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Obstructive sleep apnea (OSA)-induced endothelial cell (EC) dysfunction contributes to OSA-related cardiovascular sequelae. The mechanistic basis of endothelial impairment by OSA is unclear.The goals of this study were to identify the mechanism of OSA-induced EC dysfunction and explore the potential therapies for OSA-accelerated cardiovascular disease.The experimental methods include data mining, bioinformatics, EC functional analyses, OSA mouse models, and assessment of OSA human subjects.Using mined miRNA-seq data, we found that microRNA 210 (miR-210) conferred the greatest induction by intermittent hypoxia in ECs. Consistently, the serum level of miR-210 was higher in OSA individuals from two independent cohorts. Importantly, miR-210 level was positively correlated with the apnea-hypopnea index. RNA-seq data collected from ECs transfected with miR-210 or treated with OSA serum showed a set of genes commonly altered by miR-210 and OSA serum; which are largely involved in mitochondrion-related pathways. ECs transfected with miR-210 or treated with OSA serum showed reduced oxygen consumption rate, mitochondrial membrane potential and DNA abundance. Mechanistically, intermittent hypoxia induced sterol regulatory element-binding protein 2 (SREBP2) bound to the promoter region of miR-210, which in turn inhibited the iron-sulfur cluster assembly enzyme and led to mitochondrial dysfunction. Moreover, the SREBP2 inhibitor betulin alleviated intermittent hypoxia-increased systolic blood pressure in the OSA mouse model.

Maternally inherited mitochondrial DNA (mtDNA) encodes essential subunits of the mitochondrial oxidative phosphorylation system, but is also a major damage-associated molecular pattern (DAMP) that engages innate immune sensors when released into the cytoplasm, outside of cells or into circulation1. This function of mtDNA contributes to antiviral resistance, but unfortunately also causes pathogenic inflammation in many disease contexts2. Cells experiencing mtDNA stress due to depletion of the mtDNA-packaging protein, Transcription Factor A, Mitochondrial (TFAM), or HSV-1 infection exhibit elongated mitochondria, mtDNA depletion, enlargement of nucleoids (mtDNA-protein complexes), and activation of cGAS/STING innate immune signaling via mtDNA released into the cytoplasm3. However, the relationships between altered mitochondrial dynamics and mtDNA-mediated activation of the cGAS-STING pathway remain unclear. Here, we show that entire enlarged nucleoids are released from mitochondria that remain bound to TFAM and colocalize with cGAS. These nucleoids arise at sites of mtDNA replication due to a block in mitochondrial fission at a stage when endoplasmic reticulum (ER) actin polymerization would normally commence, which we propose is a fission checkpoint to ensure that mtDNA has completed replication and is competent for segregation into daughter mitochondria. Released nucleoids also colocalize with the early endosomal marker RAB5 as well as the late endosomal marker RAB7 in TFAM-deficient cells and in response to mtDNA stress caused by the HSV-1 UL12.5 protein. Loss of RAB7 increases interferon stimulated gene (ISG) expression. Thus, we propose that defects in mtDNA replication and/or segregation enact a late mitochondrial fission checkpoint that, if persistent, leads to selective removal of dysfunctional nucleoids by a mitochondrial-endosomal pathway. Early steps in this pathway are prone to mtDNA release and cGAS-STING activation, but the immunostimulatory mtDNA is ultimately disposed of through a mechanism involving RAB7-containing late endosomes to prevent excessive innate immune signaling. This mtDNA quality control pathway might represent a therapeutic target to prevent mtDNA-mediated inflammation and associated pathology.

The actin cytoskeleton plays multiple critical roles in cells, from cell migration to organelle dynamics. The small and transient actin structures regulating organelle dynamics are difficult to detect with fluorescence microscopy, and the limited resolution of fluorescence microscopy makes it difficult to determine whether actin filaments are directly associated with specific membranes. To address these limitations, we developed an approach using fluorescent protein-tagged actin nanobodies targeted to organelle membranes to enable live cell imaging of sub-organellar actin dynamics with unprecedented spatiotemporal resolution.

Mitochondria are essential and dynamic organelles undergoing constant fission and fusion. The primary players in mitochondrial morphology (MFN1/2, OPA1, DRP1) have been identified, but their mechanism(s) of regulation are still being elucidated. ARL2 is a regulatory GTPase that has previously been shown to play a role in the regulation of mitochondrial morphology. Here we demonstrate that ELMOD2, an ARL2 GTPase-activating protein (GAP), is necessary for ARL2 to promote mitochondrial elongation. We show that loss of ELMOD2 causes mitochondrial fragmentation and a lower rate of mitochondrial fusion, while ELMOD2 overexpression promotes mitochondrial tubulation and increases the rate of fusion in a mitofusin-dependent manner. We also show that a mutant of ELMOD2 lacking GAP activity is capable of promoting fusion, suggesting that ELMOD2 does not need GAP activity to influence mitochondrial morphology. Finally, we show that ELMOD2, ARL2, Mitofusins 1 and 2, Miros 1 and 2, and mitochondrial phospholipase D (mitoPLD) all localize to discrete, regularly spaced puncta along mitochondria. These results suggest that ELMOD2 is functioning as an effector downstream of ARL2 and upstream of the mitofusins to promote mitochondrial fusion. Our data provide insights into the pathway by which mitochondrial fusion is regulated in the cell.

