Quantitative Electron Microscopy Research Articles

Scutellarein alleviates chronic obstructive pulmonary disease through inhibition of ferroptosis by chelating iron and interacting with arachidonate 15-lipoxygenase.

Ferroptosis, an iron-dependent cell death characterized by lethal lipid peroxidation, is involved in chronic obstructive pulmonary disease (COPD) pathogenesis. Therefore, ferroptosis inhibition represents an attractive strategy for COPD therapy. Herein, we identified natural flavonoid scutellarein as a potent ferroptosis inhibitor for the first time, and characterized its underlying mechanisms for inhibition of ferroptosis and COPD. In vitro, the anti-ferroptotic activity of scutellarein was investigated through CCK8, real-time quantitative polymerase chain reaction (RT-qPCR), Western blotting, flow cytometry, and transmission electron microscope (TEM). In vivo, COPD was induced by lipopolysaccharide (LPS)/cigarette smoke (CS) and assessed by changes in histopathological, inflammatory, and ferroptotic markers. The mechanisms were investigated by RNA-sequencing (RNA-seq), electrospray ionization mass spectra (ESI-MS), local surface plasmon resonance (LSPR), drug affinity responsive target stability (DARTS), cellular thermal shift assay (CETSA), and molecular dynamics. Our results showed that scutellarein significantly inhibited Ras-selective lethal small molecule (RSL)-3-induced ferroptosis and mitochondria injury in BEAS-2B cells, and ameliorated LPS/CS-induced COPD in mice. Furthermore, scutellarein also repressed RSL-3- or LPS/CS-induced lipid peroxidation, GPX4 down-regulation, and overactivation of Nrf2/HO-1 and JNK/p38 pathways. Mechanistically, scutellarein inhibited RSL-3- or LPS/CS-induced Fe2+ elevation through directly chelating Fe2+ . Moreover, scutellarein bound to the lipid peroxidizing enzyme arachidonate 15-lipoxygenase (ALOX15), which resulted in an unstable state of the catalysis-related Fe2+ chelating cluster. Additionally, ALOX15 overexpression partially abolished scutellarein-mediated anti-ferroptotic activity. Our findings revealed that scutellarein alleviated COPD by inhibiting ferroptosis via directly chelating Fe2+ and interacting with ALOX15, and also highlighted scutellarein as a candidate for the treatment of COPD and other ferroptosis-related diseases.

On the role of Ag additions on the initial solute hardening and competitive precipitation of Al-Cu-Mg alloys

The initial solute clustering is known to significantly affect the subsequent precipitation of various strengthening precipitates that determine the mechanical properties of Al-Cu-Mg-Ag alloys. In this work, the influence of Ag variations on the initial cluster hardening and microstructure evolution of Al-Cu-Mg-Ag alloys is investigated by quantitative transmission electron microscopy (TEM), electron backscatter diffraction (EBSD) and atom probe tomography (APT) analysis. Our statistical data and calculation results indicate that the overwhelming Mg-Ag co-clustering by increasing Ag from 0.41 to 0.97 indeed enables a higher nucleation rate of Ω phase and contributes to a denser Ω precipitation generating a stronger age-hardening response at 180 ℃. Analysis of the contribution of individual precipitates to the yield strength highlights the positive role of increasing Ag on improving the cluster hardening capacity of Mg-Ag co-clusters by increasing its order hardening level. Compared with Cu-Mg co-clusters and GP zones, Mg-Ag co-clusters exhibit a greatest strengthening contribution to yield strength of the material as the bulk Ag content increases. Except for GP zones, Mg-Ag and Cu-Mg co-clusters prefer to exhibit a strong order hardening rather than modulus hardening in initially aged alloys. In addition, the average grain sizes, as well as the proportions of low angle grain boundaries (LAGBs) and high angle grain boundaries (HAGBs), reveal no distinct differences in both alloys, suggesting the independence of grain structure on the bulk Ag content.

Robust Local Thickness Estimation of Sub-Micrometer Specimen by 4D-STEM.

