrial phenotype. We find that M1 macrophages are more active with a higher number of mitochondria undergoing either a fission or fusion event compared to both M0 and M2 cells. The M1-like elongated mitochondrial phenotype can also be seen under atherogenic lipid-loading conditions. Exciting preliminary data suggests inhibiting mitochondrial fission (i.e. by knocking down three critical proteins, Drp1, Mff & Fis1) or inhibiting mitochondrial fusion (i.e. by knocking down fusion proteins Opa1, Mfn1 & Mfn2) impacts macrophages ability to polarizing into M1 or M2 phenotypes. Together these data suggest that the changes in mitochondrial dynamics direct the polarization of these macrophages and thus could be targeted to promote regression. Regression is an untapped area of atherosclerosis research, and mitochondria dynamics may be a new target to direction macrophage polarization within a lesion.

Mitochondrial bioenergetics and enzymatic antioxidant defense differ in Paraná pine cell lines with contrasting embryogenic potential

Araucaria angustifolia is classified as a critically endangered species by the International Union for Conservation of Nature. This threat is worsened by the inefficiency of methods for ex-situ conservation and propagation. In conifers, somatic embryogenesis (SE) associated with cryopreservation is an efficient method to achieve germplasm conservation and mass clonal propagation. However, the efficiency of SE is highly dependent on genotype responsivity to the artificial stimulus used in vitro during cell line proliferation and later during somatic embryo development. In this study, we evaluated the activity of antioxidant enzymes and characterized mitochondrial functions during the proliferation of embryogenic cells of A. angustifolia responsive (SE1) and non-responsive (SE6) to the development of somatic embryos. The activities of the antioxidant enzymes GR (EC 1.6.4.2), MDHAR (EC 1.6.5.4), and POX (EC 1.11.1.7) were increased in SE1 culture, while in SE6 culture, only the activity of DHAR (EC 1.8.5.1) was significantly higher. Additionally, SE6 culture presented a higher number of mitochondria, which agreed with the increased rate of oxygen consumption compared to responsive SE1 culture; however, the mitochondrial volume was lower. Although the ATP levels did not differ, the NAD(P)H levels were higher in SE1 cells. NDs, AOX, and UCP were less active in responsive SE1 than in non-responsive cells. Our results show significant differences between SE1 and SE6 embryogenic cells regarding mitochondrial functions and antioxidant enzyme activities, which may be intrinsic to the in vitro proliferation phase of both cell lines, possessing a crucial role for the induction of in vitro maturation process.

136 GameteGuard in maturation medium improves bovine embryo quality and quantity

The number of invitro-produced (IVP) bovine embryos generated in the United States increased 11% from 2017 to 2018, demonstrating a demand for new technology to rapidly and efficiently improve herd genetics. Unfortunately, embryo production on a per oocyte basis remains low, limiting the number of high-quality commercial embryos available for transfer or freezing. Reactive oxygen species (ROS) are a major contributor to low embryo production rates of invitro gametes and are especially problematic in maturation media. This is due to high numbers of mitochondria in the bovine oocyte producing ROS exacerbated by the invitro environment. GameteGuard® is an organic plant-based additive that provides protection against reactive oxygen species. The aims of this research were to evaluate the effects of GameteGuard addition to invitro bovine embryo production media to (1) mitigate the impact of ROS on oocyte maturation and competence, and (2) increase the production of high-quality embryos in our IVP system. Four rounds of invitro embryo production (Colorado State University protocol and media) with or without maturation medium supplemented with GameteGuard were performed (n=1175 total oocytes). Oocytes were aspirated from abattoir ovaries; those with homogeneous ooplasm and multiple layers of even cumulus cells were matured using control (n=585) or GameteGuard-supplemented (n=590) maturation medium. Oocytes were fertilized with frozen/thawed semen from proven IVF bulls (n=4) and then cultured in a two-step media system to Day 7. Blastocysts were evaluated on Day 7 according to IETS embryo grading standards. A subset of 80 blastocysts were fixed and stained after grading using an In Situ Cell Death Detection Kit (Fluorescein; Millipore Sigma) and Hoechst 33342 (Invitrogen) as apoptotic and counter-stains, respectively. Stained blastocysts were assessed by immunofluorescence microscopy to obtain the percentage of apoptotic cells and total cell count. Because multiple bulls were used, a linear mixed model was required for data analysis. Treatment was considered the fixed effect and bulls as a random effect to determine the impact of the treatment on stage and quality of blastocysts. Cleavage rates were not different (P>0.5) but GameteGuard supplementation of maturation medium resulted in a 22% increase in grade-1 embryos per total blastocysts produced (P<0.01; n=79/106 control vs. n=120/137 GameteGuard grade 1 blastocysts). Additionally, the percent of grade 2 and 3 embryos per total blastocysts decreased, (P<0.01; 27%, n=27/106 control and 11%, n=14/137 GameteGuard grade 2 and 3 blastocysts), suggesting that supplementation with GameteGuard promoted development of better-quality embryos overall. The percentage of apoptotic cells was also significantly higher in control blastocysts compared with treated blastocysts (P<0.02; 11.4%, n=38 control and 12.8%, n=42 GameteGuard). GameteGuard supplementation of the maturation medium significantly increased the production of high-quality IVP bovine embryos, thereby providing potential marketable gain for embryo producers.