Funding Acknowledgements Type of funding sources: None. Introduction. Cardiovascular assessment of Marfan syndrome (MS) patients has normally focused on the aortic root and vascular manifestations due to the high risk of aortic dissection. Although primary myocardial impairment has long been suspected, the evidence has been controversial. Advanced in CMR may support the early detection of cardiac dysfunction. Beyond left ventricle ejection fraction (EF) and myocardial strain (S), a new parameter is emerging, the hemodynamic forces (HF) exchanged between the blood flowing in the heart and the myocardium. The application of these techniques to MS could be useful in demonstrating the presence of primary myocardial impairment. Aim. The aim of this study is to explore myocardial function in MS through the evaluation in cine CMR of EF, S and cardiac HF exchanged between the blood and the myocardium and compare these data with those of a control group (C). Methods. We retrospectively analysed CMR cine images of MS (diagnosed according revised Ghent criteria) without valvular disease or previous cardio surgery, and C, in standard long-axis projections, to define endocardial borders for subsequent quantification of left ventricular volumes, EF, longitudinal, circumferential and radial S, apex-to-base and lateral-to-septum HF (expressed in mN and as a percentage of gravity acceleration). The analysis were performed on Medical Imaging Systems (QStrain version 1.3.0.79; MEDIS) (Figure 1). Results. 108 MS and 44 C had a good quality study, suitable for MEDIS analysis. The mean age was 33 ± 13 ys in MS, 35 ± 12 ys in C; 39% were male in MS, 50% in C. The results of left ventricular function were: EF 63 ± 7% in MS vs 66 ± 5% in C group, p .008, global longitudinal S -24.5 ± 4.1% in MS vs -26.2 ± 4.1 in C, p .014; global circumferential S -30.6 ± 6.3% in MS vs -33.8 ± 4.4 in C, p .002; radial S 64.5 ± 16.2% in MS vs 72.7 ± 15.9 in C, p .005; apex-to-base HF 13.2 ± 4.7% in MS vs 17.8 ± 7.6% in C, p .000; lateral-to-septum 2.6 ± 1.3% in MS vs 3.1 ± 1.4% in C, p .048. Moreover, 4.6% MS patients had mid reduced EF (40-50%); 9.2% had global longitudinal S reduction (cut off -19.3%); 7.4% had global circumferential S reduction (cut-off -21.7%). Conclusion. These data provide support for the existence of a cardiomyopathy in MS. In our opinion, the term "primary cardiomyopathy" is not appropriate to describe this condition: patients with MS have changes in aortic stiffness and probably in cardiac afterload. The HF data are the most interesting of this study, both in the validation of this new parameter and in early detection a cardiomyopathy in MS Moreover, the reduction of global circumferential S, as wall as global longitudinal S, in MS patients may help provide new elements to characterize the MS cardiomyopathy: sure enough, in literature, circumferential strain abnormalities are related to afterload increase. HF analysis is really a new challenge of cardiac imaging, as sensitive markers of subtle systolic dysfunction. Abstract Figure. Figue 1. Analisis exemple.

Coronavirus disease 2019 (COVID-19) includes the cardiovascular complications in addition to respiratory disease. SARS-CoV-2 infection impairs endothelial function and induces vascular inflammation, leading to endotheliitis. SARS-CoV-2 infection relies on the binding of Spike glycoprotein (S protein) to angiotensin converting enzyme 2 (ACE2) in the host cells. We show here that S protein alone can damage vascular endothelial cells (ECs) in vitro and in vivo, manifested by impaired mitochondrial function, decreased ACE2 expression and eNOS activity, and increased glycolysis. The underlying mechanism involves S protein downregulation of AMPK and upregulation of MDM2, causing ACE2 destabilization. Thus, the S protein-exerted vascular endothelial damage via ACE2 downregulation overrides the decreased virus infectivity.

Charcot-Marie-Tooth (CMT) disease is the most commonly inherited neurological disorder, defined by progressive deterioration of the peripheral nerves. Clinical manifestations of CMT mutations are typically limited to peripheral neurons, the longest cells in the body. Currently, mutations in at least 80 different genes are associated with CMT and new mutations are regularly being discovered. A large portion of the proteins mutated in axonal CMT have documented roles in mitochondrial mobility. This suggests that trafficking defects may be a common underlying mechanism in CMT. This review will focus on the potential role of altered mitochondrial mobility in the pathogenesis of axonal CMT, highlighting the challenges and opportunities to this “impaired mobility” model of the disease.

Point-scanning imaging systems are among the most widely used tools for high-resolution cellular and tissue imaging, benefitting from arbitrarily defined pixel sizes. The resolution, speed, sample preservation, and signal-to-noise ratio (SNR) of point-scanning systems are difficult to optimize simultaneously. We show these limitations can be mitigated via the use of Deep Learning-based supersampling of undersampled images acquired on a point-scanning system, which we term point-scanning super-resolution (PSSR) imaging. We designed a “crappifier” that computationally degrades high SNR, high pixel resolution ground truth images to simulate low SNR, low-resolution counterparts for training PSSR models that can restore real-world undersampled images. For high spatiotemporal resolution fluorescence timelapse data, we developed a “multi-frame” PSSR approach that utilizes information in adjacent frames to improve model predictions. In conclusion, PSSR facilitates point-scanning image acquisition with otherwise unattainable resolution, speed, and sensitivity. All the training data, models, and code for PSSR are publicly available at 3DEM.org. Editor’s summary Point-scanning super-resolution imaging uses deep learning to supersample undersampled images and enable time-lapse imaging of subcellular events. An accompanying “crappifier” rapidly generates quality training data for robust performance.