A quantitative four-dimensional scanning transmission electron microscopy (4D-STEM) imaging technique (q4STEM) for local thickness estimation across amorphous specimen such as obtained by focused ion beam (FIB)-milling of lamellae for (cryo-)TEM analysis is presented. This study is based on measuring spatially resolved diffraction patterns to obtain the angular distribution of electron scattering, or the ratio of integrated virtual dark and bright field STEM signals, and their quantitative evaluation using Monte Carlo simulations. The method is independent of signal intensity calibrations and only requires knowledge of the detector geometry, which is invariant for a given instrument. This study demonstrates that the method yields robust thickness estimates for sub-micrometer amorphous specimen using both direct detection and light conversion 2D-STEM detectors in a coincident FIB-SEM and a conventional SEM. Due to its facile implementation and minimal dose reauirements, it is anticipated that this method will find applications for in situ thickness monitoring during lamella fabrication of beam-sensitive materials.

Frustrating Surface Segregation by Nanoconfinement: Boosting Electrochemical Ozone Production over a B13C2-Encapsulated PtNi Alloy Electrocatalyst

A major challenge that limits the applications of nanostructured electrocatalysts is precise surface structure regulation. The critical performance-impeding factors for the important electrochemical ozone production (EOP) lie in the leaching-induced poor stability as well as the competing oxygen evolution and ozone production reactions over the most promising platinum-based electrocatalysts. Although composition diversification by alloying appears to be a prevailing strategy to optimize platinum-based electrocatalysts, a practical restriction turns out to be the inevitable surface segregation and termination of platinum-enriched structures due to their lower surface energies. In this work, we introduce the nanoconfinement of intermetallic platinum–nickel nanostructures encapsulated by boron carbide, which effectively frustrates the surface segregation of alloy nanostructures and well maintains the pristine termination of the alloy. Precise atomic-level structural elucidation and model construction of the encapsulated alloy nanostructures are achieved by quantitative electron microscopy. The composite nanoalloy with a unique surface termination evokes synergetic catalytic effects that promote the charge transfer between the surface and adsorbed oxygen intermediates, which entails outstanding EOP performance with a high Faraday efficiency of 14.8% in neutral media and long-term stability of up to 120 h as a qualified electrocatalyst for the EOP electrolyzer devices. More importantly, the current work paves a new route to overwhelm the thermodynamically limited surface structures of bare nanoalloy catalysts through diverse nanoconfinement strategies.

Long-term effects of canagliflozin treatment on the skeleton of aged UM-HET3 mice.

Sodium glucose cotransporter-2 inhibitors (SGLT2is) promote urinary glucose excretion and decrease plasma glucose levels independent of insulin. Canagliflozin (CANA) is an SGLT2i, which is widely prescribed, to reduce cardiovascular complications, and as a second-line therapy after metformin in the treatment of type 2 diabetes mellitus. Despite the robust metabolic benefits, reductions in bone mineral density (BMD) and cortical fractures were reported for CANA-treated subjects. In collaboration with the National Institute on Aging (NIA)-sponsored Interventions Testing Program (ITP), we tested skeletal integrity of UM-HET3 mice fed control (137 mice) or CANA-containing diet (180ppm, 156 mice) from 7 to 22months of age. Micro-computed tomography (micro-CT) revealed that CANA treatment caused significant thinning of the femur mid-diaphyseal cortex in both male and female mice, did not affect trabecular bone architecture in the distal femur or the lumbar vertebra-5 in male mice, but was associated with thinning of the trabeculae at the distal femur in CANA-treated female mice. In male mice, CANA treatment is associated with significant reductions in cortical bone volumetric BMD by micro-CT, and by quantitative backscattered scanning electron microscopy. Raman microspectroscopy, taken at the femur mid-diaphyseal posterior cortex, showed significant reductions in the mineral/matrix ratio and an increased carbonate/phosphate ratio in CANA-treated male mice. These data were supported by thermogravimetric assay (TGA) showing significantly decreased mineral and increased carbonate content in CANA-treated male mice. Finally, the sintered remains of TGA were subjected to X-ray diffraction and showed significantly higher fraction of whitlockite, a calcium orthophosphate mineral, which has higher resorbability than hydroxyapatite. Overall, long-term CANA treatment compromised bone morphology and mineral composition of bones, which likely contribute to increased fracture risk seen with this drug.