Sound production, hearing sensitivity, and in-depth study of the sound-producing muscles in the cowfish (Lactoria cornuta).

The ability to produce sounds has been reported in various Ostraciidae but not deeply studied. In some Ostracion species, two different sound-producing muscles allow these boxfishes to produce two different kinds of sounds in a sequence. This study investigates sound production in another Indo-Pacific species, the longhorn cowfish Lactoria cornuta that also possesses two pairs of sonic muscles associated with the swim bladder: extrinsic sonic muscles (ESMs) and intrinsic sonic muscles (ISMs). The cowfish produces two kinds of sounds called hums and clicks. Hums are made of trains of low amplitude pulses that last for long periods of time, suggesting that they are produced by fatigue-resistant muscles, whereas clicks correspond to shorter sounds with greater amplitude than the hums, suggesting that they result from more powerful contractions. Ultra-structural differences are found between extrinsic and intrinsic sonic muscles. According to features such as long sarcomeres, long I-bands, a high number of mitochondria, and a proliferation of sarcoplasmic reticulum (SR), ESMs would be able to produce fast, strong, and short contractions corresponding to clicks (the shortest sounds with the greatest amplitude). ISMs have the thinnest cells, the smallest number of myofilaments that have long I-bands, the highest volume of mitochondria, and well-developed SR supporting these muscles; these features should generate fast and prolonged contractions that could correspond to the hums that can be produced over long periods of time. A concluding figure shows clear comparisons of the different fibers that were studied in L. cornuta. This study also compared the call features of each sound with the cowfish's hearing ability and supports L. cornuta was more sensitive to frequencies ranging between at least 100 and 400Hz with thresholds of 128-143dBre1µPa over this range, meaning that they are sensitive to the frequencies produced by conspecifics.

Hypoxic Regulation of Mitochondrial Metabolism and Mitophagy in Nucleus Pulposus Cells Is Dependent on HIF-1α–BNIP3 Axis

Nucleus pulposus (NP) cells reside in an avascular and hypoxic microenvironment of the intervertebral disc and are predominantly glycolytic due to robust HIF-1 activity. It is generally thought that NP cells contain few functional mitochondria compared with cells that rely on oxidative metabolism. Consequently, the contribution of mitochondria to NP cell metabolism and the role of hypoxia and HIF-1 in mitochondrial homeostasis is poorly understood. Using mitoQC reporter mice, we show for the first time to our knowledge that NP cell mitochondria undergo age-dependent mitophagy in vivo. Mechanistically, in vitro studies suggest that, under hypoxic conditions, mitochondria in primary NP cells undergo HIF-1α-dependent fragmentation, controlled by modulating the levels of key proteins DRP1 and OPA1 that are involved in mitochondrial fission and fusion, respectively. Seahorse assays and steady state metabolic profiling coupled with [1-2-13 C]-glucose flux analysis revealed that in hypoxia, HIF-1α regulated metabolic flux through coordinating glycolysis and the mitochondrial TCA cycle interactions, thereby controlling the overall biosynthetic capacity of NP cells. We further show that hypoxia and HIF-1α trigger mitophagy in NP cells through the mitochondrial translocation of BNIP3, an inducer of receptor-mediated mitophagy. Surprisingly, however, loss of HIF-1α in vitro and analysis of NP-specific HIF-1α null mice do not show a decrease in mitophagic flux in NP cells but a compensatory increase in NIX and PINK1-Parkin pathways with higher mitochondrial number. Taken together, our studies provide novel mechanistic insights into the complex interplay between hypoxia and HIF-1α signaling on the mitochondrial metabolism and quality control in NP cells. © 2020 American Society for Bone and Mineral Research.

