Continuous wave electron paramagnetic resonance L-band spectrometer with direct digitalization using time-locked subsampling.

This article describes a novel digital L-band EPR spectrometer. The spectrometer uses direct digital detection with time-locked subsampling (TLSS). The device consists of a microwave bridge equipped with a microwave source based on direct digital synthesis (DDS) and a digital receiver. DDS technology combined with an ultra-low noise 1 GHz master clock allowed the development of a digitally controlled microwave source with exceptionally good phase noise characteristics. The obtained level of phase noise is as low as -140 dBc/Hz at 30.5 kHz from the carrier frequency of 1.15 GHz, which is important when registering the EPR dispersion signal. The receiver is equipped with a high-speed A/D converter that enables direct digitalization of the L-band microwave signal. The obtained discrete data are then buffered and averaged in a programmable logic FPGA device. Data packets from FPGA are transferred to a DSP microcontroller that correlates them with the appropriate reference signals. This detection algorithm requires time locking of the generator and the receiver, which is ensured by clocking both devices from the same reference source. This procedure allows the simultaneous detection of the absorption and dispersion signals at the magnetic field modulation frequency and at any of its harmonics. The software to control the spectrometer was designed in the LabView programming environment. The program also allows further data processing. To the best of our knowledge, the described spectrometer is one of the first full implementation of the direct digital detection technique which could replace conventional analog CW spectrometers that utilize magnetic field modulation. For an 11 µm aqueous TEMPOL solution, the new spectrometer obtained a S/N ratio greater than 160 for an EPR spectrum registered in 69 s.

Nox1/4 inhibition exacerbates age dependent perivascular inflammation and fibrosis in a model of spontaneous hypertension.

Hypertension is associated with oxidative stress and perivascular inflammation, critical contributors to perivascular fibrosis and accelerated vascular ageing. Oxidative stress can promote vascular inflammation, creating options for potential use of NADPH oxidase inhibitors in pharmacological targeting of perivascular inflammation and its consequences. Accordingly, we characterized age-related changes in oxidative stress and immune cell infiltration in normotensive (WKY) and spontaneously hypertensive rats (SHRs). Subsequently, we used pharmacological inhibitors of Nox1 (ML171) and Nox1/Nox4 (GKT137831; 60 mg/kg), to modulate NADPH oxidase activity at the early stage of spontaneous hypertension and investigated their effects on perivascular inflammation and fibrosis. RESULTS: Ageing was associated with a progressive increase of blood pressure as well as an elevation of the total number of leukocytes, macrophages and NK cells infiltrating perivascular adipose tissue (PVAT) in SHRs but not in WKY. At 1 month of age, when blood pressure was not yet different, only perivascular NK cells were significantly higher in SHR. Spontaneous hypertension was also accompanied by the higher perivascular T cell accumulation, although this increase was age independent. Aortic Nox1 and Nox2 mRNA expression increased with age only in SHR but not in WKY, while age-related increase of Nox4 mRNA in the vessels has been observed in both groups, it was more pronounced in SHRs. At early stage of hypertension (3-months) the most pronounced differences were observed in Nox1 and Nox4. Surprisingly, GKT137831, dual inhibitor of Nox1/4, therapy increased both blood pressure and perivascular macrophage infiltration. Mechanistically, this was linked to increased expression of proinflammatory chemokines expression (CCL2 and CCL5) in PVAT. This inflammatory response translated to increased perivascular fibrosis. This effect was likely Nox4 dependent as the Nox1 inhibitor ML171 did not affect the development of spontaneous hypertension, perivascular macrophage accumulation, chemokine expression nor adventitial collagen deposition. In summary, spontaneous hypertension promotes ageing-associated perivascular inflammation which is exacerbated by Nox4 but not Nox1 pharmacological inhibition.

Identification of transcription factors responsible for dysregulated networks in human osteoarthritis cartilage by global gene expression analysis.

