A current view of mitochondria damage and the diversity of lipopigment inclusions in neuronal ceroid lipofuscinose type 2 from rectal biopsy.

Neuronal ceroid lipofuscinoses (NCLs) are a growing group of neurodegenerative storage diseases, in which specific features are sought to facilitate the creation of a universal diagnostic algorithm in the future. In our ultrastructural studies, the group of NCLs was represented by the CLN2 disease caused by a defect in the TPP1 gene encoding the enzyme tripeptidyl-peptidase 1. A 3.5-year-old girl was affected by this disease. Due to diagnostic difficulties, the spectrum of clinical, enzymatic, and genetic tests was extended to include analysis of the ultrastructure of cells from a rectal biopsy. The aim of our research was to search for pathognomonic features of CLN2 and to analyse the mitochondrial damage accompanying the disease. In the examined cells of the rectal mucosa, as expected, filamentous deposits of the curvilinear profile (CVP) type were found, which dominated quantitatively. Mixed deposits of the CVP/fingerprint profile (FPP) type were observed less frequently in the examined cells. A form of inclusions of unknown origin, not described so far in CLN2 disease, were wads of osmophilic material (WOMs). They occurred alone or co-formed mixed deposits. In addition, atypically damaged mitochondria were observed in muscularis mucosae. Their deformed cristae had contact with inclusions that looked like CVPs. Considering the confirmed role of the c subunit of the mitochondrial ATP synthase in the formation of filamentous lipopigment deposits in the group of NCLs, we suggest the possible significance of other mitochondrial proteins, such as mitochondrial contact site and cristae organizing system (MICOS), in the formation of these deposits. The presence of WOMs in the context of searching for ultrastructural pathognomonic features in CLN2 disease also requires further research.

Exploring the neuroprotective role of artesunate in mouse models of anti-NMDAR encephalitis: insights from molecular mechanisms and transmission electron microscopy.

BACKGROUND: The pathway involving PTEN-induced putative kinase 1 (PINK1) and PARKIN plays a crucial role in mitophagy, a process activated by artesunate (ART). We propose that patients with anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis exhibit insufficient mitophagy, and ART enhances mitophagy via the PINK1/PARKIN pathway, thereby providing neuroprotection. METHODS: Adult female mice aged 8-10 weeks were selected to create a passive transfer model of anti-NMDAR encephalitis. We conducted behavioral tests on these mice within a set timeframe. Techniques such as immunohistochemistry, immunofluorescence, and western blotting were employed to assess markers including PINK1, PARKIN, LC3B, p62, caspase3, and cleaved caspase3. The TUNEL assay was utilized to detect neuronal apoptosis, while transmission electron microscopy (TEM) was used to examine mitochondrial autophagosomes. Primary hippocampal neurons were cultured, treated, and then analyzed through immunofluorescence for mtDNA, mtROS, TMRM. RESULTS: In comparison to the control group, mitophagy levels in the experimental group were not significantly altered, yet there was a notable increase in apoptotic neurons. Furthermore, markers indicative of mitochondrial leakage and damage were found to be elevated in the experimental group compared to the control group, but these markers showed improvement following ART treatment. ART was effective in activating the PINK1/PARKIN pathway, enhancing mitophagy, and diminishing neuronal apoptosis. Behavioral assessments revealed that ART ameliorated symptoms in mice with anti-NMDAR encephalitis in the passive transfer model (PTM). The knockdown of PINK1 led to a reduction in mitophagy levels, and subsequent ART intervention did not alleviate symptoms in the anti-NMDAR encephalitis PTM mice, indicating that ART's therapeutic efficacy is mediated through the activation of the PINK1/PARKIN pathway. CONCLUSIONS: At the onset of anti-NMDAR encephalitis, mitochondrial damage is observed; however, this damage is mitigated by the activation of mitophagy via the PINK1/PARKIN pathway. This regulatory feedback mechanism facilitates the removal of damaged mitochondria, prevents neuronal apoptosis, and consequently safeguards neural tissue. ART activates the PINK1/PARKIN pathway to enhance mitophagy, thereby exerting neuroprotective effects and may achieve therapeutic goals in treating anti-NMDAR encephalitis.