Checkpoint molecules coordinately restrain hyperactivated effector T cells in the tumor microenvironment

ABSTRACT The immune checkpoint blockade (ICB) immunotherapy has prolonged overall survival for cancer patients but the response rates are low. The resistance to ICB is likely due to compensatory upregulation of additional immune inhibitory molecules. In this study, we first systematically examined Tim-3 expression in immune cells in mouse tumors and found that Tim-3 was specifically up-regulated in a large number of Treg, conventional CD4+, CD8+ T cells, dendritic cell 1 (DC1), and macrophage 1 (M1) in the tumor microenvironment (TME). Interestingly, Tim-3+ T cells in the TME were phenotypically effector but not “exhausted” T cells because Tim-3+ PD-1+ CD8+ T cells had a higher number of mitochondria, greater levels of glycolysis, and higher tumor-specific cytolytic activities compared to Tim-3− PD-1− CD8+ T cells. The combination treatment with Tim-3 and PD-1 mAbs resulted in a synergistic antitumor activity but also increased the expression of Lag-3 and GITR in TIL, demonstrating cross-regulation between multiple checkpoint molecules. Furthermore, we found that the antitumor efficacy with triple combination of Tim-3, PD-1, and Lag3 mAbs was much greater than any two antibodies. Mechanistically, we demonstrated that simultaneous targeting of Tim-3, PD-1, and Lag-3 cooperatively increased the levels of granzyme B and tumor-specific cytolytic activities of CD8+ TIL. Our data indicate that multiple checkpoint molecules are coordinately upregulated to inhibit the function of hyperactivated T cells in the TME and requirement for the simultaneous blockade of PD-1, Tim-3 and Lag3 for cancer treatment.

Processes exacerbating apoptosis in non-alcoholic steatohepatitis.

Non-alcoholic fatty liver disease (NAFLD) is a significant public health concern, owing to its high prevalence, progressive nature and lack of effective medical therapies. NAFLD is a complex and multifactorial disease involving the progressive and concerted action of factors that contribute to the development of liver inflammation and eventually fibrosis. Here, we summarize fundamental molecular mechanisms underlying the pathogenesis of non-alcoholic steatohepatitis (NASH), how they are interrelated and possible translation to clinical applications. We focus on processes triggering and exacerbating apoptotic signalling in the liver of NAFLD patients and their metabolic and pathological implications. Indeed, liver injury and inflammation are cardinal histopathological features of NASH, a duo in which derailment of apoptosis is of paramount importance. In turn, the liver houses a very high number of mitochondria, crucial metabolic unifiers of both extrinsic and intrinsic signals that converge in apoptosis activation. The role of lifestyle options is also dissected, highlighting the management of modifiable risk factors, such as obesity and harmful alcohol consumption, influencing apoptosis signalling in the liver and ultimately NAFLD progression. Integrating NAFLD-associated pathologic mechanisms in the cell death context could provide clues for a more profound understating of the disease and pave the way for novel rational therapies.

Ultrastructure on the Light Organ of Tropical Synchronize Firefly, Pteroptyx tener

The ultrastructure of the light organ Pteroptyx tener was examined using a Carl Zeiss Axioscope microscope and transmission electron microscope (TEM). The light organ of the male and female P. tener comprises of two layers namely the photogenic layer and the dorsal (reflector) layer. Photogenic layer consists of several elements, i.e. photocyte, differentiated zone, cylinder, trachea and trachea end cell. The photocyte is the main source of the emitted light. This layer is packed with photocyte granules except at a differentiated zone that have low granules but high number of mitochondria. The reflector layer comprised of trachea and the cytoplasm of the cells in this layer is densely packed with spherical uric acid granule. The finding of this study has shown that P. tener have similar general histology structure of light organ of fireflies species that produce sharp flashes. However, the cylinder and trachea end cell may have significant capacities in controlling the luminescent creation in the fireflies that need to be investigate further.

Ultrastructural morphology of goblet cells of the conjunctiva of dogs.

To describe the morphology of goblet cells of the eyelid conjunctiva in dogs using transmission and scanning electron microscopy. Ten dogs, both male and female of different breeds, with no ocular changes were examined (20 eyes). Ten samples of conjunctiva were collected and processed for scanning and transmission electron microscopy (SEM and TEM), while another 10 samples were stained with Schiff's periodic stain (SPA) and alcian blue, pH 2.5, and analyzed using light microscopy. Scanning electron microscopy revealed several points of mucus extrusion in the free apical portion of the goblet cells as well as a wide distribution of lymphoid follicles and macrophages intermingling with the microvilli of palpebral epithelium cells. TEM revealed normal goblet cells that were predominantly oval with wide cytoplasm of different diameters, and large vesicles with heterogeneous granules and free edges, suggesting the release of mucus content onto the conjunctival surface. Cytoplasmic organelles, such as the Golgi apparatus, endoplasmic reticulum, and a high number of mitochondria were also observed. All the samples were positive for SPA and alcian blue staining. This is the first study to evaluate the goblet cells of the eyelid conjunctiva in healthy dogs using electron microscopy techniques. These results are useful for comparing the palpebral conjunctiva of dogs without ocular changes to palpebral conjunctiva of dogs and other species with ocular changes.