On the origin of carrier localization in AlInAsSb digital alloy

We compared the photoluminescence (PL) properties of AlInAsSb digital alloy samples with different periods grown on GaSb (001) substrates by molecular beam epitaxy. Temperature-dependent S-shape behavior is observed and explained using a thermally activated redistribution model within a Gaussian distribution of localized states. There are two different mechanisms for the origin of the PL intensity quenching for the AlInAsSb digital alloy. The high-temperature activation energy E 1 is positively correlated with the interface thickness, whereas the low-temperature activation energy E 2 is negatively correlated with the interface thickness. A quantitative high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) study shows that the interface quality improves as the interface thickness increases. Our results confirm that E 1 comes from carrier trapping at a state in the InSb interface layer, while E 2 originates from the exciton binding energy due to the roughness of the AlAs interface layer.

Quantitative Scanning Transmission Electron Microscopy for Materials Science: Imaging, Diffraction, Spectroscopy, and Tomography

Scanning transmission electron microscopy (STEM) is one of the most powerful characterization tools in materials science research. Due to instrumentation developments such as highly coherent electron sources, aberration correctors, and direct electron detectors, STEM experiments can examine the structure and properties of materials at length scales of functional devices and materials down to single atoms. STEM encompasses a wide array of flexible operating modes, including imaging, diffraction, spectroscopy, and 3D tomography experiments. This review outlines many common STEM experimental methods with a focus on quantitative data analysis and simulation methods, especially those enabled by open source software. The hope is to introduce both classic and new experimental methods to materials scientists and summarize recent progress in STEM characterization. The review also discusses the strengths and weaknesses of the various STEM methodologies and briefly considers promising future directions for quantitative STEM research.

N-3 PUFA Promotes Ferroptosis in PCOS GCs by Inhibiting YAP1 through Activation of the Hippo Pathway.

Polycystic ovary syndrome (PCOS) is an endocrine disorder characterized by hyperandrogenemia with multiple suspended sinus follicles, thickened cortical tissue, and excessive proliferation of ovarian granulosa cells that severely affects the fertility and quality of life of women. The addition of n-3 PUFA to the diet may slightly reduce body weight and greatly alleviate disturbed blood hormone levels in PCOS mice. We treated KGN as a cell model for n-3 PUFA addition and showed that n-3 PUFA inhibited the proliferation of GCs and promoted ferroptosis in ovarian granulosa cells. We used CCK-8, fluorescence quantitative transmission electron microscopy experiments and ferroptosis marker gene detection and other methods. Furthermore, n-3 PUFA was found to promote YAP1 exocytosis by activating Hippo and weakening the cross-talk between YAP1 and Nrf2 by activating the Hippo signaling pathway. In this study, we found that n-3 PUFA inhibited the over proliferation of granulosa cells in ovarian follicles by activating Hippo, promoting YAP1 exocytosis, weakening the cross-talk between YAP1 and Nrf2, and ultimately activating the ferroptosis sensitivity of ovarian granulosa cells. We demonstrate that n-3 PUFA can alleviate the hormonal and estrous cycle disorder with PCOS by inhibiting the YAP1-Nrf2 crosstalk that suppresses over proliferating ovarian granulosa cells and promotes iron death in GCs. These findings reveal the molecular mechanisms by which n-3 PUFA attenuates PCOS and identify YAP1-Nrf2 as a potential therapeutic target for regulation granulosa cells in PCOS.

Inhibition of Autophagy Maintains ESC Pluripotency and Inhibits Primordial Germ Cell Formation in Chickens.

Autophagy plays an important role in the pluripotency and differentiation of stem cells. Transcriptome data showed that the autophagy genes MAP1LC3A and MAP1LC3B were significantly upregulated in primordial germ cells (PGCs). The Kyoto Encyclopedia of Genes and Genome (KEGG) results showed that the lysosome signaling pathway, which is related to autophagy, was significantly enriched in PGCs. Quantitative RT-PCR, western blotting, and transmission electron microscopy (TEM) results showed that autophagy was expressed in both embryonic stem cells (ESCs) and PGCs but was significantly activated in PGCs. To explore the role of autophagy in the differentiation of chicken ESCs into PGCs, autophagy was activated and inhibited using rapamycin and bafilomycin A1, respectively. Results of qRT-PCR, flow cytometry, and indirect immunofluorescence showed that the efficiency of PGC formation significantly decreased after autophagy inhibition. Our results showed, for the first time, that autophagy plays an indispensable role in the formation of chicken PGCs, which lays the foundation for studying the mechanism of autophagy in chicken PGCs and in bird gene editing and the rescue of endangered birds.