Transcriptional and ultrastructural changes of macrophages after african swine fever virus infection.

African swine fever (ASF) is a highly impactful infectious disease in the swine industry, leading to substantial economic losses globally. The causative agent, African swine fever virus (ASFV), possesses intricate pathogenesis, warranting further exploration. In this study, we investigated the impact of ASFV infection on host gene transcription and organelle changes through macrophage transcriptome sequencing and ultrastructural transmission electron microscopy observation. According to the results of the transcriptome sequencing, ASFV infection led to significant alterations in the gene expression pattern of porcine bone marrow derived macrophages (BMDMs), with 2404 genes showing upregulation and 1579 genes downregulation. Cytokines, and chemokines were significant changes in the expression of BMDMs; there was significant activation of pattern recognition receptors such as Toll-like receptors and Nod-like receptors. According to the observation of the ultrastructure, mitochondrial damage and mitochondrial autophagy were widely present in ASFV-infected cells. The reduced number of macrophage pseudopodia suggested that virus-induced structural changes may compromise pathogen recognition, phagocytosis, and signal communication in macrophages. Additionally, the decreased size and inhibited acidification of secondary lysosomes in macrophages implied suppressed phagocytosis. Overall, ASFV infection resulted in significant changes in the expression of cytokines and chemokines, accompanied by the activation of NLR and TLR signaling pathways. We reported for the first time that ASFV infection led to a reduction in pseudopodia numbers and a decrease in the size and acidification of secondary lysosomes.

Novel Ultrastructural Insights into the Clear-Cell Carcinoma of the Pancreas: A Case Report.

Pancreatic cancer, most frequently as ductal adenocarcinoma (PDAC), is the third leading cause of cancer death. Clear-cell primary adenocarcinoma of the pancreas (CCCP) is a rare, aggressive, still poorly characterized subtype of PDAC. We report here a case of a 65-year-old male presenting with pancreatic neoplasia. A histochemical examination of the tumor showed large cells with clear and abundant intracytoplasmic vacuoles. The clear-cell foamy appearance was not related to the hyperproduction of mucins. Ultrastructural characterization with transmission electron microscopy revealed the massive presence of mitochondria in the clear-cell cytoplasm. The mitochondria showed disordered cristae and various degrees of loss of structural integrity. Immunohistochemistry staining for NADH dehydrogenase [ubiquinone] 1 alpha subcomplex, 4-like 2 (NDUFA4L2) proved specifically negative for the clear-cell tumor. Our ultrastructural and molecular data indicate that the clear-cell nature in CCCP is linked to the accumulation of disrupted mitochondria. We propose that this may impact on the origin and progression of this PDAC subtype.

A Three-Dimensional Technique for The Visualization of Mitochondrial Ultrastructural Changes in Pancreatic Cancer Cells.

Understanding the dynamic features of the cell organelle ultrastructure, which is not only rich in unknown information but also sophisticated from a three-dimensional (3D) perspective, is critical for mechanistic studies. Electron microscopy (EM) offers good imaging depth and allows for the reconstruction of high-resolution image stacks to investigate the ultrastructural morphology of cellular organelles even at the nanometer scale; therefore, 3D reconstruction is gaining importance due to its incomparable advantages. Scanning electron microscopy (SEM) provides a high-throughput image acquisition technology that allows for reconstructing large structures in 3D from the same region of interest in consecutive slices. Therefore, the application of SEM in large-scale 3D reconstruction to restore the true 3D ultrastructure of organelles is becoming increasingly common. In this protocol, we suggest a combination of serial ultrathin section and 3D reconstruction techniques to study mitochondrial cristae in pancreatic cancer cells. The details of how these techniques are performed are described in this protocol in a step-by-step manner, including the osmium-thiocarbohydrazide-osmium (OTO) method, the serial ultrathin section imaging, and the visualization display.

Structural basis of mitochondrial protein import by the TIM23 complex.

