Machine learning-based 3D segmentation of mitochondria in polarized epithelial cells.

Mitochondria are dynamic organelles that alter their morphological characteristics in response to functional needs. Therefore, mitochondrial morphology is an important indicator of mitochondrial function and cellular health. Reliable segmentation of mitochondrial networks in microscopy images is a crucial initial step for further quantitative evaluation of their morphology. However, 3D mitochondrial segmentation, especially in cells with complex network morphology, such as in highly polarized cells, remains challenging. To improve the quality of 3D segmentation of mitochondria in super-resolution microscopy images, we took a machine learning approach, using 3D Trainable Weka, an ImageJ plugin. We demonstrated that, compared with other commonly used methods, our approach segmented mitochondrial networks effectively, with improved accuracy in different polarized epithelial cell models, including differentiated human retinal pigment epithelial (RPE) cells. Furthermore, using several tools for quantitative analysis following segmentation, we revealed mitochondrial fragmentation in bafilomycin-treated RPE cells.

Expression of two major isoforms of MYO7A in the retina: Considerations for gene therapy of Usher syndrome type 1B.

Usher syndrome type 1B (USH1B) is a deaf-blindness disorder, caused by mutations in the MYO7A gene, which encodes the heavy chain of an unconventional actin-based motor protein. Here, we examined the two retinal isoforms of MYO7A, IF1 and IF2. We compared 3D models of the two isoforms and noted that the 38-amino acid region that is present in IF1 but absent from IF2 affects the C lobe of the FERM1 domain and the opening of a cleft in this potentially important protein binding domain. Expression of each of the two isoforms of human MYO7A and pig and mouse Myo7a was detected in the RPE and neural retina. Quantification by qPCR showed that the expression of IF2 was typically âˆ¼ 7-fold greater than that of IF1. We discuss the implications of these findings for any USH1B gene therapy strategy. Given the current incomplete knowledge of the functions of each isoform, both isoforms should be considered for targeting both the RPE and the neural retina in gene augmentation therapies.

Measurement of the

The ^{18}O(α,γ)^{22}Ne reaction is critical for AGB star nucleosynthesis due to its connection to the abundances of several key isotopes, such as ^{21}Ne and ^{22}Ne. However, the ambiguous resonance energy and spin-parity of the dominant 470 keV resonance leads to substantial uncertainty in the ^{18}O(α,γ)^{22}Ne reaction rate for the temperature of interest. We have measured the resonance energies and strengths of the low-energy resonances in ^{18}O(α,γ)^{22}Ne at the Jinping Underground Nuclear Astrophysics experimental facility (JUNA) with improved precision. The key 470 keV resonance energy has been measured to be E_{α}=474.0±1.1 keV, with such high precision achieved for the first time. The spin-parity of this resonance state is determined to be 1^{-}, removing discrepancies in the resonance strengths in earlier studies. The results significantly improve the precision of the ^{18}O(α,γ)^{22}Ne reaction rates by up to about 10 times compared with the previous data at typical AGB temperatures of 0.1-0.3 GK. We demonstrate that such improvement leads to precise ^{21}Ne abundance predictions, with an impact on probing the origin of meteoritic stardust SiC grains from AGB stars.

Deep Underground Laboratory Measurement of

The ^{13}C(α,n)^{16}O reaction is the main neutron source for the slow-neutron-capture process in asymptotic giant branch stars and for the intermediate process. Direct measurements at astrophysical energies in above-ground laboratories are hindered by the extremely small cross sections and vast cosmic-ray-induced background. We performed the first consistent direct measurement in the range of E_{c.m.}=0.24 to 1.9 MeV using the accelerators at the China Jinping Underground Laboratory and Sichuan University. Our measurement covers almost the entire intermediate process Gamow window in which the large uncertainty of the previous experiments has been reduced from 60% down to 15%, eliminates the large systematic uncertainty in the extrapolation arising from the inconsistency of existing datasets, and provides a more reliable reaction rate for the studies of the slow-neutron-capture and intermediate processes along with the first direct determination of the alpha strength for the near-threshold state.

Regulation of Partial and Reversible Endothelial-to-Mesenchymal Transition in Angiogenesis.

During development and in several diseases, endothelial cells (EC) can undergo complete endothelial-to-mesenchymal transition (EndoMT or EndMT) to generate endothelial-derived mesenchymal cells. Emerging evidence suggests that ECs can also undergo a partial EndoMT to generate cells with intermediate endothelial- and mesenchymal-character. This partial EndoMT event is transient, reversible, and supports both developmental and pathological angiogenesis. Here, we discuss possible regulatory mechanisms that may control the EndoMT program to dictate whether cells undergo complete or partial mesenchymal transition, and we further consider how these pathways might be targeted therapeutically in cancer.