Analysis of Titin in Red and White Muscles: Crucial Role on Muscle Contractions Using a Fish Model.

Several studies have compared molecular components between red and white skeletal muscles in mammals. However, mammalian skeletal muscles are composed of mixed types of muscle fibers. In the current study, we analyzed and compared the distributions of titin, lipid, phosphate ions, and fatty acid levels in red and white muscles using a fish model (Tilapia), which is rich in red and white muscles, and these are well separated. Oil-red O staining showed that red muscle had more-abundant lipids than did white muscle. A time-of-flight secondary-ion mass spectrometric (TOF-SIMS) analysis revealed that red muscle possessed high levels of palmitic acid and oleic acid, but white muscle contained more phosphate ions. Moreover, elastica-van Gieson (EVG) and Mito-Tracker green FM staining showed that collagen and elastic fibers were highly, respectively, distributed in connective tissues and mitochondria in red muscle. An electron micrographic analysis indicated that red muscle had a relatively higher number of mitochondria and longer sarcomere lengths and Z-line widths, while myofibril diameters were thicker in white muscle. Myofibrillar proteins separated by SDS-PAGE showed that the major giant protein, titin, was highly expressed in white muscle than in red muscle. Furthermore, ratios of titin to myosin heavy chain (MHC) (titin/MHC) were about 1.3 times higher in white muscle than red muscle. We postulated that white muscle is fit for short and strong contractile performance due to high levels of titin and condensed sarcomeres, whereas red muscle is fit for low intensity and long-lasting activity due to high levels of lipids and mitochondria and long sarcomeres.

Melatonin protects hippocampal HT22 cells from the effects of serum deprivation specifically targeting mitochondria.

Neurons contain a high number of mitochondria, these neuronal cells produce elevated levels of oxidative stress and live for a long time without proliferation; therefore, mitochondrial homeostasis is crucial to their health. Investigations have recently focused on mitochondrial dynamics revealing the ability of these organelles to change their distribution and morphology. It is known that mitochondrial fission is necessary for the transmission of mitochondria to daughter cells during mitosis and mitochondrial fragmentation has been used as an indicator of cell death and mitochondrial dysfunction. Oxidative stress is a trigger able to induce changes in the mitochondrial network. The aim of the present study was to determine the effects of melatonin on the mitochondrial network in HT22 serum-deprived cells. Our results showed that serum deprivation increased reactive oxygen species (ROS) content, promoted the activation of plasma membrane voltage-dependent anion channels (VDACs) and affected the expression of pDRP1 and DRP1 fission proteins. Moreover, parallel increases in apoptotic and autophagic features were found. Damaged and dysfunctional mitochondria are deleterious to the cell; hence, the degradation of such mitochondria through mitophagy is crucial to cell survival. Our results suggest that melatonin supplementation reduces cell death and restores mitochondrial function through the regulation of autophagy.

Trimethylamine-N-Oxide (TMAO)-Induced Impairment of Cardiomyocyte Function and the Protective Role of Urolithin B-Glucuronide.

One of the most recently proposed candidates as a potential trigger for cardiovascular diseases is trimethylamine-N-oxide (TMAO). Possible direct effects of TMAO on myocardial tissue, independent of vascular damage, have been only partially explored so far. In the present study, we assessed the detrimental direct effects of TMAO on cardiomyocyte contractility and intracellular calcium dynamics, and the ability of urolithin B-glucuronide (Uro B-gluc) in counteracting TMAO-induced cell damage. Cell mechanics and calcium transients were measured, and ultrastructural analysis was performed in ventricular cardiomyocytes isolated from the heart of normal adult rats. Cells were either untreated, exposed to TMAO, or to TMAO and Uro B-gluc. TMAO exposure worsened cardiomyocyte mechanics and intracellular calcium handling, as documented by the decrease in the fraction of shortening (FS) and the maximal rate of shortening and re-lengthening, associated with reduced efficiency in the intracellular calcium removal. Ultrastructurally, TMAO-treated cardiomyocytes also exhibited glycogen accumulation, a higher number of mitochondria and lipofuscin-like pigment deposition, suggesting an altered cellular energetic metabolism and a higher rate of protein oxidative damage, respectively. Uro B-gluc led to a complete recovery of cellular contractility and calcium dynamics, and morphologically to a reduced glycogen accumulation. We demonstrated for the first time a direct negative role of TMAO on cardiomyocyte functional properties and the ability of Uro B-gluc in counteracting these detrimental effects.