OBJECTIVE: Osteoarthritis (OA) is the most prevalent joint disease. As disease-modifying therapies are not available, novel therapeutic targets need to be discovered and prioritized for their importance in mediating the abnormal phenotype of cells in OA-affected joints. Here, we generated a genome-wide molecular profile of OA to elucidate regulatory mechanisms of OA pathogenesis and to identify possible therapeutic targets using integrative analysis of mRNA-sequencing data obtained from human knee cartilage. DESIGN: RNA-sequencing (RNA-seq) was performed on 18 normal and 20 OA human knee cartilage tissues. RNA-seq datasets were analysed to identify genes, pathways and regulatory networks that were dysregulated in OA. RESULTS: RNA-seq data analysis revealed 1332 differentially expressed (DE) genes between OA and non-OA samples, including known and novel transcription factors (TFs). Pathway analysis identified 15 significantly perturbed pathways in OA with ECM-related, PI3K-Akt, HIF-1, FoxO and circadian rhythm pathways being the most significantly dysregulated. We selected DE TFs that are enriched for regulating DE genes in OA and prioritized these TFs by creating a cartilage-specific interaction subnetwork. This analysis revealed eight TFs, including JUN, Early growth response (EGR)1, JUND, FOSL2, MYC, KLF4, RELA, and FOS that both target large numbers of dysregulated genes in OA and are themselves suppressed in OA. CONCLUSIONS: We identified a novel subnetwork of dysregulated TFs that represent new mediators of abnormal gene expression and promising therapeutic targets in OA.

Initial steps in defining the environment of the prepuce of the bull by measuring pH and temperature.

OBJECTIVE: To determine the baseline pH and temperature of the preputial cavity of bulls. METHODS: We enrolled 55 bulls ranging in age from 15 to 84 months. The preputial temperature and pH were measured by insertion of temperature and pH probes, respectively, into the preputial orifice prior to routine breeding soundness examinations. Information was obtained from owners regarding the diet of each bull and categorised as one of three categories: forage only, grain supplemented or silage supplemented. RESULTS: The average temperature of the prepuce was 37.81°C ± 1.76 and the median pH of the prepuce was 8.45 (6.35-9.46). Preputial temperatures of the bull weakly correlated with ambient temperatures (rs = -0.29, P = 0.028). The preputial pH of silage-fed bulls was significantly lower than that of bulls fed forage only (P = 0.025) or grain-supplemented diets (P = 0.002). The median preputial pH of bulls fed a silage-based diet was 7.6 (6.3-8.9) compared with a median pH 8.7 (7.8-9.1) for bulls fed forage-based diets or a median of 8.5 (7.7-9.4) for those given grain-supplemented diets. CONCLUSION: Diet and ambient temperature can, respectively, affect pH and the temperature in the prepuce. Further studies to describe and understand the microbiota of the prepuce and penis may assist in developing treatments for diseases of the genital tract in bulls.

Selenium status parameters in patients with polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in women of reproductive age. To date, no systematic study of interactions between selenium status parameters (SSPs: serum selenium concentration, plasma glutathione peroxidase, GPX3, plasma selenoprotein P, SELENOP), sex hormones, thyroid function parameters, and other laboratory parameters in patients with PCOS has been undertaken. Therefore we aimed to compare such parameters in women with PCOS and in the control groups, and to investigate the multidimensional interactions between various parameters in PCOS patients and in controls. The subjects were diagnosed either with PCOS (n=28, 25.4±5.2 y) or with PCOS+Hashimoto disease (n=13, 27.3±5.6 y). Female patients having normal menses were recruited into the first control group (n=70, 26.8±7.3 y) or to the second control group comprising women only with Hashimoto disease (n=10, 26.2±6.9 y). No apparent differences in SSPs between control subjects and patients with PCOS, also complicated with Hashimoto disease, were identified, though such differences were noticeable for total testosterone (tT), sex hormone binding globulin, free androgen index, dehydroepiandrosterone sulfate (DHEAS), and insulin profile. The correlation between tT and DHEAS was found the strongest. The other group of mutually highly and positively correlated parameters consisted of GPX3, follicle stimulating hormone, free triiodothyronine and free thyroxine. All the latter parameters correlated negatively with vitamin D3. SSPs took part in interactions with thyroid hormones, sex hormones and some other parameters, but only for GPX3 such interactions were statistically significant. The significance of these findings remains open for further investigation, particularly in patients with PCOS and/or Hashimoto disease.

Limitation of tuning the antibody-antigen reaction by changing the value of pH and its consequence for hyperthermia.

