Sub-diffraction Limit Localization of Proteins in Volumetric Space Using Bayesian Restoration of Fluorescence Images from Ultrathin Specimens

Photon diffraction limits the resolution of conventional light microscopy at the lateral focal plane to 0.61λ/NA (λ = wavelength of light, NA = numerical aperture of the objective) and at the axial plane to 1.4nλ/NA² (n = refractive index of the imaging medium, 1.51 for oil immersion), which with visible wavelengths and a 1.4NA oil immersion objective is ∼220 nm and ∼600 nm in the lateral plane and axial plane respectively. This volumetric resolution is too large for the proper localization of protein clustering in subcellular structures. Here we combine the newly developed proteomic imaging technique, Array Tomography (AT), with its native 50–100 nm axial resolution achieved by physical sectioning of resin embedded tissue, and a 2D maximum likelihood deconvolution method, based on Bayes'' rule, which significantly improves the resolution of protein puncta in the lateral plane to allow accurate and fast computational segmentation and analysis of labeled proteins. The physical sectioning of AT allows tissue specimens to be imaged at the physical optimum of modern high NA plan-apochormatic objectives. This translates to images that have little out of focus light, minimal aberrations and wave-front distortions. Thus, AT is able to provide images with truly invariant point spread functions (PSF), a property critical for accurate deconvolution. We show that AT with deconvolution increases the volumetric analytical fidelity of protein localization by significantly improving the modulation of high spatial frequencies up to and potentially beyond the spatial frequency cut-off of the objective. Moreover, we are able to achieve this improvement with no noticeable introduction of noise or artifacts and arrive at object segmentation and localization accuracies on par with image volumes captured using commercial implementations of super-resolution microscopes.     

Vagal Withdrawal and Susceptibility to Cardiac Arrhythmias in Rats with High Trait Aggressiveness

Personality characteristics, e.g. aggressiveness, have long been associated with an increased risk of cardiac disease. However, the underlying mechanisms remain unclear. In this study we used a rodent model for characterizing cardiac autonomic modulation in rats that differ widely in their level of aggressive behavior. To reach this goal, high-aggressive (HA, n = 10) and non-aggressive (NA, n = 10) rats were selected from a population (n = 121) of adult male Wild-type Groningen rats on the basis of their latency time to attack (ALT, s) a male intruder in a resident-intruder test lasting 600 s. In order to obtain information on their cardiac autonomic modulation, ECG recordings were subsequently obtained via radiotelemetry at rest, during stressful stimuli and under autonomic pharmacological manipulations, and analyzed by means of time- and frequency-domain indexes of heart rate variability. During resting conditions, HA rats (ALT<90 s) displayed reduced heart rate variability, mostly in terms of lower vagal modulation compared to NA rats (ALT>600 s). Exposure to stressful stimuli (i.e. restraint and psychosocial stress) provoked similar tachycardic responses between the two groups. However, under stress conditions HA rats displayed a reduced vagal antagonism and an increased incidence of tachyarrhythmias compared to NA rats. In addition, beta-adrenergic pharmacological stimulation induced a much larger incidence of ventricular tachyarrhythmias in HA rats compared to NA counterparts. These findings are consistent with the view that high levels of aggressive behavior in rats are associated to signs of cardiac autonomic impairment and increased arrhythmogenic susceptibility that may predict vulnerability to cardiac morbidity and mortality.     

Single beam optical trapping integrated in a confocal microscope for biological applications.

Confocal microscopy is very useful in biology because of its three dimensional imaging capacities and has proven to be an excellent tool to study the 3D organization of, for instance, cell structures. This property of confocal microscopy makes it also very suitable for observation during guidance of the three dimensional manipulation of single cells or cell elements. Therefore we decided to integrate a confocal microscope and a single beam optical manipulator into a single instrument. The advantage of optical manipulation over mechanical techniques is that it is non-invasive and therefore may be applied on living (micro-) organisms and cells. The creation of an effective single beam optical trap requires the use of a high numerical aperture (N.A.) objective to focus the laser beam. In this paper we briefly discuss the vertical or axial force exerted on a sphere in a single beam trap. The axial force on a sphere placed on the optical axis, caused by reflection and refraction, is calculated applying a electromagnetic vector diffraction theory to determine the field distribution in the focal region. One of the results is that the particle also experiences a vertical trapping force towards the focusing lens when it is in the strongly convergent part of the field in addition to the known negative signed trapping force in the divergent part of the field. Further we describe an instrumental approach to realize optical trapping in which the optical trap position is controlled by moving the focusing objective only.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of forces and displacements along the axial direction in an optical trap