Integrating RNA-seq and scRNA-seq to explore the mechanism of macrophage ferroptosis associated with COPD.

Aims: Our study focused on whether macrophages ferroptosis is associated with the pathogenesis of chronic obstructive pulmonary disease (COPD) or not. Main methods: We first identified macrophage module genes by weighted gene co-expression network analysis (WGCNA) in RNA sequencing (RNA-seq) date from COPD, and then identified macrophage marker genes by comprehensive analysis of single-cell RNA sequencing (scRNA-seq) data from COPD macrophages. There were 126 macrophage marker genes identified, and functional enrichment analyses indicated that ferroptosis pathway genes were significantly enriched. Secondly, we identified eight macrophage ferroptosis related genes and based on these eight genes, we performed co-expression analysis and drug prediction. Thirdly, two biomarkers (SOCS1 and HSPB1) were screened by the least absolute shrinkage and selection operator (LASSO), random forest (RF), and support vector machine-recursive feature elimination (SVM-RFE) and established an artificial neural network (ANN) for diagnosis. Subsequently, the biomarkers were validated in the dataset and validation set. These two biomarkers were then subjected to single gene-gene set enrichment analysis (GSEA) and gene set variation analysis (GSVA) analysis, and the ceRNA network was constructed. Finally, we carried out molecular validation with COPD models in vitro for cell counting kit-8 (CCK8) experiments, Western blot and quantitative real-time PCR (qRT-PCR) analysis and transmission electron microscopy (TEM). Key findings: This study revealed the vital role of macrophage ferroptosis in COPD, and novel biomarkers (SOCS1 and HSPB1) may be involved in the pathogenesis of COPD by regulating macrophage ferroptosis. Significance: Taken together, our results suggest that targeting SOCS1 and HSPB1 could treat COPD by inhibiting macrophage ferroptosis.

Mineralogical and Microstructural Characterization of Cement-Stabilized Soft Soils Based on Quantitative Analyses

The effects of cement dosage (percentage by dry soil weight) and initial moisture content (wo) on cement stabilized soft estuarine clay were investigated by effective integration of multi-quantitative analyses, including quantitative X-ray diffraction (XRD), thermalgravimetric analysis (TGA), Brunauer-Emmett-Teller (BET) test and scanning electron microscopy (SEM) with image processing. These quantitative analyses are highly complementary, which greatly improved the scientificalness of this research. The results revealed that the strength development of cement stabilized soft estuarine clay with varying cement dosages, and initial moisture content was ascribed to the combined influence of both soil fabric improvement and cementation bonding enhancement. The increase of wo resulted in larger clay particle spaces, which can counteract the enhancement of soil structure due to the rising hydration products. The hydration degrees of samples with fixed cement dosage but different wo are quite similar, while clay particle spaces were enlarged with an increase of initial moisture content. Thus, the same quantity of hydration products may bind more soil particles when pores are small at lower wo, thus increasing unconfined compressive strength (UCS). Furthermore, BET and quantitative SEM results illustrate that hydration degree plays a dominant role in the development of intra-aggregate pores (<0.2 μm), while wo affects inter-aggregate pores (0.2 μm–2 μm) more.

Mitochondrial haplotype modulates genome expression and mitochondrial structure/function in cardiomyocytes following volume overload.

Mitochondrial DNA (mtDNA) haplotype regulates mitochondrial structure/function and reactive oxygen species in aortocaval fistula (ACF) in mice. Here, we unravel the mitochondrial haplotype effects on cardiomyocyte mitochondrial ultrastructure and transcriptome response to ACF in vivo. Phenotypic responses and quantitative transmission electron microscopy (TEM) and RNA sequence at 3 days were determined after sham surgery or ACF in vivo in cardiomyocytes from wild-type (WT) C57BL/6J (C57n:C57mt) and C3H/HeN (C3Hn:C3Hmt) and mitochondrial nuclear exchange mice (C57n:C3Hmt or C3Hn:C57mt). Quantitative TEM of cardiomyocyte mitochondria C3HWT hearts have more electron-dense compact mitochondrial cristae compared with C57WT. In response to ACF, mitochondrial area and cristae integrity are normal in C3HWT; however, there is mitochondrial swelling, cristae lysis, and disorganization in both C57WT and MNX hearts. Tissue analysis shows that C3HWT hearts have increased autophagy, antioxidant, and glucose fatty acid oxidation-related genes compared with C57WT. Comparative transcriptomic analysis of cardiomyocytes from ACF was dependent upon mtDNA haplotype. C57mtDNA haplotype was associated with increased inflammatory/protein synthesis pathways and downregulation of bioenergetic pathways, whereas C3HmtDNA showed upregulation of autophagy genes. In conclusion, ACF in vivo shows a protective response of C3Hmt haplotype that is in large part driven by mitochondrial nuclear genome interaction.NEW & NOTEWORTHY The results of this study support the effects of mtDNA haplotype on nuclear gene expression in cardiomyocytes. Currently, there is no acceptable therapy for volume overload due to mitral regurgitation. The findings of this study could suggest that mtDNA haplotype activates different pathways after ACF warrants further investigations on human population of heart disease from different ancestry backgrounds.

