Chronic Mineral Dysregulation Promotes Vascular Smooth Muscle Cell Adaptation and Extracellular Matrix Calcification

In chronic kidney disease (CKD) vascular calcification occurs in response to deranged calcium and phosphate metabolism and is characterized by vascular smooth muscle cell (VSMC) damage and attrition. To gain mechanistic insights into how calcium and phosphate mediate calcification, we used an ex vivo model of human vessel culture. Vessel rings from healthy control subjects did not accumulate calcium with long-term exposure to elevated calcium and/or phosphate. In contrast, vessel rings from patients with CKD accumulated calcium; calcium induced calcification more potently than phosphate (at equivalent calcium-phosphate product). Elevated phosphate increased alkaline phosphatase activity in CKD vessels, but inhibition of alkaline phosphatase with levamisole did not block calcification. Instead, calcification in CKD vessels most strongly associated with VSMC death resulting from calcium- and phosphate-induced apoptosis; treatment with a pan-caspase inhibitor ZVAD ameliorated calcification. Calcification in CKD vessels was also associated with increased deposition of VSMC-derived vesicles. Electron microscopy confirmed increased deposition of vesicles containing crystalline calcium and phosphate in the extracellular matrix of dialysis vessel rings. In contrast, vesicle deposition and calcification did not occur in normal vessel rings, but we observed extensive intracellular mitochondrial damage. Taken together, these data provide evidence that VSMCs undergo adaptive changes, including vesicle release, in response to dysregulated mineral metabolism. These adaptations may initially promote survival but ultimately culminate in VSMC apoptosis and overt calcification, especially with continued exposure to elevated calcium

Automating pitted red blood cell counts using deep neural network analysis: a new method for measuring splenic function in sickle cell anaemia

The spleen plays an important role in the body's defence against bacterial infections. Measuring splenic function is of interest in multiple conditions, including sickle cell anaemia (SCA), where spleen injury occurs early in life. Unfortunately, there is no direct and simple way of measuring splenic function, and it is rarely assessed in clinical or research settings. Manual counts of pitted red blood cells (RBCs) observed with differential interference contrast (DIC) microscopy is a well-validated surrogate biomarker of splenic function. The method, however, is both user-dependent and laborious. In this study, we propose a new automated workflow for counting pitted RBCs using deep neural network analysis. Secondly, we assess the durability of fixed RBCs for pitted RBC counts over time. We included samples from 48 children with SCA and 10 healthy controls. Cells were fixed in paraformaldehyde and examined using an oil-immersion objective, and microscopy images were recorded with a DIC setup. Manual pitted RBC counts were performed by examining a minimum of 500 RBCs for pits, expressing the proportion of pitted RBCs as a percentage (%PIT). Automated pitted RBC counts were generated by first segmenting DIC images using a Zeiss Intellesis deep learning model, recognising and segmenting cells and pits from background. Subsequently, segmented images were analysed using a small ImageJ macro language script. Selected samples were stored for 24 months, and manual pitted RBC counts performed at various time points. When comparing manual and automated pitted RBC counts, we found the two methods to yield comparable results. Although variability between the measurements increased with higher %PIT, this did not change the diagnosis of asplenia. Furthermore, we found no significant changes in %PIT after storing samples for up to 24 months and under varying temperatures and light exposures. We have shown that automated pitted RBC counts, produced using deep neural network analysis, are comparable to manual counts, and that fixed samples can be stored for long periods of time without affecting the %PIT. Automating pitted RBC counts makes the method less time consuming and results comparable across laboratories

Diet restriction in migraine, based on IgG against foods: A clinical double-blind, randomised, cross-over trial

Introduction: It is well-known that specific foods trigger migraine attacks in some patients. We aimed to investigate the effect of diet restriction, based on IgG antibodies against food antigens on the course of migraine attacks in this randomised, double blind, cross-over, headache-diary based trial on 30 patients diagnosed with migraine without aura

The significance of spleen size in children with sickle cell anemia

It is well established that splenic dysfunction occurs in early childhood in sickle cell anemia (SCA), although the determinants and consequences of splenic injury are not fully understood. In this study, we examined spleen size and splenic function in 100 children with SCA aged 0–16 years at King's College Hospital in London. Spleen size was assessed by abdominal ultrasound (US) and splenic function by pitted red blood cells (PIT counts). In our cohort, 5.6% of children aged 6–10 years and 19.4% of children aged 11–16 years had no visible spleen on US (autosplenectomy). Splenomegaly was common in all age groups, with 28% of children overall having larger spleens than the average for their age. Only one child had a PIT count suggesting preserved splenic function. We found no correlation between hemoglobin F levels and spleen size, nor was there any difference in spleen size between children treated with or without hydroxyurea. Although there was a trend toward increased spleen length in children with co-inherited α-thalassemia, this did not reach statistical significance. Finally, we found a strong association between erythrocyte deformability measured with oxygen gradient ektacytometry, spleen size, and PIT counts. In conclusion, our results do not agree with the general perception that most children with SCA undergo autosplenectomy within the first decade of life and indicate that loss of erythrocyte deformability contributes to loss of splenic filtration capacity in SCA, as well as phenotypical variations in spleen size