Krüppel-like factor 5 (KLF5) is a basic transcription factor that regulates diverse cellular processes during tumor development. Acetylation of KLF5 at lysine 369 (K369) reverses its function from promoting to suppressing cell proliferation and tumor growth. In this study, we examined the regulation of KLF5 by histone deacetylases in the prostate cancer cell line DU 145. While confirming the functions of HDAC1/2 in KLF5 deacetylation and the promotion of cell proliferation, we found that the knockdown of HDAC1/2 upregulated KLF5 protein but not KLF5 mRNA, and the increase in KLF5 protein level by silencing HDAC1/2 was at least in part due to decreased proteasomal degradation. Deacetylase activity was required for HDAC1/2-mediated KLF5 degradation, and mutation of KLF5 to an acetylation-mimicking form prevented its degradation, even though the mutation did not affect the binding of KLF5 with HDAC1/2. Mutation of K369 to arginine, which prevents acetylation, did not affect the binding of KLF5 to HDAC1 or the response of KLF5 to HDAC1/2-promoted degradation. These findings provide a novel mechanistic association between the acetylation status of KLF5 and its protein stability. They also suggest that maintaining KLF5 in a deacetylated form may be an important mechanism by which KLF5 and HDACs promote cell proliferation and tumor growth.

Point scanning imaging systems (e.g. scanning electron or laser scanning confocal microscopes) are perhaps the most widely used tools for high resolution cellular and tissue imaging. Like all other imaging modalities, the resolution, speed, sample preservation, and signal-to-noise ratio (SNR) of point scanning systems are difficult to optimize simultaneously. In particular, point scanning systems are uniquely constrained by an inverse relationship between imaging speed and pixel resolution. Here we show these limitations can be mitigated via the use of deep learning-based super-sampling of undersampled images acquired on a point-scanning system, which we termed point-scanning super-resolution (PSSR) imaging. Oversampled, high SNR ground truth images acquired on scanning electron or Airyscan laser scanning confocal microscopes were "crappified" to generate semi-synthetic training data for PSSR models that were then used to restore real-world undersampled images. Remarkably, our EM PSSR model could restore undersampled images acquired with different optics, detectors, samples, or sample preparation methods in other labs. PSSR enabled previously unattainable 2 nm resolution images with our serial block face scanning electron microscope system. For fluorescence, we show that undersampled confocal images combined with a multiframe PSSR model trained on Airyscan timelapses facilitates Airyscan-equivalent spatial resolution and SNR with ~100x lower laser dose and 16x higher frame rates than corresponding high-resolution acquisitions. In conclusion, PSSR facilitates point-scanning image acquisition with otherwise unattainable resolution, speed, and sensitivity.

In cancer cells metabolic changes and mitochondrial morphology are coupled. It is known that the cytoskeleton and molecular motors are directly involved in regulating mitochondrial morphology. Here we show that myosin-Va, an actin-based molecular motor, is required for the malignant properties of melanoma cells and localizes to mitochondria in these cells. Knockdown of myosin-Va increases cellular respiration rates and ROS production and decreases glucose uptake and lactate secretion. In addition, knockdown of myosin-Va results in reduced mitochondrial fission and correspondingly elongated mitochondria. We show that myosin-Va interacts with the mitochondrial outer membrane protein Spire1C, an actin-regulatory protein implicated in mitochondrial fission, and that Spire1C recruits myosin-Va to mitochondria. Finally, we show that during mitochondrial fission myosin-Va localization to mitochondria increases, and that myosin-Va localizes to mitochondrial fission sites immediately adjacent to Drp1 punctae. We conclude that myosin-Va facilitates mitochondrial fission. These data implicate myosin-Va as a target for the Warburg effect in melanoma cells.

The actin cytoskeleton plays multiple critical roles in cells, from cell migration to organelle dynamics. The small and transient actin structures regulating organelle dynamics are difficult to detect with fluorescence microscopy. We developed an approach using fluorescent protein-tagged actin nanobodies targeted to organelle membranes to enable live cell imaging of sub-organellar actin dynamics with unprecedented spatiotemporal resolution. These probes reveal that ER-associated actin drives fission of multiple organelles including mitochondria, endosomes, lysosomes, peroxisomes, and the Golgi.

Purpose Cone rod dystrophy (CRD) is a group of inherited retinopathies characterized by the loss of cone and rod photoreceptor cells, which results in poor vision. This study aims to clinically and genetically characterize the segregating CRD phenotype in two large, consanguineous Pakistani families. Methods Funduscopy, optical coherence tomography (OCT), electroretinography (ERG), color vision, and visual acuity assessments were performed to evaluate the retinal structure and function of the affected individuals. Exome sequencing was performed to identify the genetic cause of CRD. Furthermore, the mutation's effect was evaluated using purified, bacterially expressed ADP-ribosylation factor-like protein 3 (ARL3) and mammalian cells. Results Fundus photography and OCT imaging demonstrated features that were consistent with CRD, including bull's eye macular lesions, macular atrophy, and central photoreceptor thinning. ERG analysis demonstrated moderate to severe reduction primarily of photopic responses in all affected individuals, and scotopic responses show reduction in two affected individuals. The exome sequencing revealed a novel homozygous variant (c.296G>T) in ARL3, which is predicted to substitute an evolutionarily conserved arginine with isoleucine within the encoded protein GTP-binding domain (R99I). The functional studies on the bacterial and heterologous mammalian cells revealed that the arginine at position 99 is essential for the stability of ARL3. Conclusions Our study uncovers an additional CRD gene and assigns the CRD phenotype to a variant of ARL3. The results imply that cargo transportation in photoreceptors as mediated by the ARL3 pathway is essential for cone and rod cell survival and vision in humans.