Effect of small Sn addition on the initial strengthening and microstructural evolution of an Al-Cu-Mg-Ag alloy

The influence of small Sn addition on the mechanical properties and microstructure of an Al-Cu-Mg-Ag alloy is systematically investigated in this work. The age-hardening level of the material at 180 ℃, as well as the strength properties during exposing at 200 ℃, is found to decrease with small Sn addition. With Sn, the morphology of the tensile fracture surface gradually evolves from a transgranular mode to an intergranular one during the prolonged exposing at 200 ℃. Quantitative transmission electron microscopy (TEM) analysis proposes the negative role of Sn on the dense precipitation of Ω phase by accelerating the θ' formation, thereby leading to a loss of strength properties and the lower thermal stability of Sn-added alloy. This detrimental effect by Sn, as revealed by atom probe tomography (APT), is primarily ascribed to the suppressed Mg-Ag co-clustering, which reduces the nucleation rate of Ω phase. Our calculation also indicates that a reduction of the cluster hardening by Sn is mainly responsible for the observed loss in the strength of underaged alloy. The contributions of modulus hardening and order hardening to the cluster hardening are also quantified for each cluster. A similar recrystallized grain size in Sn-free and Sn-added alloys eliminates the grain refinement effect by Sn.

Comprehensive Associations between Acidosis and the Skeleton in Patients with Kidney Disease.

Renal osteodystrophy (ROD) contributes substantially to morbidity in CKD, including increased fracture risk. Metabolic acidosis (MA) contributes to the development of ROD, but an up-to-date skeletal phenotype in CKD-associated acidosis has not been described. We comprehensively studied associations between acidosis and bone in patients with CKD using advanced methods to image the skeleton and analyze bone-tissue, along with biochemical testing. Cross-sectionally, acidosis was associated with higher markers of bone remodeling and female-specific impairments in cortical and trabecular bone quality. Prospectively, acidosis was associated with cortical expansion and trabecular microarchitectural deterioration. At the bone-tissue level, acidosis was associated with deficits in bone mineral content. Future work investigating acidosis correction on bone quality is warranted. Renal osteodystrophy is a state of impaired bone quality and strength. Metabolic acidosis (MA) is associated with alterations in bone quality including remodeling, microarchitecture, and mineralization. No studies in patients with CKD have provided a comprehensive multimodal skeletal phenotype of MA. We aim to describe the structure and makeup of bone in patients with MA in the setting of CKD using biochemistry, noninvasive imaging, and histomorphometry. The retrospective cross-sectional analyses included 180 patients with CKD. MA was defined as bicarbonate ≤22 mEq/L. We evaluated circulating bone turnover markers and skeletal imaging with dual energy x-ray absorptiometry and high-resolution peripheral computed tomography. A subset of 54 participants had follow-up. We assessed associations between baseline and change in bicarbonate with change in bone outcomes. Histomorphometry, microCT, and quantitative backscatter electron microscopy assessed bone biopsy outcomes in 22 participants. The mean age was 68±10 years, 54% of participants were male, and 55% were White. At baseline, acidotic subjects had higher markers of bone turnover, lower areal bone mineral density at the radius by dual energy x-ray absorptiometry, and lower cortical and trabecular volumetric bone mineral density and impaired trabecular microarchitecture. Over time, acidosis was associated with opposing cortical and trabecular effects: cortical expansion but trabecular deterioration. Bone-tissue analyses showed reduced tissue mineral density with increased heterogeneity of calcium distribution in acidotic participants. MA is associated with multiple impairments in bone quality. Future work should examine whether correction of acidosis improves bone quality and strength in patients with CKD.