X-ray Absorption and Reflection in Active Galactic Nuclei

X-ray spectroscopy offers an opportunity to study the complex mixture of emitting and absorbing components in the circumnuclear regions of active galactic nuclei, and to learn about the accretion process that fuels AGN and the feedback of material to their host galaxies. We describe the spectral signatures that may be studied and review the X-ray spectra and spectral variability of active galaxies, concentrating on progress from recent Chandra, XMM-Newton and Suzaku data for local type 1 AGN. We describe the evidence for absorption covering a wide range of column densities, ionization and dynamics, and discuss the growing evidence for partial-covering absorption from data at energies > 10 keV. Such absorption can also explain the observed X-ray spectral curvature and variability in AGN at lower energies and is likely an important factor in shaping the observed properties of this class of source. Consideration of self-consistent models for local AGN indicates that X-ray spectra likely comprise a combination of absorption and reflection effects from material originating within a few light days of the black hole as well as on larger scales. It is likely that AGN X-ray spectra may be strongly affected by the presence of disk-wind outflows that are expected in systems with high accretion rates, and we describe models that attempt to predict the effects of radiative transfer through such winds, and discuss the prospects for new data to test and address these ideas.Comment: Accepted for publication in the Astronomy and Astrophysics Review. 58 pages, 9 figures. V2 has fixed an error in footnote

Gravitational Waves from Gravitational Collapse

Gravitational wave emission from the gravitational collapse of massive stars has been studied for more than three decades. Current state of the art numerical investigations of collapse include those that use progenitors with realistic angular momentum profiles, properly treat microphysics issues, account for general relativity, and examine non--axisymmetric effects in three dimensions. Such simulations predict that gravitational waves from various phenomena associated with gravitational collapse could be detectable with advanced ground--based and future space--based interferometric observatories.Comment: 68 pages including 13 figures; revised version accepted for publication in Living Reviews in Relativity (http://www.livingreviews.org

The Fate of Porous Hydroxyapatite Granules Used in Facial Skeletal Augmentation

Facial appearance is largely determined by the morphology of the underlying skeleton. Hydroxyapatite is one of several materials available to enhance projection of the facial skeleton. This study evaluated the long-term maintenance of augmented bony projection when porous hydroxyapatite granules are used on the facial skeleton. Ten female patients aged 28–58 years were studied following aesthetic augmentation of the facial skeleton at 24 sites using porous hydroxyapatite granules. Postoperative CT scans at 3 months served as the baseline measurement and compared with scans taken at 1 and 2 years, with the thickness of the hydroxyapatite measured in axial and coronal planes. Thickness of original bone plus overlay of hydroxyapatite, thickness of the overlying soft tissue, and the overall projection (bone plus soft tissue) were recorded. It was found that 99.7% of the hydroxyapatite was maintained at 2 years, with no statistical difference (t test) from the baseline measurement. The overall projection (bony and soft tissue) was maintained as there was no evidence of native bone resorption or soft tissue atrophy. Radiographic results confirmed that the use of porous hydroxyapatite granules for enhancement of the facial skeleton is not only a predictable procedure, but maintains full bony projection at 2 years

Simultaneous genotyping of multiple polymorphisms in human serotonin transporter gene and detection of novel allelic variants

The serotonin transporter, called SLC6A4, SERT or 5-HTT, modulates neurotransmission by removal of serotonin from the synapse of serotonergic neurons, facilitating serotonin reuptake into the presynaptic terminus. Selective serotonin reuptake inhibitors block the action of the serotonin transporter and are used to treat depression and other neuropsychiatric disorders. Three polymorphisms in the 5-HTT gene have been implicated in treatment response and neuropsychiatric disorders. A 44-bp promoter ins/del polymorphism (5-HTTLPR) produces primarily long and/or short alleles due to either 14 (short) or 16 (long) repeats of variably conserved 20–23 bp units. Also implicated, a 17–18 bp variable number tandem repeat found in intron2 (StIn2) is expressed as triallelic content with 9, 10, or 12 repeats (StIn2.9, StIn2.10 or StIn2.12). Finally, a single nucleotide polymorphism rs25531 located within the promoter polymorphic-linked region alters the function of the long promoter allele. We developed a PCR-based fragment analysis assay, which is analyzed on an ABI sequencer, whereby we are able to detect all three genotypes simultaneously. Using this technique, we identified novel sequences, which demonstrate promoter repeat regions containing (1) a 17 repeat with rs25531 A/G polymorphism, (2) two with 18-repeat units, (3) one with 20-repeat units and (4) a 24-repeat sequence. The novel repeats were confirmed by direct sequencing of gel-purified amplicons

From L-Dopa to Dihydroxyphenylacetaldehyde: A Toxic Biochemical Pathway Plays a Vital Physiological Function in Insects

One protein in Aedes aegypti, classified into the aromatic amino acid decarboxylase (AAAD) family based on extremely high sequence homology (∼70%) with dopa decarboxylase (Ddc), was biochemically investigated. Our data revealed that this predicted AAAD protein use L-dopa as a substrate, as does Ddc, but it catalyzes the production of 3,4-dihydroxylphenylacetaldehyde (DHPAA) directly from L-dopa and apparently has nothing to do with the production of any aromatic amine. The protein is therefore named DHPAA synthase. This subsequently led to the identification of the same enzyme in Drosophila melanogaster, Anopheles gambiae and Culex quinquefasciatus by an initial prediction of putative DHPAA synthase based on sequence homology and subsequent verification of DHPAA synthase identity through protein expression and activity assays. DHPAA is highly toxic because its aldehyde group readily reacts with the primary amino groups of proteins, leading to protein crosslinking and inactivation. It has previously been demonstrated by several research groups that Drosophila DHPAA synthase was expressed in tissues that produce cuticle materials and apparent defects in regions of colorless, flexible cuticular structures have been observed in its gene mutants. The presence of free amino groups in proteins, the high reactivity of DHPAA with the free amino groups, and the genetically ascertained function of the Drosophila DHPAA synthase in the formation of colorless, flexible cuticle, when taken together, suggest that mosquito and Drosophila DHPAA synthases are involved in the formation of flexible cuticle through their reactive DHPAA-mediated protein crosslinking reactions. Our data illustrate how a seemingly highly toxic pathway can serve for an important physiological function in insects