We present measurements of the forces on, and displacements of, an optically trapped bead along the propagation direction of the trapping laser beam (the axial direction). In a typical experimental configuration, the bead is trapped in an aqueous solution using an oil-immersion, high-numerical-aperture objective. This refractive index mismatch complicates axial calibrations due to both a shift of the trap center along the axial direction and spherical aberrations. In this work, a known DNA template was unzipped along the axial direction and its characteristic unzipping force-extension data were used to determine 1), the location of the trap center along the axial direction; 2), the axial displacement of the bead from the trap center; and 3), the axial force exerted on the bead. These axial calibrations were obtained for trap center locations up to approximately 4 microm into the aqueous solution and with axial bead displacements up to approximately 600 nm from the trap center. In particular, the axial trap stiffness decreased substantially when the trap was located further into the aqueous solution. This approach, together with conventional lateral calibrations, results in a more versatile optical trapping instrument that is accurately calibrated in all three dimensions.     

Parapapillary Atrophy: Histological Gamma Zone and Delta Zone

Background

To examine histomorphometrically the parapapillary region in human eyes.

Methodology/Principal Findings

The histomorphometric study included 65 human globes (axial length:21–37 mm). On anterior-posterior histological sections, we measured the distance Bruch''s membrane end (BME)-optic nerve margin (“Gamma zone”), BME-retinal pigment epithelium (RPE) (“Beta zone”), BME-beginning of non-occluded choriocapillaris, and BME-beginning of photoreceptor layer. “Delta zone” was defined as part of gamma zone in which blood vessels of at least 50 µm diameter were not present over a length of >300 µm. Beta zone (mean length:0.35±0.52 mm) was significantly (P = 0.01) larger in the glaucoma group than in the non-glaucomatous group. It was not significantly (P = 0.28) associated with axial length. Beta zone was significantly (P = 0.004) larger than the region with occluded choriocapillaris. Gamma zone (mean length:0.63±1.25 mm) was associated with axial length (P<0.001;r² = 0.73) with an increase starting at an axial length of 26.5 mm. It was not significantly (P = 0.24) associated with glaucomatous optic neuropathy. Delta zone (present only in eyes with axial length of ≥27 mm) was associated with axial length (P = 0.001) and scleral flange length (P<0.001) but not with glaucoma (P = 0.73).

Conclusions/Significance

Parapapillary gamma zone (peripapillary sclera without overlying choroid, Bruch''s membrane and deep retinal layers) was related with axial globe elongation and was independent of glaucoma. Delta zone (no blood vessels >50 µm diameter within gamma zone) was present only in highly axially elongated globes and was not related with glaucoma. Beta zone (Bruch''s membrane without RPE) was correlated with glaucoma but not with globe elongation. Since the region with occluded choriocapillaris was smaller than beta zone, complete loss of RPE may have occurred before complete choriocapillaris closure.     

Enhancing the Strength of an Optical Trap by Truncation

Optical traps (tweezers) are beginning to be used with increasing efficacy in diverse studies in the biological and biomedical sciences. We report here results of a systematic study aimed at enhancing the efficiency with which dielectric (transparent) materials can be optically trapped. Specifically, we investigate how truncation of the incident laser beam affects the strength of an optical trap in the presence of a circular aperture. Apertures of various sizes have been used by us to alter the beam radius, thereby changing the effective numerical aperture and intensity profile. We observe significant enhancement of the radial and axial trap stiffness when an aperture is used to truncate the beam compared to when no aperture was used, keeping incident laser power constant. Enhancement in trap stiffness persists even when the beam intensity profile is modulated. The possibility of applying truncation to multiple traps is explored; to this end a wire mesh is utilized to produce multiple trapping that also alters the effective numerical aperture. The use of a mesh leads to reduction in trap stiffness compared to the case when no wire mesh is used. Our findings lead to a simple-to-implement and inexpensive method of significantly enhancing optical trapping efficiency under a wide range of circumstances.     