Rods and rings (RRs) are large linear- or circular-shaped structures typically described as polymers of IMPDH (inosine monophosphate dehydrogenase). They have been observed across a wide variety of cell types and species and can be induced to form by inhibitors of IMPDH. RRs are thought to play a role in the regulation of de novo guanine nucleotide synthesis; however, the function and regulation of RRs is poorly understood. Here we show that the regulatory GTPase, ARL2, a subset of its binding partners, and several resident proteins at the endoplasmic reticulum (ER) also localize to RRs. We also have identified two new inducers of RR formation: AICAR and glucose deprivation. We demonstrate that RRs can be disassembled if guanine nucleotides can be generated by salvage synthesis regardless of the inducer. Finally, we show that there is an ordered addition of components as RRs mature, with IMPDH first forming aggregates, followed by ARL2, and only later calnexin, a marker of the ER. These findings suggest that RRs are considerably more complex than previously thought and that the function(s) of RRs may include involvement of a regulatory GTPase, its effectors, and potentially contacts with intracellular membranes.

Mitochondria are essential, dynamic organelles that regularly undergo both fusion and fission in response to cellular conditions, though mechanisms of the regulation of their dynamics are incompletely understood. We provide evidence that increased activity of the small GTPase ARL2 is strongly correlated with an increase in fusion, while loss of ARL2 activity results in a decreased rate of mitochondrial fusion. Strikingly, expression of activated ARL2 can partially restore the loss of fusion resulting from deletion of either mitofusin 1 (MFN1) or mitofusin 2 (MFN2), but not deletion of both. We only observe the full effects of ARL2 on mitochondrial fusion when it is present in the intermembrane space (IMS), as constructs driven to the matrix or prevented from entering mitochondria are essentially inactive in promoting fusion. Thus, ARL2 is the first regulatory (small) GTPase shown to act inside mitochondria or in the fusion pathway. Finally, using high-resolution, structured illumination microscopy (SIM), we find that ARL2 and mitofusin immunoreactivities present as punctate staining along mitochondria that share a spatial convergence in fluorescence signals. Thus, we propose that ARL2 plays a regulatory role in mitochondrial fusion, acting from the IMS and requiring at least one of the mitofusins in their canonical role in fusion of the outer membranes.

ARL2 is among the most highly conserved proteins, predicted to be present in the last eukaryotic common ancestor, and ubiquitously expressed. Genetic screens in multiple model organisms identified ARL2, and its cytosolic binding partner cofactor D (TBCD), as important in tubulin folding and microtubule dynamics. Both ARL2 and TBCD also localize to centrosomes, making it difficult to dissect these effects. A growing body of evidence also has found roles for ARL2 inside mitochondria, as a regulator of mitochondrial fusion. Other studies have revealed roles for ARL2, in concert with its closest paralog ARL3, in the traffic of farnesylated cargos between membranes and specifically to cilia and photoreceptor cells. Details of each of these signaling processes continue to emerge. We summarize those data here and speculate about the potential for cross-talk or coordination of cell regulation, termed higher order signaling, based upon the use of a common GTPase in disparate cell functions.

Pyridoxal 5′-phosphate (PLP) is an essential cofactor for nearly 60 Escherichia coli enzymes but is a highly reactive molecule that is toxic in its free form. How PLP levels are regulated and how PLP is delivered to target enzymes are still open questions. The COG0325 protein family belongs to the fold-type III class of PLP enzymes and binds PLP but has no known biochemical activity although it occurs in all kingdoms of life. Various pleiotropic phenotypes of the E. coli COG0325 (yggS) mutant have been reported, some of which were reproduced and extended in this study. Comparative genomic, genetic and metabolic analyses suggest that these phenotypes reflect an imbalance in PLP homeostasis. The E. coli yggS mutant accumulates the PLP precursor pyridoxine 5′-phosphate (PNP) and is sensitive to an excess of pyridoxine but not of pyridoxal. The pyridoxine toxicity phenotype is complemented by the expression of eukaryotic yggS orthologs. It is also suppressed by the presence of amino acids, specifically isoleucine, threonine and leucine, suggesting the PLP-dependent enzyme transaminase B (IlvE) is affected. These genetic results lay a foundation for future biochemical studies of the role of COG0325 proteins in PLP homeostasis.

An antiserum to beta-thromboglobulin was used in immunofluorescence to detect normal and malignant megakaryocytes. In normal peripheral blood and bone marrow smears, only platelets and megakaryocytes were specifically stained by this antiserum. Among 25 cases of acute lymphoid and non-lymphoid leukaemia, only 2 exhibited a clear positivity in a % of blasts, thus proving their megakaryocytic origin. This method is proposed as a simple and useful tool to detect normal and malignant megakaryocytes on smears.

Summary: Adenine nucleotides (ATP and ADP) and enzyme activities of Adenylate Kinase (AK) and Phosphoglycerate Kinase (PGK) were assayed on semen samples from normal and infertile men. In accordance with the values obtained, samples could be classified into two groups: 1) one group with levels of either adenine nucleotides and enzymes assumed as normal; 2) a second group with high levels of all the parameters. The latter group comprised the samples with low sperm density (less than 40 million/ml). When the samples were subdivided according to sperm motility, the above distinction into two groups appeared less evident. Moreover, comparing biochemical parameters with morphological findings some contrasts were seen. Nevertheless, we suggest comparing both biochemical and morphological data for a more detailed classification of human semen abnormalities. Zusammenfassung: Adenin-Nukleotide (ATP und ADP) und die enzymatischen Wirkungen der Adenylat-Kinase (AK) und Phosphoglyzerat-Kinase (PGK) wurden jeweils in einer Probe normalen mannlichen Samens und einer Probe unfruchtbaren Samens unter-sucht. Aufgrund der erzielten Werte kann man zwei Gruppen unterscheiden: 1) eine Gruppe mit normalen ATP- und ADP-Werten sowie normalen enzymatischen Wer-ten, 2) eine zweite Gruppe mit hohen Werten aller in Erwagung gezogenen Parameter. Die zweite Gruppe bezieht sich auf Proben mit niedriger Spermatozoendichte (weniger als 40 Millionen/m1). Wenn man die Proben nach der Spermamotilitat unterteilt, erscheint der Unterschied zwischen den zwei Gruppen weniger deutlich. Wenn man die biochemischen Parameter mit den morphologischen Ergebnissen vergleicht, resultieren einige Unterschiede. Trotz-dem empfehlen wir, die gemeinsame, vergleichende Betrachtung der biochemischen und morphologischen Werte zu verwenden, um eine prazise Klassifikation der pathologischen Veranderungen des mannlichen Samens ausarbeiten zu konnen.