Distribution of the isoelectric point (pI) was calculated for the hypervariable regions of Fab fragments of the antibody molecules, which structure is annotated in the structural antibody database SabDab. The distribution is consistent with the universal for all organisms dividing the proteome into two sets of acidic and basic proteins. It shows the additional fine structure in a form of the narrow-sized peaks of pI values. This is an explanation why a small change of the environmental pH can have a strong effect on the antibody-antigen affinity. To show this, a typical enzyme-linked immunospecific assay experiment for testing the reaction of goat anti-human IgA antibodies with human IgA immunoglobulins of saliva as antigens was modified in such a way that Fe3O4magnetic nanoparticles were added to PBS buffer. The magnetic nanoparticles were remotely heated by the radio frequency magnetic field providing the local change of temperature and pH. It was observed that short times of the heating were significantly increasing the antibody-antigen binding strength while it was not the case for a longer time. The finding discussed in the study can be useful for biopharmaceuticals using antibodies, the immunoassay techniques as well as for control over the use of hyperthermia.

Statistical shape modeling of proximal femoral shape deformities in Legg-Calvé-Perthes disease and slipped capital femoral epiphysis.

INTRODUCTION: The current understanding of morphological deformities of the hip such as femoroacetabular impingement (FAI), Legg-Calvé-Perthes disease (LCPD), and slipped capital femoral epiphysis (SCFE) is based on two-dimensional metrics, primarily involving the femoral head, that only partially describe the complex skeletal morphology. OBJECTIVE: This study aimed to improve the three-dimensional (3-D) understanding of shape variations during normal growth, and in LCPD and SCFE, through statistical shape modeling. DESIGN: Thirty-two patients with asymptomatic, LCPD, and SCFE hips, determined from physical and radiographic examinations, were scanned using 3-D computed tomography (CT) at a voxel size of (0.5-0.9 mm)(2) in-plane and 0.63 mm slice thickness. Statistical shape modeling was performed on segmented proximal femoral surfaces to determine modes of variation and shape variables quantifying 3-D shape. In addition, conventional variables were determined for all femora. RESULTS: Proximal femur shape was described by eight modes of variation and corresponding shape variables. Statistical shape variables were distinct with age and revealed coordinated, growth-associated differences in neck length-to-width ratio, femoral head medialization, and trochanter protrusion. After size and age-based shape adjustment, diseased proximal femora were characterized by shape variables distinct from those of asymptomatic hips. The shape variables defined morphology in health and disease, and were correlated with certain conventional variables of shape, including neck-shaft angle, head diameter, and neck diameter. CONCLUSION: 3-D quantitative analyses of proximal femoral bone shape during growth and in disease are useful for furthering the understanding of normal and abnormal shape deviations which affect cartilage biomechanics and risk of developing osteoarthritis.

Comparison of cartilage histopathology assessment systems on human knee joints at all stages of osteoarthritis development.

OBJECTIVE: To compare the MANKIN and OARSI cartilage histopathology assessment systems using human articular cartilage from a large number of donors across the adult age spectrum representing all levels of cartilage degradation. DESIGN: Human knees (n=125 from 65 donors; age range 23-92) were obtained from tissue banks. All cartilage surfaces were macroscopically graded. Osteochondral slabs representing the entire central regions of both femoral condyles, tibial plateaus, and the patella were processed for histology and Safranin O - Fast Green staining. Slides representing normal, aged, and osteoarthritis (OA) tissue were scanned and electronic images were scored online by five observers. Statistical analysis was performed for inter- and intra-observer variability, reproducibility and reliability. RESULTS: The inter-observer variability among five observers for the MANKIN system showed a similar good Intra-class correlation coefficient (ICC>0.81) as for the OARSI system (ICC>0.78). Repeat scoring by three of the five readers showed very good agreement (ICC>0.94). Both systems showed a high reproducibility among four of the five readers as indicated by the Spearman's rho value. For the MANKIN system, the surface represented by lesion depth was the parameter where all readers showed an excellent agreement. Other parameters such as cellularity, Safranin O staining intensity and tidemark had greater inter-reader disagreement. CONCLUSION: Both scoring systems were reliable but appeared too complex and time consuming for assessment of lesion severity, the major parameter determined in standardized scoring systems. To rapidly and reproducibly assess severity of cartilage degradation, we propose to develop a simplified system for lesion volume.