The ApoA-I mimetic peptide 4F attenuates in vitro replication of SARS-CoV-2, associated apoptosis, oxidative stress and inflammation in epithelial cells.

An oral antiviral against SARS-CoV-2 that also attenuates inflammatory instigators of severe COVID-19 is not available to date. Herein, we show that the apoA-I mimetic peptide 4 F inhibits Spike mediated viral entry and has antiviral activity against SARS-CoV-2 in human lung epithelial Calu3 and Vero-E6 cells. In SARS-CoV-2 infected Calu3 cells, 4 F upregulated inducers of the interferon pathway such as MX-1 and Heme oxygenase 1 (HO-1) and downregulated mitochondrial reactive oxygen species (mito-ROS) and CD147, a host protein that mediates viral entry. 4 F also reduced associated cellular apoptosis and secretion of IL-6 in both SARS-CoV-2 infected Vero-E6 and Calu3 cells. Thus, 4 F attenuates in vitro SARS-CoV-2 replication, associated apoptosis in epithelial cells and secretion of IL-6, a major cytokine related to COVID-19 morbidity. Given established safety of 4 F in humans, clinical studies are warranted to establish 4 F as therapy for COVID-19.

[Study on pharmacokinetics and tissue distribution characteristics of salidroside in mice].

The present study aims to establish a method for determination of salidroside in plasma and tissues of mice, and to study the pharmacokinetics and distribution characteristics of salidroside in mice. After intragastrically administered of salidroside at doses of 12.30, 36.90 and 73.80 mg·kg~(-1) to mice, plasma and tissue samples were collected, and the concentration of salidroside in each sample was measured by UPLC-MS/MS to study plasma drug parameters and tissue distribution of salidroside. Salidroside showed a good linear relationship in the plasma and tissues of mice in the concentration range of 43.40-5 556 ng·mL~(-1). The intraday and interday precision are less than 15%, and the accuracy is between 79.50% and 98.20%. The tissue distribution study found that salidroside had higher plasma concentration, and the plasma concentration reached the maximum at 0.5 h. After 6 h, no salidroside was detected in the plasma, indicating that salidroside has good solubility and absorption fast, clearance is also fast. In tissues, the concentration of liver and kidney tissues is higher, indicating that salidroside liver and kidney are the main metabolic and excretory organs. The established method is easy to operate and has good precision. It is suitable for salidroside in mice. Pharmacokinetics and tissue distribution studies provide a reference for the mechanism of action and drug development of salidroside.

Slug regulates the Dll4-Notch-VEGFR2 axis to control endothelial cell activation and angiogenesis.

Slug (SNAI2), a member of the well-conserved Snail family of transcription factors, has multiple developmental roles, including in epithelial-to-mesenchymal transition (EMT). Here, we show that Slug is critical for the pathological angiogenesis needed to sustain tumor growth, and transiently necessary for normal developmental angiogenesis. We find that Slug upregulation in angiogenic endothelial cells (EC) regulates an EMT-like suite of target genes, and suppresses Dll4-Notch signaling thereby promoting VEGFR2 expression. Both EC-specific Slug re-expression and reduced Notch signaling, either by γ-secretase inhibition or loss of Dll4, rescue retinal angiogenesis in SlugKO mice. Conversely, inhibition of VEGF signaling prevents excessive angiogenic sprouting of Slug overexpressing EC. Finally, endothelial Slug (but not Snail) is activated by the pro-angiogenic factor SDF1α via its canonical receptor CXCR4 and the MAP kinase ERK5. Altogether, our data support a critical role for Slug in determining the angiogenic response during development and disease.

Quantification of cristae architecture reveals time-dependent characteristics of individual mitochondria.

Recent breakthroughs in live-cell imaging have enabled visualization of cristae, making it feasible to investigate the structure-function relationship of cristae in real time. However, quantifying live-cell images of cristae in an unbiased way remains challenging. Here, we present a novel, semi-automated approach to quantify cristae, using the machine-learning Trainable Weka Segmentation tool. Compared with standard techniques, our approach not only avoids the bias associated with manual thresholding but more efficiently segments cristae from Airyscan and structured illumination microscopy images. Using a cardiolipin-deficient cell line, as well as FCCP, we show that our approach is sufficiently sensitive to detect perturbations in cristae density, size, and shape. This approach, moreover, reveals that cristae are not uniformly distributed within the mitochondrion, and sites of mitochondrial fission are localized to areas of decreased cristae density. After a fusion event, individual cristae from the two mitochondria, at the site of fusion, merge into one object with distinct architectural values. Overall, our study shows that machine learning represents a compelling new strategy for quantifying cristae in living cells.