Mitochondria import nearly all of their approximately 1,000-2,000 constituent proteins from the cytosol across their double-membrane envelope1-5. Genetic and biochemical studies have shown that the conserved protein translocase, termed the TIM23 complex, mediates import of presequence-containing proteins (preproteins) into the mitochondrial matrix and inner membrane. Among about ten different subunits of the TIM23 complex, the essential multipass membrane protein Tim23, together with the evolutionarily related protein Tim17, has long been postulated to form a protein-conducting channel6-11. However, the mechanism by which these subunits form a translocation path in the membrane and enable the import process remains unclear due to a lack of structural information. Here we determined the cryo-electron microscopy structure of the core TIM23 complex (heterotrimeric Tim17-Tim23-Tim44) from Saccharomyces cerevisiae. Contrary to the prevailing model, Tim23 and Tim17 themselves do not form a water-filled channel, but instead have separate, lipid-exposed concave cavities that face in opposite directions. Our structural and biochemical analyses show that the cavity of Tim17, but not Tim23, forms the protein translocation path, whereas Tim23 probably has a structural role. The results further suggest that, during translocation of substrate polypeptides, the nonessential subunit Mgr2 seals the lateral opening of the Tim17 cavity to facilitate the translocation process. We propose a new model for the TIM23-mediated protein import and sorting mechanism, a central pathway in mitochondrial biogenesis.

Rapid Cryopurification of the Yeast Mitochondrial Ribosome.

Cryogenic milling, or cryomilling, involves the use of liquid nitrogen to lower the temperature of the biological material and/or the milling process. When applied to the study of subcellular or suborganellar structures and processes, it allows for their rapid extraction from whole cells frozen in the physiological state of choice. This approach has proven to be useful for the study of yeast mitochondrial ribosomes. Following cryomilling of 100 mL of yeast culture, conveniently tagged mitochondrial ribosomes can be immunoprecipitated and purified in native conditions. These ribosomes are suitable for the application of downstream approaches. These include mitoribosome profiling to analyze the mitochondrial translatome or mass spectrometry analyses to assess the mitoribosome proteome in normal growth conditions or under stress, as described in this method.

Mitochondria in cancer stem cells: Achilles heel or hard armor.

Previous studies have shown that mitochondria play core roles in not only cancer stem cell (CSC) metabolism but also the regulation of CSC stemness maintenance and differentiation, which are key regulators of cancer progression and therapeutic resistance. Therefore, an in-depth study of the regulatory mechanism of mitochondria in CSCs is expected to provide a new target for cancer therapy. This article mainly introduces the roles played by mitochondria and related mechanisms in CSC stemness maintenance, metabolic transformation, and chemoresistance. The discussion mainly focuses on the following aspects: mitochondrial morphological structure, subcellular localization, mitochondrial DNA, mitochondrial metabolism, and mitophagy. The manuscript also describes the recent clinical research progress on mitochondria-targeted drugs and discusses the basic principles of their targeted strategies. Indeed, an understanding of the application of mitochondria in the regulation of CSCs will promote the development of novel CSC-targeted strategies, thereby significantly improving the long-term survival rate of patients with cancer.

Spatial mapping of mitochondrial networks and bioenergetics in lung cancer.

Mitochondria are critical to the governance of metabolism and bioenergetics in cancer cells1. The mitochondria form highly organized networks, in which their outer and inner membrane structures define their bioenergetic capacity2,3. However, in vivo studies delineating the relationship between the structural organization of mitochondrial networks and their bioenergetic activity have been limited. Here we present an in vivo structural and functional analysis of mitochondrial networks and bioenergetic phenotypes in non-small cell lung cancer (NSCLC) using an integrated platform consisting of positron emission tomography imaging, respirometry and three-dimensional scanning block-face electron microscopy. The diverse bioenergetic phenotypes and metabolic dependencies we identified in NSCLC tumours align with distinct structural organization of mitochondrial networks present. Further, we discovered that mitochondrial networks are organized into distinct compartments within tumour cells. In tumours with high rates of oxidative phosphorylation (OXPHOSHI) and fatty acid oxidation, we identified peri-droplet mitochondrial networks wherein mitochondria contact and surround lipid droplets. By contrast, we discovered that in tumours with low rates of OXPHOS (OXPHOSLO), high glucose flux regulated perinuclear localization of mitochondria, structural remodelling of cristae and mitochondrial respiratory capacity. Our findings suggest that in NSCLC, mitochondrial networks are compartmentalized into distinct subpopulations that govern the bioenergetic capacity of tumours.