New tricks with old dogs: personalised medicine and clinical trials.

We provide a didactic example of how clinical trials can accommodate individualised patient information relative to design and analysis.

Macroscopic and histopathologic analysis of human knee menisci in aging and osteoarthritis.

OBJECTIVE: Meniscus lesions following trauma or associated with osteoarthritis (OA) have been described, yet meniscus aging has not been systematically analyzed. The objectives of this study were to (1) establish standardized protocols for representative macroscopic and microscopic analysis, (2) improve existing scoring systems, and (3) apply these techniques to a large number of human menisci. DESIGN: Medial and lateral menisci from 107 human knees were obtained and cut in two different planes (triangle/cross section and transverse/horizontal section as well) in three separate locations (middle portion, anterior and posterior horns). All sections included vascular and avascular regions and were graded for (1) surface integrity, (2) cellularity, (3) matrix/fiber organization and collagen alignment, and (4) Safranin-O staining intensity. The cartilage in all knee compartments was also scored. RESULTS: The new macroscopic and microscopic grading systems showed high inter-reader and intra-reader intraclass correlation coefficients. The major age-related changes in menisci in joints with no or minimal OA included increased Safranin-O staining intensity, decreased cell density, the appearance of acellular zones, and evidence of mucoid degeneration with some loss of collagen fiber organization. The earliest meniscus changes occurred predominantly along the inner rim. Menisci from OA joints showed severe fibrocartilaginous separation of the matrix, extensive fraying, tears and calcification. Abnormal cell arrangements included decreased cellularity, diffuse hypercellularity along with cellular hypertrophy and abnormal cell clusters. In general, the anterior horns of both medial and lateral menisci were less affected by age and OA. CONCLUSIONS: New standardized protocols and new validated grading systems allowed us to conduct a more systematic evaluation of changes in aging and OA menisci at a macroscopic and microscopic level. Several meniscus abnormalities appear to be specific to aging in the absence of significant OA. With aging the meniscal surface can be intact but abnormal matrix organization and cellularity were observed within the meniscal substance. The increased Safranin-O staining appears to represent a shift from fibroblastic to chondrocytic phenotype during aging and early degeneration.

Selective MMP-inhibition with Ro 28-2653 in acute experimental stroke--a magnetic resonance imaging efficacy study.

Blood-brain-barrier (BBB) breakdown due to matrix metalloproteinase (MMP) activity following stroke is often associated with cerebral edema, larger infarct volumes and bad outcome. In the present study we examined a novel MMP-inhibitor (Ro 28-2653) with high selectivity for MMP2, MMP9 and membrane type 1-MMP in an acute stroke model comparing two different treatment regimens. We subjected rats to 90 min of focal cerebral ischemia followed by 3 days or 7 days of reperfusion, respectively, using the middle cerebral artery (MCA) filament occlusion technique. Ro 28-2653 was administered daily in a vehicle solution for 2 days or 6 days after ischemia, respectively. We assessed the behavior with a functional neuroscore and infarct volumes as well as blood-brain-barrier (BBB) breakdown with magnetic resonance imaging (MRI) after 3 and 7 days. Infarct edema volumes, BBB breakdown and behavior at 3 days were significantly attenuated in rats treated for 2 days with Ro 28-2653 as compared to vehicle and untreated controls. After 6 days of treatment however, infarct and BBB breakdown volumes as well as behavior did not differ significantly between the groups at 7 days. The new high selective MMP-inhibitor Ro 28-2653 significantly reduced brain injury only when administered in the first 2 days after focal cerebral ischemia. Prolonged treatment for 6 days did not show any beneficial effects possibly due to interference with protective restorative processes.

Sonographic monitoring of mass effect in stroke patients treated with hypothermia. Correlation with intracranial pressure and matrix metalloproteinase 2 and 9 expression.