On Cross-Sectional Associations of Leukocyte Telomere Length with Cardiac Systolic,Diastolic and Vascular Function: The Asklepios Study

Background

Systemic telomere length has been associated with measures of diastolic function, vascular stiffness and left ventricular mass mainly in smaller, patient-specific settings and not in a general population. In this study we describe the applicability of these findings in a large, representative population.

Methods and Results

Peripheral blood leukocyte telomere length (PBL TL) was measured using telomere restriction fragment analysis in the young to middle-aged (>2500 volunteers, ∼35 to 55 years old) Asklepios study population, free from overt cardiovascular disease. Subjects underwent extensive echocardiographic, hemodynamic and biochemical phenotyping. After adjusting for relevant confounders (age, sex, systolic blood pressure, heart rate, body mass index and use of antihypertensive drugs) we found no associations between PBL TL and left ventricular mass index (P = 0.943), ejection fraction (P = 0.933), peak systolic septal annular motion (P = 0.238), pulse wave velocity (P = 0.971) or pulse pressure (P = 0.999). In contrast, our data showed positive associations between PBL TL and parameters of LV filling: the transmitral flow early (E) to late (A) velocity ratio (E/A-ratio; P<0.001), the ratio of early (e′) to late (a′) mitral annular velocities (e′/a′-ratio; P = 0.012) and isovolumic relaxation time (P = 0.015). Interestingly, these associations were stronger in women than in men and were driven by associations between PBL TL and the late diastolic components (A and a′).

Conclusions

In a generally healthy, young to middle-aged population, PBL TL is not related to LV mass or systolic function, but might be associated with an altered LV filling pattern, especially in women.     

Peripapillary Arterial Circle of Zinn-Haller: Location and Spatial Relationships with Myopia

Purpose

To measure histomorphometrically the location of the peripapillary arterial circle of Zinn-Haller (ZHAC) and assess its associations with axial length.

Methods

Using a light microscope, we measured the distance from the ZHAC to the peripapillary ring (optic disc border), the merging point of the dura mater with the posterior sclera (“dura-sclera point”), and the inner scleral surface. In the parapapillary region, we differentiated between beta zone (presence of Bruch''s membrane, absence of retinal pigment epithelium) and gamma zone (absence of Bruch''s membrane). The peripapillary scleral flange as roof of the orbital cerebrospinal fluid space was the connection between the end of the lamina cribrosa and the posterior full-thickness sclera starting at the dura-sclera point.

Results

The study included 101 human globes (101 patients) with a mean axial length of 26.7±3.7 mm (range: 20.0–39.0 mm). The distance between the ZHAC and the peripapillary ring increased significantly with longer axial length (P<0.001; correlation coefficient r = 0.49), longer parapapillary gamma zone (P<0.001;r = 0.85), longer (P<0.001;r = 0.73) and thinner (P<0.001;r = −0.45) peripapillary scleral flange, and thinner sclera posterior to the equator (P<0.001). ZHAC distance to the peripapillary ring was not significantly associated with length of parapapillary beta zone (P = 0.33). Including only non-highly myopic eyes (axial length <26.5 mm), the ZHAC distance to the disc border was not related with axial length (P = 0.84). In non-highly myopic eyes, the ZHAC was located close to the dura-sclera point. With increasing axial length and decreasing thickness of the peripapillary scleral flange, the ZHAC was located closer to the inner scleral surface.

Conclusions

The distance between the ZHAC and the optic disc border is markedly enlarged in highly myopic eyes. Since the ZHAC is the main arterial source for the lamina cribrosa blood supply, the finding may be of interest for the pathogenesis of the increased glaucoma susceptibility in highly myopic eyes.     

Subfoveal Choroidal Thickness and Glaucoma. The Beijing Eye Study 2011

Purpose

To examine subfoveal choroidal thickness (SFCT) in eyes with glaucoma, using enhanced depth imaging spectral domain optical coherence tomography.