Mitochondria to Bitter Melon: Understanding the 3D Ultrastructure of the Cell Via 2D Thin Section Reconstruction and the History of Mitochondrial Visualization.

The origin of histology-the study of microscopic anatomy-is intimately connected with the development of the light microscope and improvements in lens design and manufacture.However, knowledge of the ultrastructure of the cell was hampered by the very nature of light microscopy, which, due to the physical properties of the visible electromagnetic spectrum, could never provide the magnification and resolution for study of the granules seen in cells, which we now know as the organelles. When the electron microscope was developed in the 1930s, a beam of electrons replaced light as the source of illumination, and the inner details of the cell could be observed directly. With thin sections obtained by transmission electron microscopy, cell biologists could embark on the task of reconstructing 3D microstructure via the painstaking stacking of the individual slices.The three-dimensional visualization of the mitochondrion was particularly challenging, as its convoluted structure could be interpreted in several ways based on differences observed by George Palade at the Rockefeller Institute for Medical Research (NYC), and Fritiof Sjöstrand at the Karolinska Institutet (Stockholm). Palade's interpretation was eventually accepted as correct due to its alignment with the findings of biochemists investigating the cascade of molecular interactions known as the Krebs cycle, responsible for the production of cellular energy in the form of adenosine triphosphate (ATP). However, it can also be argued that Palade's visualization via a physical model of the mitochondrion, which he built with sheets of wax, photographed, and published in 1953, better enabled colleagues to comprehend its unique inner structures known as cristae.To teach undergraduate science students about this pivotal moment in cell biology and add to their understanding of the reconstruction process, a pedagogical exercise was created in which students are provided with outline drawings of various organic objects cut in random planes of section. Working individually at first, and then in groups, they are tasked with collaborating to devise an accurate description of the shape and texture of the object. After their observations are presented to the class, they are shown a photo of the object prior to its sectioning to determine if their observations were correct.

Characterisation of cold-induced mitochondrial fission in porcine aortic endothelial cells.

BACKGROUND: Previously, we observed that hypothermia, widely used for organ preservation, elicits mitochondrial fission in different cell types. However, temperature dependence, mechanisms and consequences of this cold-induced mitochondrial fission are unknown. Therefore, we here study cold-induced mitochondrial fission in endothelial cells, a cell type generally displaying a high sensitivity to cold-induced injury. METHODS: Porcine aortic endothelial cells were incubated at 4-25 °C in modified Krebs-Henseleit buffer (plus glucose to provide substrate and deferoxamine to prevent iron-dependent hypothermic injury). RESULTS: Cold-induced mitochondrial fission occurred as early as after 3 h at 4 °C and at temperatures below 21 °C, and was more marked after longer cold incubation periods. It was accompanied by the formation of unusual mitochondrial morphologies such as donuts, blobs, and lassos. Under all conditions, re-fusion was observed after rewarming. Cellular ATP content dropped to 33% after 48 h incubation at 4 °C, recovering after rewarming. Drp1 protein levels showed no significant change during cold incubation, but increased phosphorylation at both phosphorylation sites, activating S616 and inactivating S637. Drp1 receptor protein levels were unchanged. Instead of increased mitochondrial accumulation of Drp1 decreased mitochondrial localization was observed during hypothermia. Moreover, the well-known Drp1 inhibitor Mdivi-1 showed only partial protection against cold-induced mitochondrial fission. The inner membrane fusion-mediating protein Opa1 showed a late shift from the long to the fusion-incompetent short isoform during prolonged cold incubation. Oma1 cleavage was not observed. CONCLUSIONS: Cold-induced mitochondrial fission appears to occur over almost the whole temperature range relevant for organ preservation. Unusual morphologies appear to be related to fission/auto-fusion. Fission appears to be associated with lower mitochondrial function/ATP decline, mechanistically unusual, and after cold incubation in physiological solutions reversible at 37 °C.

Enrichment of the exocytosis protein STX4 in skeletal muscle remediates peripheral insulin resistance and alters mitochondrial dynamics via Drp1.