Severe stroke leads to subsequent cerebral oedema. Patients with severe stroke develop midline shift (MLS) which can be measured by transcranial duplex sonography (TCD). We measured MLS with TCD in 30 patients with large infarction in the territory of the middle cerebral artery (MCA). All of the examined patients had intracranial pressure (ICP) measure devices and the ICP at the time of the TCD was recorded. MLS was also determined on CT scan on day 4. Ten of the 30 patients were treated with hypothermia. We also determined matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9 (MMP9) in serum by zymography. MLS measured by TCD correlated significantly with MLS on CT. In addition there was a strong correlation between the ICP measured at the time of TCD and MLS. In patients treated with hypothermia MLS was less pronounced. MMP9 and MMP2 showed a characteristic time course and had strong associations with MLS. We confirm earlier reports that TCD is a reliable noninvasive method for serially monitoring patients with intracranial lesions. Hypothermia reduces MMP9 activity as well as MLS. TCD may reduce the need for repetitive CT scans in neurological critically ill patients.

Mapping of angiogenic markers for targeting of vectors to tumor vascular endothelial cells.

The vasculature of mouse breast tumor spheroids grown on mammary fat pad tissue in an intravital microscopy (IVM) viewing chamber was shown to derive from infiltrating angiogenic mammary vessels. The receptors tissue factor (TF), alpha V beta 3 integrin and Tie-2 were expressed on the vascular endothelium in the periphery but not in the center of the tumor spheroids nor in the mammary tissue nor in smooth muscle tissue, whereas Tie-1 and PCAM-1 were expressed extensively in the entire tumor and in the vascular endothelium of the entire tumor nodule and in normal mammary tissue. TF is a specific target for adenoviral vector-mediated cancer immunotherapy. Subcutaneous injection of the AdfVII/IgG(1)Fc vector leads to the release into the system circulation of a fVII/IgG(1)Fc immunoconjugate molecule that binds specifically and tightly to TF on vascular endothelial cells and tumor cells, activating a cytolytic immune response against the targeted cells. We show that a single administration of the AdfVII/IgG(1)Fc vector destroys the peripheral but not the central vasculature of a tumor spheroid, causing partial tumor regression; additional administrations prevent regeneration of the peripheral vasculature and regrowth of the tumor. These findings indicate that a critical parameter for optimizing tumor damage is the schedule for successive administrations of the AdfVII/IgG(1)Fc, which should coincide with the regeneration of the peripheral vasculature and continue until the tumor is destroyed.

Vascular matrix adhesion and the blood-brain barrier.

The integrity of the cerebral microvasculature depends on the interaction between its component cells and the extracellular matrix, as well as reorganized cell-cell interactions. In the central nervous system, matrix adhesion receptors are expressed in the microvasculature and by neurons and their supporting glial cells. Cells within cerebral microvessels express both the integrin and dystroglycan families of matrix adhesion receptors. However, the functional significance of these receptors is only now being explored. Endothelial cells and astrocytes within cerebral capillaries co-operate to generate and maintain the basal lamina and the unique barrier functions of the endothelium. Integrins and the dystroglycan complex are found on the matrix-proximate faces of both endothelial cells and astrocyte end-feet. Pericytes rest against the basal lamina. In the extravascular compartment, select integrins are expressed on neurons, microglial cells and oligodendroglia. Significant alterations in both cellular adhesion receptors and their matrix ligands occur during focal cerebral ischaemia, which support their functional significance in the normal state. We propose that matrix adhesion receptors are essential for the maintenance of the integrity of the blood-brain permeability barrier and that modulation of these receptors contributes to alterations in the barrier during brain injury.

Angiotensin type 1 receptor blockage improves ischemic injury following transient focal cerebral ischemia.

Following cerebral ischemia, i.v. infusion of angiotensin II increases cerebral edema and mortality. Angiotensin type 1 receptor blockage should therefore improve acute cerebral ischemia. Left middle cerebral artery occlusion (120 min) followed by reperfusion was performed with the thread method under halothane anesthesia in Sprague-Dawley rats. Olmesartan (angiotensin type 1 receptor blocker; 0.01 or 0.1mumol/kg/h) was infused i.p. for 7 days following middle cerebral artery occlusion followed by reperfusion. Stroke index score, infarct volume, specific gravity, and brain angiotensin II and matrix metalloproteinases were quantified in the ischemic and non-ischemic hemispheres. Olmesartan treatment improved stroke index score, infarct volume, and cerebral edema in our cerebral ischemia model. In particular, stroke index score, infarct volume, and cerebral edema were reduced even with a low dose of olmesartan that did not decrease blood pressure. Paralleling these effects on cerebral ischemia, olmesartan treatment also reduced the reactive upregulation in brain angiotensin II, matrix metalloproteinase-2, matrix metalloproteinase-9, and membrane type 1-matrix metalloproteinase in the ischemic area. Angiotensin type 1 receptor stimulation may be one of the important factors that cause cerebral edema following cerebral ischemia, and that its inhibition may be of therapeutic advantage in cerebral ischemia.