Methods

The population-based Beijing Eye Study 2011 included 3468 individuals with a mean age of 64.6±9.8 years (range: 50–93 years). A detailed ophthalmic examination was performed including spectral-domain optical coherence tomography (SD-OCT) with enhanced depth imaging for measurement of SFCT, and assessment of fundus photographs for presence of glaucoma. In addition, the group of patients with chronic angle-closure glaucoma (ACG) from the Beijing Eye Study (n = 37) was merged with a group of patients with chronic ACG from the Tongren hospital (n = 52).

Results

Assessments of SFCT and glaucoma were available for 3232 (93.2%) subjects. After adjusting for age, axial length, gender, anterior chamber and lens thickness, SFCT was not significantly associated with presence of glaucoma (P = 0.08; regression coefficient B:−15.7). As a corollary, in logistic regression analysis with adjustment for age, axial length and intraocular pressure, presence of glaucoma was not significantly associated with SFCT (P = 0.20). If only open-angle glaucoma was considered, multivariate analysis revealed no significant association between SFCT and presence of open-angle glaucoma (P = 0.44). As a corollary, in logistic regression analysis, open-angle glaucoma was not significantly associated with SFCT (P = 0.91). In a similar manner if only ACG was taken into account, SFCT was not significantly associated with the presence of ACG (P = 0.27) in multivariate analysis. As a corollary in binary regression analysis, presence of ACG was not significantly associated with SFCT (P = 0.27).

Conclusions

In multivariate analysis with adjustment for age, axial length, gender, anterior chamber and lens thickness, neither OAG nor ACG was associated with an abnormal SFCT.     

Vitamin D Deficiency in a Multiethnic Healthy Control Cohort and Altered Immune Response in Vitamin D Deficient European-American Healthy Controls

Objective

In recent years, vitamin D has been shown to possess a wide range of immunomodulatory effects. Although there is extensive amount of research on vitamin D, we lack a comprehensive understanding of the prevalence of vitamin D deficiency or the mechanism by which vitamin D regulates the human immune system. This study examined the prevalence and correlates of vitamin D deficiency and the relationship between vitamin D and the immune system in healthy individuals.

Methods

Healthy individuals (n = 774) comprised of European-Americans (EA, n = 470), African–Americans (AA, n = 125), and Native Americans (NA, n = 179) were screened for 25-hydroxyvitamin D [25(OH)D] levels by ELISA. To identify the most noticeable effects of vitamin D on the immune system, 20 EA individuals with severely deficient (<11.3 ng/mL) and sufficient (>24.8 ng/mL) vitamin D levels were matched and selected for further analysis. Serum cytokine level measurement, immune cell phenotyping, and phosphoflow cytometry were performed.

Results

Vitamin D sufficiency was observed in 37.5% of the study cohort. By multivariate analysis, AA, NA, and females with a high body mass index (BMI, >30) demonstrate higher rates of vitamin D deficiency (p<0.05). Individuals with vitamin D deficiency had significantly higher levels of serum GM-CSF (p = 0.04), decreased circulating activated CD4⁺ (p = 0.04) and CD8⁺ T (p = 0.04) cell frequencies than individuals with sufficient vitamin D levels.

Conclusion

A large portion of healthy individuals have vitamin D deficiency. These individuals have altered T and B cell responses, indicating that the absence of sufficient vitamin D levels could result in undesirable cellular and molecular alterations ultimately contributing to immune dysregulation.     

Properties and parameters of the electron beam injected into the mirror magnetic trap of a plasma accelerator

The parameters of the injector of an axial plasma beam injected into a plasma accelerator operating on the basis of gyroresonance acceleration of electrons in the reverse magnetic field are determined. The trapping of the beam electrons into the regime of gyroresonance acceleration is numerically simulated by the particle- in-cell method. The optimal time of axial injection of the beam into a magnetic mirror trap is determined. The beam parameters satisfying the condition of efficient particle trapping into the gyromagnetic autoresonance regime are found.     

Can Camera Traps Monitor Komodo Dragons a Large Ectothermic Predator?