Constraining the External Capture to the

The ^{12}C(α,γ)^{16}O reaction is one of the most crucial reactions in nuclear astrophysics. The E2 external capture to the ^{16}O ground state (GS) has not been emphasized in previous analyses but may make a significant contribution to the ^{12}C(α,γ)^{16}O cross section depending on the value of the GS asymptotic normalization coefficient (ANC). In the present work, we determine this ANC to be 337±45 fm^{-1/2} through the ^{12}C(^{11}B,^{7}Li)^{16}O reaction using a high-precision magnetic spectrograph. This sheds light on the existing large discrepancy of more than 2 orders of magnitude between the previously reported ANC values. Based on the new ANC, we experimentally constrain the GS external capture and show that through interference with the high energy tail of the 2^{+} subthreshold state, a substantial enhancement in the GS S_{E2}(300) factor can be obtained (70±7 keV b) compared to that of a recent review (45 keV b), resulting in an increase of the total S factor from 140 to 162 keV b, which is now in good agreement with the value obtained by reproducing supernova nucleosynthesis calculations with the solar-system abundances. This work emphasizes that the external capture contribution for the ground state transition cannot be neglected in future analyses of the ^{12}C(α,γ)^{16}O reaction.

Consensus guidelines for the use and interpretation of angiogenesis assays.

The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference.

Expression of Ski and its role in astrocyte proliferation and migration.

Ski, as an evolutionarily conserved protein, is a versatile transcriptional regulator which widely distributes in various tissues and species. Recently, we have demonstrated for the first time that Ski was strikingly up-regulated in reactive astrocytes after spinal cord injury (SCI) in vivo, which indicates that maybe Ski is a new molecule that controls astrocytes' biological properties after SCI. However, the accurate distributions and functions of Ski in astrocytes after central nervous system (CNS) injury are still unclear. Astrocytes were collected from rats' cerebral cortex. To elucidate the expression and role of Ski in reactive astrocytes, we performed an activated astrocytes model induced by LPS and scratch injury in vitro. Our results showed that Ski gradually increased and reached a peak at 4days, then declined at 6days after induction by LPS. Up-regulation of Ski was accompanied with the increase in proliferation-related proteins including PCNA, CDK4 and CyclinD1. Furthermore, immunofluorescent staining analysis also demonstrated a highly positive relationship between Ski and GFAP, PCNA in astrocytes. These results indicated that Ski might play an important role in astrocyte proliferation. To further explore the role of Ski, astrocytes were transfected with Ski-specific small interfering RNA (siRNA). We found that the primary activated astrocytes' proliferation decreased significantly after transfection with Ski-specific siRNA. Surprisingly, Ski knockdown also weakened the primary astrocyte migration. Based on the above, we could conclude that Ski might play a crucial role in astrocyte proliferation and migration. This discovery might contribute to a promising therapeutic intervention in CNS injury.

3D surface analysis of hippocampal microvasculature in the irradiated brain.

Cranial irradiation used to control CNS malignancies can also disrupt the vasculature and impair neurotransmission and cognition. Here we describe two distinct methodologies for quantifying early and late radiation injury in CNS microvasculature. Intravascular fluorescently labeled lectin was used to visualize microvessels in the brain of the irradiated mouse 2 days post exposure and RECA-1 immunostaining was similarly used to visualize microvessels in the brain of the irradiated rat 1-month post exposure. Confocal microscopy, image deconvolution and 3-dimensional rendering methods were used to define vascular structure in a ∼4 × 10(7) µm(3) defined region of the brain. Quantitative analysis of these 3D images revealed that irradiation caused significant short- and long-term reductions in capillary density, diameter and volume. In mice, irradiation reduced mean vessel volume from 2,250 to 1,470 µm(3) and mean vessel diameter from 5.0 to 4.5 µm, resulting in significant reductions of 34% and 10%, in the hippocampus respectively. The number of vessel branch points and area was also found to also drop significantly in mice 2 days after irradiation. For rats, immunostaining revealed a significant, three-fold drop in capillary density 1 month after exposure compared to controls. Such radiation-induced disruption of the CNS microvasculature may be contributory if not causal to any number of neurocognitive side effects that manifest in cancer patients following cranial radiotherapy. This study demonstrates the utility of two distinct methodologies for quantifying these important adverse effects of radiotherapy. Environ. Mol. Mutagen. 57:341-349, 2016. © 2016 Wiley Periodicals, Inc.

Identification of the porcine sialoadhesin gene promoter region and its cell-specific expression in porcine alveolar macrophage cells.