Plasma B-type natriuretic peptide (BNP) concentration was evaluated in end-stage renal disease patients to verify if measurements before or after the session could furnish different information. BNP levels in plasma from 52 hemodialysis (HD) patients were measured both before and after the first session of the week. Echocardiographic studies were also performed and patients were followed over a period of 28 months. BNP removal from plasma was influenced by equilibrated Kt/V and patient characteristics. Initial plasma BNP concentration was correlated both with cardiac systolic function (LVEF) and mortality rate, independent of blood sample timing (before or after HD). A relative risk of death of 2.67 was found for plasma BNP levels above 335 pg/mL or 232 pg/mL, before and after HD, respectively. Higher BNP levels were observed in patients with higher burden of comorbidity, as measured by the Charlson Comorbidity Index; however, statistical significance was obtained only for BNP measured before HD. In conclusion, measurement of plasma BNP could give a valuable risk stratification of HD patients while cutting costs, by confining echocardiographic studies only to cases with BNP levels above the established cutoff values.

Background: Patients with chronic renal failure on maintenance haemodialysis (HD) are at high risk of atherothrombotic events; an enhanced oxidant stress might have a major role. The decrease of human paraoxonase (PON1), an anti-oxidant high-density lipoprotein (HDL)-linked enzyme, is a possible mechanism for developing cardiovascular disease. To ascertain the causes of low PON1 in such patients, we investigated the contribution of both PON1 gene polymorphism and individual pattern of HDL. Methods: On 74 HD patients (47 M and 27 F) and on 92 healthy individuals (HS, 48 M and 44 F), we studied PON1 activity, PON1 genotype (55 and 192 PON1 allelic polymorphisms) and the lipid profile, including the HDL subfractions. Results: We observed in HD patients the following significant differences: (1) decreased median PON1 activity (73.5 vs. 110 U/l); (2) decreased mean HDL concentration (1.05±0.18 vs. 1.55±0.41 mmol/l); (3) decreased mean HDL3 concentration (0.79±0.21 vs. 1.28±0.24 mmol/l). Total HDL retained about 70% of serum activity, almost completely carried (95%) by the HDL3. Finally, PON1 activity remained significantly low in HD vs. HS after matching for the allelic polymorphism. Conclusions: The reduction of the HDL3, not the genetic PON1 polymorphism, seems the most important determinant of PON1 activity reduction in HD.

Mitochondria are essential, dynamic organelles that respond to a number of stressors with changes in morphology that are linked to several mitochondrial functions, though the mechanisms involved are poorly understood. We show that the levels of the regulatory GTPase ARL2 and its GAP, ELMOD2, are specifically increased at mitochondria in immortalized mouse embryo fibroblasts deleted for Mitofusin 2 (MFN2), but not MFN1. Elevated ARL2 and ELMOD2 in MEFs deleted for MFN2 could be reversed by re-introduction of MFN2, but only when the mitochondrial fragmentation in these MEFs was also reversed, demonstrating that reversal of elevated ARL2 and ELMOD2 requires the fusogenic activity of MFN2. Other stressors with links to mitochondrial morphology were investigated and several, including glucose or serum deprivation, also caused increases in ARL2 and ELMOD2. In contrast, a number of pharmacological inhibitors of energy metabolism caused increases in ARL2 without affecting ELMOD2 levels. Together we interpret these data as evidence of two ARL2-sensitive pathways in mitochondria, one affecting ATP levels that is independent of ELMOD2 and the other leading to mitochondrial fusion involving MFN2 that does involve ELMOD2.

MOONS is a new Multi-Object Optical and Near-infrared Spectrograph selected by ESO as a third generation instrument for the Very Large Telescope (VLT). The grasp of the large collecting area offered by the VLT (8.2m diameter), combined with the large multiplex and wavelength coverage (optical to near-IR: 0.8μm - 1.8μm) of MOONS will provide the European astronomical community with a powerful, unique instrument able to pioneer a wide range of Galactic, Extragalactic and Cosmological studies and provide crucial follow-up for major facilities such as Gaia, VISTA, Euclid and LSST. MOONS has the observational power needed to unveil galaxy formation and evolution over the entire history of the Universe, from stars in our Milky Way, through the redshift desert, and up to the epoch of very first galaxies and re-ionization of the Universe at redshift z>8-9, just few million years after the Big Bang. On a timescale of 5 years of observations, MOONS will provide high quality spectra for >3M stars in our Galaxy and the local group, and for 1-2M galaxies at z>1 (SDSS-like survey), promising to revolutionise our understanding of the Universe. The baseline design consists of ~1000 fibers deployable over a field of view of ~500 square arcmin, the largest patrol field offered by the Nasmyth focus at the VLT. The total wavelength coverage is 0.8μm-1.8μm and two resolution modes: medium resolution and high resolution. In the medium resolution mode (R~4,000-6,000) the entire wavelength range 0.8μm-1.8μm is observed simultaneously, while the high resolution mode covers simultaneously three selected spectral regions: one around the CaII triplet (at R~8,000) to measure radial velocities, and two regions at R~20,000 one in the J-band and one in the H-band, for detailed measurements of chemical abundances. © (2014) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only.