Counting AP2 using quantitative correlative light and electron microscopy (quant-CLEM).

Clathrin-mediated-endocytosis (CME) constitutes a ubiquitous and essential mechanism for the internalization of membrane segments or extracellular cargo in eukaryotic cells. Together with the pathway's namesake protein clathrin, dozens of other proteins contribute to the binding of specific cargo, coating the plasma membrane, inducing membrane curvature and eventual budding of vesicles. Facilitated by their characteristic honeycomb structure, clathrin-coated membranes have proven an ideal subject for correlative light and electron microscopy (CLEM) imaging. Combination of this approach with super-resolution light microscopy allowed mapping the precise locations of numerous key proteins during different stages of vesicle formation.

Direct laser writing in YAG single crystal: Evolution from amorphization to nanograting formation and phase transformation

Femtosecond laser writing is a versatile and effective technique for high-precision 3D micromachining of crystals for applications in photonics. Here, we demonstrate the femtosecond laser-assisted control over the structure and phase composition in yttrium aluminum garnet (YAG) single crystal. Increasing the energy of the laser pulse enables achieving extremely high temperature and pressure in the laser beam waist region, which leads to pronounced plastic deformation of the crystal structure and even to its complete amorphization inside forming nanogratings or a phase transition from the garnet phase to the perovskite phase. The main types of laser-induced modifications in YAG single crystal are described using quantitative phase microscopy, quantitative birefringence imaging and transmission high-resolution electron microscopy. For the first time, the mechanism of plastic deformation responsible for the formation of dislocations and amorphous phase is experimentally confirmed. The results obtained contribute to the deeper understanding of fundamentals of the direct laser writing in crystals which are essential for the optimization of selective etching processes and fabrication of photonic crystal waveguides.

Paclitaxel, but Not Cisplatin, Affects Satellite Glial Cells in Dorsal Root Ganglia of Rats with Chemotherapy-Induced Peripheral Neurotoxicity

Chemotherapy-induced peripheral neurotoxicity is one of the most common dose-limiting toxicities of several widely used anticancer drugs such as platinum derivatives (cisplatin) and taxanes (paclitaxel). Several molecular mechanisms related to the onset of neurotoxicity have already been proposed, most of them having the sensory neurons of the dorsal root ganglia (DRG) and the peripheral nerve fibers as principal targets. In this study we explore chemotherapy-induced peripheral neurotoxicity beyond the neuronocentric view, investigating the changes induced by paclitaxel (PTX) and cisplatin (CDDP) on satellite glial cells (SGC) in the DRG and their crosstalk. Rats were chronically treated with PTX (10 mg/Kg, 1qwx4) or CDDP (2 mg/Kg 2qwx4) or respective vehicles. Morpho-functional analyses were performed to verify the features of drug-induced peripheral neurotoxicity. Qualitative and quantitative immunohistochemistry, 3D immunofluorescence, immunoblotting, and transmission electron microscopy analyses were also performed to detect alterations in SGCs and their interconnections. We demonstrated that PTX, but not CDDP, produces a strong activation of SGCs in the DRG, by altering their interconnections and their physical contact with sensory neurons. SGCs may act as principal actors in PTX-induced peripheral neurotoxicity, paving the way for the identification of new druggable targets for the treatment and prevention of chemotherapy-induced peripheral neurotoxicity.

Heat treatment-induced autophagy promotes breast cancer cell invasion and metastasis via TGF-β2-mediated epithelial-mesenchymal transitions.