Autoimmune thrombocytopenia: flow cytometric determination of platelet-associated autoantibodies against platelet-specific receptors.

Immune thrombocytopenic purpura (ITP) is an autoimmune disorder characterized by antibody-induced platelet destruction. Despite its clinical importance, the diagnosis of ITP is one of exclusion, thus, inevitably associated with potential difficulties. We here describe a feasible diagnostic method using the commonly available technique of flow cytometry. An antigen-specific assay for platelet-associated antibody was developed and tested in 62 adult patients with chronic ITP, 14 patients with thrombocytopenia of decreased production and 60 healthy controls. The method is based on flow cytometric (FCM) detection of autoantibodies reacting with specific platelet receptors immobilized on microbeads. The average fluorescence level in the ITP group calculated as a ratio to normal was 4.07 (range 0.8-31.0), in the non-ITP thrombocytopenic patients 0.9 (range 0.7-1.2), and in the healthy controls 1.0 (range 0.7-1.3). The average assay coefficient of variation was 0.218 [95% confidence interval (CI) 0.213, 0.221]. The difference between the ITP patients and both groups was highly significant (P < 0.001), using a stringent non-parametric analysis. A comparison of the FCM assay with the radioactive immunobead assay previously reported on the same cohort of patients showed significant correlation (R2 = 0.71, 95% CI 0.39, 0.53). The overall performance of the FCM assay in discriminating between ITP patients and normals was estimated by the receiver operating characteristic (ROC) plot, showing an area under the curve of 0.96 (maximal value 1.0), with standard error of 0.033. We conclude that the present FCM assay is clinically useful for routine diagnosis and follow-up of ITP.

Characterization of edible oils using total luminescence spectroscopy.

Total luminescence spectroscopy was used to characterise and differentiate edible oils and additionally, to control one of the major problems in the oil quality--the effect of thermal and photo-oxidation. We studied several vegetable oils available on the Polish market, including soybean, rapeseed, corn, sunflower, linseed and olive oils. Total luminescence spectroscopy measurements were performed using two different sample geometries: front-face for pure oil samples and right-angle for transparent samples, diluted in n-hexane. All the samples studied as n-hexane solutions exhibit an intense peak, which appears at 320 nm in emission and 290 nm in excitation, attributed to tocopherols. Some of the oils exhibit a second long-wavelength peak, appearing at 670 nm in emission and 405 nm in excitation, belonging to pigments of the chlorophyll group. Additional bands were present in the intermediate range of excitation and emission wavelengths; however, the compounds responsible for this emission were not identified. The front-face spectra for pure oils included chlorophyll peaks for most samples, and some additional peaks in the intermediate range, while the tocopherol peaks were comparatively less intense. The results presented demonstrate the capability of the total luminescence techniques to characterise and differentiate vegetable oil products, and additionally, to characterize the effect of thermal and photo-oxidation on such products. In the photo-oxidation experiments, special attention was paid to possible involvement of singlet oxygen. Experiments were done to monitor the highly specific O2(1delta(g)) --> O2(3sigma(g)-) singlet oxygen emission at 1270 nm. Thus, total luminescence spectroscopy presents an interesting alternative to time-consuming and expensive techniques such as gas or liquid chromatography, mass spectrometry and other methods requiring wet chemistry steps.

A note on the genome scan meta-analysis statistic.

Wise and colleagues (Ann. Hum. Genet. (1999) 63: 263-72) introduced a rank-based statistical technique for meta-analysis of genome scans, the Genome Scan Meta-Analysis (GSMA) method. We provide an alternative derivation of the null distribution of the GSMA statistic, with extensions, and we suggest approximations to the distribution of the GSMA statistic that may be useful in applications.