To compare the chronic effect of several dialytic techniques (bicarbonate dialysis, BHD; acetate free biofiltration, AFB; hemodiafiltration, HDF; paired filtration dialysis, PFD) on atherosclerosis and antioxidant activity, three different indices were created. The first (atherosclerotic index = AI) is formed using the sum of three plasma substances: MDA, Hcy, and Cys (malondialdehyde, homocysteine, cysteine). The second (antioxidant activity index = AOAI) is the sum of five erythrocyte (E) parameters: E-GSH, GPx, CAT, SOD, GR (E-glutathione, E-glutathione peroxidase, E-catalase, E-superoxide dismutase, E-glutathione reductase). The third (defense index = DI) is derived from the previous two: (AOAI - AI). The indices were so expressed as AI in mmol/L, AOAI in U/g hemoglobin (Hb), and DI in arbitrary units. These indices were calculated in 20 controls and 51 chronic HD patients (26 female, 25 male) before, during, and after the first session of the week. HD patients were divided according to their dialytic technique: BHD, n = 35; AFB, n = 5 patients; HDF, n = 7 patients; or PFD = 4 patients. All patients had been treated with a given technique for at least 12 months, before entering the study. As expected, HD patients had AI values higher than controls, both before and after the session, with a mean value of 541 (before) and 331 (after), whereas controls had a mean value of 205. The AOAI was lower than controls, both before and after the session, the mean value being 1,122 (before) and 1,582 (after), that of controls being 2,424. In all cases, PFD gave the best "acute" results; at the end of a PFD session, near normal values of AI, AOAI, and DI (defensive index = AOAI - AI) were obtained.

We describe the construction and uses of a series of plasmids for directing expression to varied levels of exogenous proteins targeted to the mitochondrial matrix or intermembrane space. We found that the level of protein expression achieved, the kinetics of expression and mitochondrial import, and half-life after import can each vary with the protein examined. These factors should be considered when directing localization of an exogenous protein to mitochondria for rescue, proteomics, or other approaches. We describe the construction of a collection of plasmids for varied expression of proteins targeted to the mitochondrial matrix or intermembrane space, using previously defined targeting sequences and strength CMV promoters. The limited size of these compartments makes them particularly vulnerable to artifacts from over-expression. We found that different proteins display different kinetics of expression and import that should be considered when analyzing results from this approach. Finally, this collection of plasmids has been deposited in the Addgene plasmid repository to facilitate the ready access and use of these tools.

Besides apolipoprotein E (APOE) polymorphism, whose association with Alzheimer's disease (AD) has been confirmed in most of the numerous population samples studied, other markers have been investigated. In most cases the association firstly described was not confirmed in subsequent works. Since it is important to examine these associations in as many populations as possible, we investigated APOE, APOC1, APOC2, alpha-1 antichymotrypsin (ACT) and presenilin-1 (PS-1) polymorphisms in a series of elderly patients with late-onset sporadic AD from Northern Italy and in a sex and age-matched control group. We could not confirm the significantly higher frequency of the ACT*A allele among carriers of APOE e*4 allele described elsewhere, although a similar trend was observed. The APOC2 and the PS-1 distributions were similar between patients and controls. However, we observed a significant difference in the genotype and allele frequencies of APOE and APOC1: patients had higher e*4 and C1*2 allele frequencies. This finding confirms the important role for APOE in AD occurrence. In addition, APOC1 seems to be an interesting marker because, though in strict linkage disequilibrium with APOE, it seems to play an independent role in AD risk. In contrast to previously reported data, plasma apoE concentrations were similar in patients and in controls. An interaction between APOE and APOC1 polymorphisms and apoE levels was observed in patients: subjects carrying the APOE E3/E2 or the APOC1 2-2 genotype have higher apoE concentrations than those who do not.

OBJECTIVE: To investigate the relationship between blood pressure and the plasma fibrinolytic system and to verify whether this association was independent or mediated by one or more potential confounding factor.DESIGN: A random sample of 94 males aged 38 years subdivided into normotensives, hypertensives and those hypertensives with the highest blood pressure values.METHODS: Overall and regional obesity, blood lipids, fasting and 2-h post-load glucose, C-peptide and insulin levels, and main behavioural variables, including adipose tissue fatty acid composition (an objective index of dietary fat intake), were measured. The plasma fibrinolytic system was evaluated by determining activities and total plasma concentrations of both tissue-type plasminogen activator before and after venous occlusion, and its inhibitor plasminogen activator inhibitor type-1 (PAI-1).RESULTS: PAI-1 activity was significantly higher in the hypertensives than in the normotensives. PAI-1 antigen tended to parallel PAI-1 activity, and levels of tissue-type plasminogen activator antigen and activity tended to be lower in the hypertensives at baseline and after venous occlusion, but not significantly different from those in the normotensives. The hypertensives also had significantly higher body mass index and body fat content (measured by bio-impedance), increased plasma triglycerides, uric acid, fasting and 2-h glucose, C-peptide and insulin concentrations. In univariate linear regression analysis both systolic and diastolic blood pressures were found to be positively correlated with PAI-1 levels (r = 0.27, P < 0.01, for both). This correlation was maintained after adjustment for total body fat, fasting glucose, fasting insulin concentration or adipose tissue alpha-linolenic acid; however, it was no longer significant after adjustment for plasma 2-h insulin, 2-h C-peptide, 2-h glucose or triglyceride levels. Multivariate regression analysis revealed that only 2-h insulin and triglyceride concentration showed an independent association with PAI-1 levels.CONCLUSIONS: This study confirms that, in 38-year-old males, hypertension is associated with increased PAI-1 activity. It supports the possibility that the relationship between blood pressure and PAI-1 may reflect the overall effect of the insulin resistance syndrome (in particular hyperinsulinaemia and hypertriglyceridaemia) rather than a direct effect of blood pressure on the fibrinolytic system.