Mitochondrial dysfunction is implicated in skeletal muscle insulin resistance. Syntaxin 4 (STX4) levels are reduced in human diabetic skeletal muscle, and global transgenic enrichment of STX4 expression improves insulin sensitivity in mice. Here, we show that transgenic skeletal muscle-specific STX4 enrichment (skmSTX4tg) in mice reverses established insulin resistance and improves mitochondrial function in the context of diabetogenic stress. Specifically, skmSTX4tg reversed insulin resistance caused by high-fat diet (HFD) without altering body weight or food consumption. Electron microscopy of wild-type mouse muscle revealed STX4 localisation at or proximal to the mitochondrial membrane. STX4 enrichment prevented HFD-induced mitochondrial fragmentation and dysfunction through a mechanism involving STX4-Drp1 interaction and elevated AMPK-mediated phosphorylation at Drp1 S637, which favors fusion. Our findings challenge the dogma that STX4 acts solely at the plasma membrane, revealing that STX4 localises at/proximal to and regulates the function of mitochondria in muscle. These results establish skeletal muscle STX4 enrichment as a candidate therapeutic strategy to reverse peripheral insulin resistance.

Ferroptosis plays an important role in promoting ionizing radiation-induced intestinal injuries.

The intestinal tract is an essential component of the body's immune system, and is extremely sensitive to exposure of ionizing radiation. While ionizing radiation can effectively induce multiple forms of cell death, whether it can also promote ferroptosis in intestinal cells and the possible interrelationship between ferroptosis and intestinal immune function has not been reported so far. Here, we found that radiation-induced major ultrastructural changes in mitochondria of small intestinal epithelial cells and the changes induced in iron content and MDA levels in the small intestine were consistent with that observed during cellular ferroptosis, thus suggesting occurrence of ferroptosis in radiation-induced intestinal damage. Moreover, radiation caused a substantial increase in the expression of ferroptosis-related factors such as LPCAT3 and ALOX15 mRNA, augmented the levels of immune-related factors INF-γ and TGF-ß mRNA, and decreased the levels of IL-17 mRNA thereby indicating that ionizing radiation induced ferroptosis and impairment of intestinal immune function. Liproxstatin-1 is a ferroptosis inhibitor that was found to ameliorate radiation-induced ferroptosis and promote the recovery from immune imbalances. These findings supported the role of ferroptosis in radiation-induced intestinal immune injury and provide novel strategies for protection against radiation injury through regulation of the ferroptosis pathway.

Alginic acid induces oxidative stress-mediated hormone secretion disorder, apoptosis and autophagy in mouse granulosa cells and ovaries.

Alginic acid (AA) is a kind of polysaccharide extracted from brown seaweeds and has been widely used in food industry. Certain positive effects of AA, such as anti-inflammation and anti-allergy, have been reported. Nevertheless, as a potential chemical contaminant of the environment, its impact on female reproductive system remains to be investigated. The purpose of this study is to explore the impact of AA on ovary and to investigate the further cellular mechanism. Primarily, in vitro cultured mouse ovary granulosa cells (GCs) were treated with AA at a concentration of 10µM for 24 h. The cells and supernatant were collected and subjected to further measures. The results demonstrated that after being treated with 10µM AA for 24 h the levels of estradiol and progesterone in supernatant were down-regulated. And excessive reactive oxygen species (ROS) and declined antioxidant capacity were also determined. Additionally, a large number of apoptotic bodies and autophagic vesicles were found in the experimental cells, and the mitochondria-mediated apoptotic pathway was demonstrated to play a main role in GCs apoptosis. To further investigate the effect of AA on ovary, the female ICR mice were administered with AA (10 mg/ kg bodyweight) intraperitoneally for successive 35 days, and the estrus phase was recorded simultaneously. After exposure, the ovaries and blood samples were collected for further analysis. The results revealed that the estrus period of the mice was shortened and the interestrus period was extended after being treated with AA for 35 days. At the organismal level, the numbers of antral follicles and atresia follicles increased and the levels of pro-apoptosis and autophagy-related proteins were detected upregulated after AA treatment. Taken together, both in vivo and in vitro data suggested that AA has toxicity on female reproduction by disrupting estrogen production and inducing oxidative stress, mitochondria-mediated apoptosis and autophagy. Our results provide new scientific basis and the concern for controlling the increasing use of AA.