Camera trapping has greatly enhanced population monitoring of often cryptic and low abundance apex carnivores. Effectiveness of passive infrared camera trapping, and ultimately population monitoring, relies on temperature mediated differences between the animal and its ambient environment to ensure good camera detection. In ectothermic predators such as large varanid lizards, this criterion is presumed less certain. Here we evaluated the effectiveness of camera trapping to potentially monitor the population status of the Komodo dragon (Varanus komodoensis), an apex predator, using site occupancy approaches. We compared site-specific estimates of site occupancy and detection derived using camera traps and cage traps at 181 trapping locations established across six sites on four islands within Komodo National Park, Eastern Indonesia. Detection and site occupancy at each site were estimated using eight competing models that considered site-specific variation in occupancy (ψ)and varied detection probabilities (p) according to detection method, site and survey number using a single season site occupancy modelling approach. The most parsimonious model [ψ (site), p (site*survey); ω = 0.74] suggested that site occupancy estimates differed among sites. Detection probability varied as an interaction between site and survey number. Our results indicate that overall camera traps produced similar estimates of detection and site occupancy to cage traps, irrespective of being paired, or unpaired, with cage traps. Whilst one site showed some evidence detection was affected by trapping method detection was too low to produce an accurate occupancy estimate. Overall, as camera trapping is logistically more feasible it may provide, with further validation, an alternative method for evaluating long-term site occupancy patterns in Komodo dragons, and potentially other large reptiles, aiding conservation of this species.     

RhoA/Rho-Kinase and Nitric Oxide in Vascular Reactivity in Rats with Endotoxaemia

RhoA/Rho-kinase (RhoA/ROK) pathway promotes vasoconstriction by calcium sensitivity mechanism. LPS causes nitric oxide (NO) overproduction to induce vascular hyporeactivity. Thus, we tried to examine the role of RhoA/ROK and NO in the regulation of vascular reactivity in different time-point of endotoxaemia. Male Wistar rats were intravenously infused for 10 min with saline or E. coli endotoxin (lipopolysaccharide, LPS, 10 mg/kg) and divided to five groups (n = 8 in each group): (i) Control, sacrificed at 6 h after saline infusion; (ii) LPS1h, sacrificed at 1 h after LPS infusion; (iii) LPS2h, sacrificed at 2 h after LPS infusion; (iv) LPS4h, sacrificed at 4 h after LPS infusion; and (v) LPS6h, sacrificed at 6 h after LPS infusion. LPS1h and LPS2h were regarded as early endotoxaemia, whereas LPS4h and LPS6h were regarded as late endotoxaemia. Indeed, our results showed that LPS reproduced a biphasic hypotension and sustained vascular hyporeactivity to noradrenaline (NA) in vivo. Interestingly, this hyporeactivity did not occur in ex vivo during early endotoxaemia. This could be due to increases of aortic RhoA activity (n = 5, P<0.05) and myosin phosphatase targeting subunit 1 phosphorylation (n = 3, P<0.05). In addition, pressor response to NA and vascular reactivity in early endotoxaemia were inhibited by ROK inhibitor, Y27632. Furthermore, plasma bradykinin was increased at 10 min (24.6±13.7 ng/mL, n = 5, P<0.05) and aortic endothelial NO synthase expression was increased at 1 h (+200%. n = 3, P<0.05) after LPS. In late endotoxaemia, the vascular hyporeactivity was associated with aortic inducible NO synthase expression (n = 3, P<0.05) and an increased serum NO level (n = 8, P<0.05). Thus, an increased RhoA activity could compensate vascular hyporeactivity in early endotoxaemia, and the large NO production inhibiting RhoA activity would lead to vascular hyporeactivity eventually.     

Diffusion Tensor Metrics as Biomarkers in Alzheimer's Disease

Background

Although diffusion tensor imaging has been a major research focus for Alzheimer’s disease in recent years, it remains unclear whether it has sufficient stability to have biomarker potential. To date, frequently inconsistent results have been reported, though lack of standardisation in acquisition and analysis make such discrepancies difficult to interpret. There is also, at present, little knowledge of how the biometric properties of diffusion tensor imaging might evolve in the course of Alzheimer’s disease.