The kidney is probably the major site of production of the plasma enzyme glutathione peroxidase (GSHPx-P). For this study, GSHPx-P activity was determined in 40 healthy people, in 34 patients with differing degrees of renal impairment, and in hemodialysis patients from whom blood samples were withdrawn either before or after each session (18 patients) or throughout the dialysis session (27 patients). Hemodialysis patients were treated by means of different techniques (bicarbonate hemodialysis, hemodiafiltration, and acetate free biofiltration), and different membranes (cuprophane, polyacrylonitrite, and polymethylmethacrylate). The following results were obtained: 1) GSHPx-P activity was significantly decreased in renal impairment patients; 2) GSHPx-P activity negatively correlated with serum creatinine values in renal impairment patients (r = -0.55; p0.001); and 3) the enzyme activity slightly increased after the session in hemodialysis patients. The following conclusions can be drawn: GSHPx-P activity could be new index of renal function, because it was decreased in patients with renal failure; the decrease in GSHPx-P activity paralleled the severity of renal impairment, and was maximal in hemodialysis patients; GSHPx-P activity was slightly raised at the end of the hemodialysis session, concomitant with other enzyme activities (aspartate transaminase, alanine transaminase, and alkaline phosphatase) and total protein concentration. This seems to be attributable to the process of water loss rather than other hypothetical mechanisms, such as A) enzyme activation by either peroxide generation during blood-membrane contact, or by the removal of a hypothetical inhibitor; and B) de novo synthesis in the residual renal mass or in other sites of production.

Increased plasma levels of plasminogen activator inhibitor-1 (PAI-1), responsible for reduced fibrinolytic activity, have been shown to be an important risk factor for cardiovascular disease. PAI-1 plasma levels are influenced by several factors which have not yet been fully clarified, including dietary fat intake. The relationships of PAI-1 with other cardiovascular risk factors are still not well known. In a random sample of 38-year-old healthy men (n = 94), the association of PAI-1 plasma levels (measured as activity and antigen) with anthropometric parameters, serum lipids, fasting and 2 h insulin and glucose concentration after oral glucose-load was analysed. Furthermore, the fatty acid composition of subcutaneous adipose tissue, as an objective and reliable index of dietary fat intake, was measured. The univariate analysis showed that plasma levels of PAI-1 were significantly associated with body mass index (BMI) (r = 0.37, P < 0.001), waist/hip ratio (WHR) (r = 0.26, P < 0.01), serum triglycerides (r = 0.47, P < 0.0001), HDL/total cholesterol ratio (r = -0.35, P < 0.001), fasting and 2-h insulin (r = 0.27, P < 0.01 and r = 0.34, P < 0.001) and glucose concentrations (r = 0.25, P < 0.05 and r = 0.28, P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

To evaluate the use of equine-derived bone grafts in the treatment of bone loss in a heterogeneous clinical case series.The study population was 48 patients (29 males and 19 females; mean age, 49 years; range, 9-84); the orthopedic defect site was located on the right side in 22 and on the left side in 26 cases. The graft material was antigen-free equine-derived collagen bone cleaned with an enzymatic treatment.Clinical and radiographic healing times were virtually similar; graft osseointegration needed from two to three months longer to heal.The outcome after grafting with an equine-derived bone substitute was satisfactory. Further study is needed to demonstrate its statistically significant effectiveness in the treatment of orthopedic defects like those in this series.

Elevated plasma levels of fibrinogen, factor VII coagulant activity (F VIIc), and plasminogen activator inhibitor (PAI-1) have been reported to be strictly associated with thrombotic events and are considered to be important risk markers of atherothrombotic caridovascular disease. Therefore, we evaluated in 15 patients on continuous ambulatory peritoneal dialysis (CAPD) the plasma levels of these coagulation factors, basal insulin values, and the lipid pattern in comparison with 33 hemodialysis (HD) patients and 59 healthy subjects. In CAPD the total cholesterol and triglyceride results were significantly increased, but no difference was found in HDL cholesterol. Fibrinogen and F VIIc results were significantly higher In CAPD and HD than In the control group, probably due to an increased hepatic synthesis as a nonspecific response to the peritoneal protein loss. Elevated F VIIc activity may be caused by the presence of large negatively charged lipoproteins, in viva thrombin formation, or reduced hepatic clearance. Both PAl 1 and t-PA results were higher in CAPD, probably due to an increased synthesis by endothelial cells activated by glucose peritoneal absorption and hypertonic dialysis solutions. The contemporary elevation of fibrinogen, F VIIc, PAI-1, and t-PA suggests that CAPD patients present a hypercoagulability and hypofibrinolysis condition, which may promote the development of atherothrombotic events.