The perinuclear region concentrates disordered proteins with predicted phase separation distributed in a 3D network of cytoskeletal filaments and organelles.

Membraneless organelles have emerged during the evolution of eukaryotic cells as intracellular domains in which multiple proteins organize into complex structures to perform specialized functions without the need of a lipid bilayer compartment. Here we describe the perinuclear space of eukaryotic cells as a highly organized network of cytoskeletal filaments that facilitates assembly of biomolecular condensates. Using bioinformatic analyses, we show that the perinuclear proteome is enriched in intrinsic disorder with several proteins predicted to undergo liquid-liquid phase separation. We also analyze immunofluorescence and transmission electron microscopy images showing the association between the nucleus and other organelles, such as mitochondria and lysosomes, or the labeling of specific proteins within the perinuclear region of cells. Altogether our data support the existence of a perinuclear dense sub-micron region formed by a well-organized three-dimensional network of structural and signaling proteins, including several proteins containing intrinsically disordered regions with phase behavior. This network of filamentous cytoskeletal proteins extends a few micrometers from the nucleus, contributes to local crowding, and organizes the movement of molecular complexes within the perinuclear space. Our findings take a key step towards understanding how membraneless regions within eukaryotic cells can serve as hubs for biomolecular condensates assembly, in particular the perinuclear space. Finally, evaluation of the disease context of the perinuclear proteins revealed that alterations in their expression can lead to several pathological conditions, and neurological disorders and cancer are among the most frequent.

ATP synthase inhibitory factor subunit 1 regulates islet ß-cell function via repression of mitochondrial homeostasis.

Mitochondrial homeostasis is crucial for the function of pancreatic ß-cells. ATP synthase inhibitory factor subunit 1 (IF1) is a mitochondrial protein interacting with ATP synthase to inhibit its enzyme activity. IF1 may also play a role in maintaining ATP synthase oligomerization and mitochondrial inner membrane formation. A recent study confirmed IF1 expresses in ß-cells. IF1 knockdown in cultured INS-1E ß-cells enhances glucose-induced insulin release. However, the role of IF1 in islet ß-cells remains little known. The present study investigates islets freshly isolated from mouse lines with global IF1 knockout (IF1-/-) and overexpression (OE). The glucose-stimulated insulin secretion was increased in islets from IF1-/- mice but decreased in islets from IF1 OE mice. Transmitted Electronic Microscopic assessment of isolated islets revealed that the number of matured insulin granules (with dense core) was relatively higher in IF1-/-, but fewer in IF1 OE islets than those of controlled islets. The mitochondrial ultrastructure within ß-cells of IF1 overexpressed islets was comparable with those of wild-type mice, whereas those in IF1-/- ß-cells showed increased mitochondrial mass. Mitochondrial network analysis in cultured INS-1 ß-cells showed a similar pattern with an increased mitochondrial network in IF1 knockdown cells. IF1 overexpressed INS-1 ß-cells showed a compromised rate of mitochondrial oxidative phosphorylation with attenuated cellular ATP content. In contrast, INS-1 cells with IF1 knockdown showed markedly increased cellular respiration with improved ATP production. These results support that IF1 is a negative regulator of insulin production and secretion via inhibiting mitochondrial mass and respiration in ß-cells. Therefore, inhibiting IF1 to improve ß-cell function in patients can be a novel therapeutic strategy to treat diabetes.

Targeting PLD2 in adipocytes augments adaptive thermogenesis by improving mitochondrial quality and quantity in mice.

Phospholipase D (PLD)2 via its enzymatic activity regulates cell proliferation and migration and thus is implicated in cancer. However, the role of PLD2 in obesity and type 2 diabetes has not previously been investigated. Here, we show that during diet-induced thermogenesis and obesity, levels of PLD2 but not PLD1 in adipose tissue are inversely related with uncoupling protein 1, a key thermogenic protein. We demonstrate that the thermogenic program in adipose tissue is significantly augmented in mice with adipocyte-specific Pld2 deletion or treated with a PLD2-specific inhibitor and that these mice are resistant to high fat diet-induced obesity, glucose intolerance, and insulin resistance. Mechanistically, we show that Pld2 deletion in adipose tissue or PLD2 pharmacoinhibition acts via p62 to improve mitochondrial quality and quantity in adipocytes. Thus, PLD2 inhibition is an attractive therapeutic approach for obesity and type 2 diabetes by resolving defects in diet-induced thermogenesis.