Insufficient thermal ablation can accelerate malignant behaviors and metastases in some solid tumors, and epithelial-mesenchymal transition (EMT) and autophagy are involved in tumor metastasis. It has been found that TGF-β2 which belongs to the family of transforming growth factors often associated with cancer cell invasiveness and EMT. However, whether the interactions between autophagy and TGF-β2 induce EMT in breast cancer (BC) cells following insufficient microwave ablation (MWA) remains unclear. BC cells were treated with sublethal heat treatment to simulate insufficient MWA, and the effects of heat treatment on the BC cell phenotypes were explored. CCK-8, colony formation, flow cytometry, Transwell, and wound healing assays were performed to evaluate the influence of sublethal heat treatment on the proliferation, apoptosis, invasion, and migration of BC cells. Western blotting, real-time quantitative PCR, immunofluorescence, and transmission electron microscopy were carried out to determine the changes in markers associated with autophagy and EMT following sublethal heat treatment. Results showed that heat treatment promoted the proliferation of surviving BC cells, which was accompanied by autophagy induction. Heat treatment-induced autophagy up-regulated TGF-β2/Smad2 signaling and promoted EMT phenotype, thereby enhancing BC cells' migration and invasion abilities. An increase or decrease of TGF-β2 expression resulted in the potentiation and suppression of autophagy, as well as the enhancement and abatement of EMT. Autophagy inhibitors facilitated apoptosis and repressed proliferation of BC cells in vitro, and thwarted BC cell tumor growth and pulmonary metastasis in vivo. Heat treatment-induced autophagy promoted invasion and metastasis via TGF-β2/Smad2-mediated EMTs. Suppressing autophagy may be a suitable strategy for overcoming the progression and metastasis of residual BC cells following insufficient MWA.

Ultrastructural Axon-Myelin Unit Alterations in Multiple Sclerosis Correlate with Inflammation.

Changes in the normal-appearing white matter (NAWM) in multiple sclerosis (MS) may contribute to disease progression. Here, we systematically quantified ultrastructural and subcellular characteristics of the axon-myelin unit in MS NAWM and determined how this correlates with low-grade inflammation. Human brain tissue obtained with short postmortem delay and fixation at autopsy enables systematic quantification of ultrastructural characteristics. In this study, we performed high-resolution immunohis tochemistry and quantitative transmission electron microscopy to study inflammation and ultrastructural characteristics of the axon-myelin unit in MS NAWM (n=8) and control white matter (WM) in the optic nerve. In the MS NAWM, there were more activated and phagocytic microglia cells (HLA+ P2RY12- and Iba1+ CD68+ ) and more T cells (CD3+ ) compared to control WM, mainly located in the perivascular space. In MS NAWM compared to control WM, there were, as expected, longer paranodes and juxtaparanodes and larger overlap between paranodes and juxtaparanodes. There was less compact myelin wrapping, a lower g-ratio, and a higher frequency of axonal mitochondria. Changes in myelin and axonal mitochondrial frequency correlated positively with the number of active and phagocytic microglia and lymphocytes in the optic nerve. These data suggest that in MS NAWM myelin detachment and uncompact myelin wrapping occurs, potassium channels are unmasked at the nodes of Ranvier, and axonal energy demand is increased, or mitochondrial transport is stagnated, accompanied by increased presence of activated and phagocytic microglia and T cells. These subclinical alterations to the axon-myelin unit in MS NAWM may contribute to disease progression. ANN NEUROL 2023;93:856-870.

The protection of luteolin against diabetic cardiomyopathy in rats is related to reversing JNK-suppressed autophagy.

Increasing evidence has shown that impaired autophagy dramatically causes myocardial hypertrophy and fibrosis in the diabetic heart, ultimately leading to diabetic cardiomyopathy (DCM). Luteolin has been reported to effectively attenuate diabetic cardiovascular injury by inhibiting oxidative stress and alleviate sepsis-induced myocardial injury by enhancing autophagy. However, whether luteolin can reduce DCM through activating autophagy and the underlying mechanism remain unclear. Here, reversing the c-Jun N-terminal kinase (JNK)-suppressed autophagy pathway by which luteolin attenuates DCM was explored. Male Sprague-Dawley rats were injected with streptozotocin to induce diabetes. After 6 weeks of diabetes, rats were treated with luteolin (50, 100 and 200 mg kg-1, i.g.) for 4 weeks. Histological and functional alterations in the diabetic heart were determined using HE staining, Masson staining and echocardiography. The expressions of myocardial miR-221, JNK, and c-Jun and autophagic vesicles in diabetes were evaluated by quantitative PCR, Western blotting and electron microscopy. Luteolin significantly improved cardiac function and attenuated myocardial disorganization and fibrosis in the diabetic rat accompanying the dose-dependent down-regulation of JNK, c-Jun, miR-221 and p62, increase of LC3-II/I and autophagic vesicles, and decrease of mitochondrial swelling in the diabetic heart. These data suggest that the protection of luteolin against DCM, at least, is related to suppressing JNK/c-Jun-regulated miR-221 and the subsequent blockage of autophagy.