Porcine reproductive and respiratory syndrome (PRRS), which is caused by the PRRS virus (PRRSV), is a communicable disease. PRRS caused huge economic losses to swine breeding. The porcine alveolar macrophage (PAM) cell is the main target cell of PRRSV; therefore, it is very important to identify the specific gene promoter that controls expression in PAM cells so that the anti-PRRSV exogenous gene can be efficiently and specifically expressed in PAM cells to improve porcine resistance to PRRSV. In this study, the transcription initiation site for sialoadhesin (Siglec-1), which is a porcine alveolar macrophage-specific gene, was determined by 5' rapid amplification of cDNA end, and 88 bp of the 5'-untranslated region was cloned. Siglec-1 promoter activity was detected by a dual-luciferase reporter assay, which showed that the fragment from -173 to +81 bp had the strongest promoter activity. Additionally, the cell-specific expression of the promoter fragments was tested in a PAM cell line (CRL-2844 cells), porcine kidney 15 cell line (PK-15 cells), porcine fetal fibroblast (PEF) cells, and porcine preadipocytes. These results also showed that the fragment from -173 to +81 bp had the strongest cell-specific expression in PAM cells.

PPARα agonist prevented the apoptosis induced by glucose and fatty acid in neonatal cardiomyocytes.

OBJECTIVE: We investigated the effect of peroxisome proliferator activator receptors α (PPARα) on cardiomyocyte apoptosis induced by glucose and fatty acid, and if high glucose levels could increase fatty acid-induced apoptosis. METHODS: Cardiomyocytes were maintained in Dulbecco's Modified Eagle Medium and divided into 5 groups: Group N (control Group); Group G (exposed to 25.5 mmol/l glucose); Group L (exposed to 5 mmol/l glucose, fatty acid); Group H (exposed to 25.5 mmol/l glucose and fatty acid); Group I (exposed to 25.5 mmol/l glucose, fatty acid and Wy14643). Apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling. Immunocytochemistry staining detected PPARα's expressing, and western blotting detected PPARα and nuclear factor κB's (NF-κB) protein level. RESULTS: Exposure to fatty acid resulted in a significant increase of cardiomyocytes apoptosis, with the extension of NF-κB formation, whereas exposure to 25.5 mmol/l glucose had no influence on the apoptosis rate. However, combination with fatty acid and high glucose concentration had induced more apoptosis with the up-regulation of NF-κB formation. The fatty acid and glucose-induced effects were improved by Wy14643, with down-regulation of NF-κB formation. CONCLUSION: These results suggested that in neonatal cardiomyocytes, fatty acid and glucose in combination with fatty acid induced apoptosis via NF-κB formation and activation of apoptosis pathways; glucose in combination with fatty acid induce more apoptosis rate for the more NF- κB formation, activation of the PPARα can reverse such apoptosis effect. The results also suggest that gluco-lipotoxicity may play a central role in the development of diabetic cardiomyopathy, and PPARα-agonist may be an effective drug in treating the diabetic cardiomyopathy.

Service learning, social justice, and campus health.

Healthy campuses are critical so that students can learn and actively participate in shaping and maintaining a strong educational environment. This Viewpoint describes the commonalities between service learning, social justice, campus health, and the goals of Healthy Campus 2010, which was developed from the larger Healthy People 2010 objectives proposed by the US Department of Health and Human Services. The values, methods, and intended results of service learning are closely related to effective health promotion and disease prevention. Service learning focuses on personal and civic responsibility, thus providing students with opportunities for enhancing individual and community health. Service learning also espouses social justice and provides a vehicle for students to learn about, reflect on, and address health disparities. The author cites research concerning the effect of service learning on students in institutions of higher education and their social justice-related behaviors.

A C-terminal aldehyde insect kinin analog enhances inhibition of weight gain and induces significant mortality in Helicoverpa zea larvae.

The first reported examples of C-terminal aldehyde analogs of an insect neuropeptide are described. They are hexapeptide insect kinin analogs Boc-VFFPWG-H and Fmoc-RFFPWG-H. Activity observed for these modified analogs in an in vitro insect diuretic assay confirms that the C-terminal aldehyde group is tolerated by an insect kinin receptor. The two analogs demonstrate greatly enhanced activity over standard C-terminal amide insect kinins in a larval weight gain inhibition assay in the corn earworm Helicoverpa zea. Treatment with Boc-VFFPWG-H led to significant increases in larval mortality at doses of 500pm (45%) and 5nm (67%). Boc-VFFPWG-H represents a lead analog in the development of novel, environmentally friendly pest insect management agents based on the insect kinin class of neuropeptides.

Reappraisal of the fine structure of Alzheimer's neurofibrillary tangles.

Alzheimer's neurofibrillary tangles were studied by electron microscopy. The study includes four cases of Alzheimer's disease, two cases of atypical senile dementia, and one case of progressive supranuclear palsy. In Alzheimer's disease the tangles were composed of either straight filaments or paired helical filaments. In progressive supranuclear palsy the tangles were composed of 15 nm straight filaments or helical filaments. A few straight filaments were mixed with paired helical filaments. In atypical senile dementia, both straight and paired helical filaments comprised the tangles and one type of filaments appeared to intermingle with the other in the same neurons.