Objective – Paraoxonase-1 (PON1) is an esterase with antioxidant properties. Low PON1 enzyme activity or specific allelic polymorphisms seem to be associated with the risk of developing coronary artery disease or acute ischemic stroke (AIS). Our objective was to determine the distribution of both PON1 enzyme activity and its genotype in a group of patients with AIS. Materials and methods – PON1 activity and the relative Q192R and L55M polymorphisms in the PON1 gene were assessed on 126 survivors of a first AIS and in 92 healthy subjects. Results – The genotype distribution for PON1 Q192R and L55M polymorphisms was similar in AIS patients and healthy subjects, but patients carrying the QRLL or RRLL genotype combination had lower PON1 enzyme activity compared with healthy subjects with the same genotype. Conclusion – We postulate that lower than expected PON1 enzyme activity within specific genotypes might explain the reported association between R and L alleles and the risk of developing AIS.

Paciente masculino, 67 anos, previamente sem comorbidades, com história de cefaleia frontal há 1 ano e ptose palpebral direita associada à diminuição da acuidade visual há 4 meses. Nega febre e perda de peso. RNM de crânio (IMAGEM 1) com lesão expansiva (4x2x2cm) extra-axial com base de implantação dural na grande asa do esfenoide à direita. Realizado microcirurgia em outro serviço para tumor cerebral, onde foi verificada invasão do seio cavernoso e órbita direita, englobando artéria carótida com efeito de massa. Realizada ressecção parcial da lesão. Lâmina da biopsia na IMAGEM 2. Qual é o Diagnóstico?

It is not unusual to meet increased levels of ferritinaemia in patients apparently healthy. Among other causes of hyperferritinaemia, recently was described the Hereditary Hyperferritinemia Cataract Syndrome, a genetic condition characterized by increased serum ferritin values without iron overload and bilateral nuclear cataract, both of early onset. It has been demonstrated that single or double point mutations or deletions in the stem-loop structure of the iron regulatory element (I.R.E.) located in the 5 untranslated regions of the ferritin L-subunit gene (19q13.1) are responsible for the upregulation of ferritin. This overexpression only for the L-chain gives rise to typical piles in several tissues. When this altered ferritin accumulates in lens it causes bilateral nuclear cataracts, that is the peculiar sign of this syndrome. It is essential to differentiate true iron overload from Hereditary Hyperferritinaemia Cataract Syndrome (H.H.C.S.), because these patients rapidly develop iron deficient anaemia when venosectioned. Here we describe a case report about a 40 years old healthy female blood donor who presented isolated hyperferritinaemia without iron overload, in the absence of concomitant pathologies. Anamnestic, biochemical, instrumental and clinical investigations led us to diagnose H.H.C.S., a pathology first described in 1995. From 1995 to date about 40 cases concerning patients showing the characteristics of this syndrome from Europe, USA, and Australia were described. Biochemical, genetical and clinical investigations led finally to understand every matter of this pathology, providing conclusive and exhaustive explanations.

Patients on hemodialysis (HD) show an increased risk for developing atherothrombotic events. The oxidative modification of low density lipoproteins (LDL) play an important role in the pathogenesis of atherosclerosis. In patients with uremia (chronic renal failure and HD), the increased oxidative stress induces oxidative modification of LDL. High density lipoproteins (HDL) exhibit a double antiatherogenic role, removing both lipid peroxides from LDL and cholesterol from tissues or vascular wall. Paraoxonase 1 (PON1) is one of three enzymes shown to prevent the formation of oxidized LDL. PON1 activity is modulated by its genetic polymorphism and by non-genetic factors, such as diet, smoking, acute phase reactants, and hormones. PON1 activity has been found to be significantly decreased in uremia. The present study aimed to verify the possibility that this reduced activity could be caused by a different PON1 gene polymorphism between patients on HD and healthy subjects, but this was not the case. The main cause may be identified in the different distribution of HDL subspecies, rather than in the different PON1 allele distribution between healthy subjects and patients with uremia.

To address the needs of state of the art scientific investigations and to improve the efficiency and economy of marine data collection, an innovative system, ARAMIS (Advanced ROV package for Automatic Mobile Investigation of Sediments), has been developed with the support of EC MAST III program. ARAMIS is a scientific and technological package to be integrated with typical mid class Remotely Operated Vehicles (ROVs) to form a highly automated scientific tool for carrying out multidisciplinary missions. Several processing units and a Man-Machine Interface (MMI) connected in a Local Area Network compose the ARAMIS surface control equipment. The supervisory control system is the heart of the surface equipment. It gets and processes information and data provided by all the other processing units allowing the pilot/scientist to execute high level commands to move the ROV in an efficient, reliable and repeatable way. In this way, the navigation control capabilities of the ROV are considerably enhanced by automatic functions such as: automatic swim to a position; automatic navigation along a transect at a given bottom distance; automatic station keeping; automatic swim around a static target; automatic landing on the sea bottom; automatic obstacle detection and avoidance. This paper focuses on the ARAMIS supervisory control system and provides a synthetic description of the overall system.

Two new polymorphisms in the regulatory region of the apolipoprotein E gene, –491 A/T and –427 T/C, have been reported to be associated with the risk of Alzheimer’s disease (AD). Moreover, in vitro studies suggest that the two polymorphisms modulate the levels of apoE protein expression. We examined these two polymorphisms, as well as the MspI polymorphism in the LDL receptor gene, in a series of elderly patients with late-onset sporadic AD and in an age-matched control group but failed to find any kind of association between these genetic features and an increased risk of AD. In the same samples we investigated the relationships between various genotypes and plasma lipid levels. Since the well-known effect of the three-allelic APOE polymorphism on plasma lipid levels could mask the effect of other polymorphisms, the analyses were performed taking into account the APOE genotype. The two regulatory region polymorphisms had significant effects only on the apoE levels. The –427 TT homozygotes had lower, and the –491 AA homozygotes had higher levels of apoE than other genotypes. This result confirmed in vivo the already observed in vitro effects of –491 A/T and –427 T/C polymorphisms on APOE promoter transcription activity.