Bi-allelic variants in DNHD1 cause flagellar axoneme defects and asthenoteratozoospermia in humans and mice.

Asthenoteratozoospermia, defined as reduced sperm motility and abnormal sperm morphology, is a disorder with considerable genetic heterogeneity. Although previous studies have identified several asthenoteratozoospermia-associated genes, the etiology remains unknown for the majority of affected men. Here, we performed whole-exome sequencing on 497 unrelated men with asthenoteratozoospermia and identified DNHD1 bi-allelic variants from eight families (1.6%). All detected variants were predicted to be deleterious via multiple bioinformatics tools. Hematoxylin and eosin (H&E) staining revealed that individuals with bi-allelic DNHD1 variants presented striking abnormalities of the flagella; transmission electron microscopy (TEM) further showed flagellar axoneme defects, including central pair microtubule (CP) deficiency and mitochondrial sheath (MS) malformations. In sperm from fertile men, DNHD1 was localized to the entire flagella of the normal sperm; however, it was nearly absent in the flagella of men with bi-allelic DNHD1 variants. Moreover, abundance of the CP markers SPAG6 and SPEF2 was significantly reduced in spermatozoa from men harboring bi-allelic DNHD1 variants. In addition, Dnhd1 knockout male mice (Dnhd1‒/‒) exhibited asthenoteratozoospermia and infertility, a finding consistent with the sperm phenotypes present in human subjects with DNHD1 variants. The female partners of four out of seven men who underwent intracytoplasmic sperm injection therapy subsequently became pregnant. In conclusion, our study showed that bi-allelic DNHD1 variants cause asthenoteratozoospermia, a finding that provides crucial insights into the biological underpinnings of this disorder and should assist with counseling of affected individuals.

DNA-Unresponsive Platinum(II) Complex Induces ERS-Mediated Mitophagy in Cancer Cells.

Mitophagy is a selective autophagic process that degrades dysfunctional mitochondria. Monofunctional platinum(II) complexes are candidates for anticancer drugs with the potential to circumvent the drug resistance and side effects of cisplatin and its analogues, but their mechanism of action is elusive. Complex Mono-Pt kills cancer cells through a mitophagic pathway. The mechanism involves the stimulation of endoplasmic reticulum stress (ERS) and activation of the unfolded protein response. Mono-Pt severely impairs the structure and function of mitochondria, including disruption of morphological integrity, dissipation of membrane potential, elevation of reactive oxygen species, inhibition of mtDNA transcription, and reduction of adenosine triphosphate (ATP), which ultimately leads to mitophagy. Mono-Pt does not react with nuclear DNA but exhibits potent antiproliferative activity against cancer cells, thus breaking the DNA-binding paradigm and classical structure-activity rules for platinum drugs. The ERS-mediated mitophagy provides an alternative mechanism for platinum complexes, which broadens the way for developing new platinum anticancer drugs.

Computer Aided Image Processing to Facilitate Determination of Congruence in Manual Classification of Mitochondrial Morphologies in Toxoplasma gondii Tissue Cysts.

Toxoplasma gondii is a parasite that chronically infects about a third of the world's population. During chronic infection, the parasite resides within tissue cysts in the form of poorly understood bradyzoites which can number in the thousands. Our prior work showed that these bradyzoites are metabolically active exhibiting heterogeneous replication potential. The morphological plasticity of the mitochondrion potentially informs about parasite metabolic state. We developed an image processing based program to assist manual classification of mitochondrial morphologies by trained operators to collect data and statistics from the manual classification of shapes. We sought to determine whether certain morphologies were readily classifiable and the congruence among manual classifiers, i.e. the degree to which different operators would place the same objects within the same class. Results from three operators classifying mitochondrial morphologies from 5 tissue cyst images showed that among the four classes, one (Blobs) were the easiest to classify. There was remarkable congruence between 2 of the 3 operators in classifying the objects (96%), while the agreement among all 3 operators was somewhat modest (57%). Such information would be valuable for biologists studying these parasites as well as in development of fully automated methods of morphological classification.