Methods

The biomarker question was addressed in this study by adopting a standardised protocol both for the whole brain (tract-based spatial statistics), and for a region of interest: the midline corpus callosum. In order to study the evolution of tensor changes, cross-sectional data from very mild (N = 21) and mild (N = 22) Alzheimer’s disease patients were examined as well as a longitudinal cohort (N = 16) that had been rescanned at 12 months.

Findings and Significance

The results revealed that increased axial and mean diffusivity are the first abnormalities to occur and that the first region to develop such significant differences was mesial parietal/splenial white matter; these metrics, however, remained relatively static with advancing disease indicating they are suitable as ‘state-specific’ markers. In contrast, increased radial diffusivity, and therefore decreased fractional anisotropy–though less detectable early–became increasingly abnormal with disease progression, and, in the splenium of the corpus callosum, correlated significantly with dementia severity; these metrics therefore appear ‘stage-specific’ and would be ideal for monitoring disease progression. In addition, the cross-sectional and longitudinal analyses showed that the progressive abnormalities in radial diffusivity and fractional anisotropy always occurred in areas that had first shown an increase in axial and mean diffusivity. Given that the former two metrics correlate with dementia severity, but the latter two did not, it would appear that increased axial diffusivity represents an upstream event that precedes neuronal loss.     

Regional Differences in Dynamic Cerebral Autoregulation in the Healthy Brain Assessed by Magnetic Resonance Imaging

A novel method is described for mapping dynamic cerebral blood flow autoregulation to assess autoregulatory efficiency throughout the brain, using magnetic resonance imaging (MRI). Global abnormalities in autoregulation occur in clinical conditions, including stroke and head injury, and are of prognostic significance. However, there is limited information about regional variations. A gradient-echo echo-planar pulse sequence was used to scan the brains of healthy subjects at a rate of 1 scan/second during a transient decrease in arterial blood pressure provoked by a sudden release of pressure in bilateral inflated thigh cuffs. The signal decrease and subsequent recovery were analyzed to provide an index of autoregulatory efficiency (MRARI). MRI time-series were successfully acquired and analyzed in eleven subjects. Autoregulatory efficiency was not uniform throughout the brain: white matter exhibited faster recovery than gray (MRARI = 0.702 vs. 0.672, p = 0.009) and the cerebral cortex exhibited faster recovery than the cerebellum (MRARI = 0.669 vs. 0.645, p = 0.016). However, there was no evidence for differences between different cortical regions. Differences in autoregulatory efficiency between white matter, gray matter and the cerebellum may be a result of differences in vessel density and vasodilation. The techniques described may have practical importance in detecting regional changes in autoregulation consequent to disease.     

Ten-Year Cumulative Incidence of Diabetic Retinopathy. The Beijing Eye Study 2001/2011

Objective

To assess the cumulative 10-year incidence of diabetic retinopathy (DR) and its associated factors in a population living in Greater Beijing.

Methods

The population-based longitudinal Beijing Eye Study, which included 4439 subjects (age in 2001: 40+years) in 2001, was repeated in 2011 with 2695 subjects participating (66.4% of the survivors). The study participants underwent a detailed ophthalmic examination. Fundus photographs were examined for the new development of DR.

Results

After excluding individuals with DR at baseline (n = 87) or no sufficient fundus photographs in 2011 (n = 6), the study included 2602 subjects with a mean age of 64.6±9.7 years (median: 64.0 years; range: 50 to 93 years). In the 10-year period, 109 subjects (39 men) developed new DR with an incidence of 4.2% (95% confidence interval (CI): 3.45,5.03). In multiple logistic regression analysis, incident DR was significantly associated with higher HbA1c value (P<0.001; Odds Ratio (OR): 1.73; 95% Confidence Interval (CI): 1.35,2.21), longer duration of diabetes mellitus (P<0.001; OR: 1.16; 95% CI: 1.10,1.22), higher serum concentration of creatinine (P = 0.02; OR: 1.01; 95% CI: 1.002,1.022), lower educational level (P = 0.049; OR: 0.74; 95% CI: 0.55,0.99), higher estimated cerebrospinal fluid pressure (P = 0.038; OR: 1.10; 95% CI: 1.01,1.22), and shorter axial length (P<0.001; OR: 0.48; 95% CI: 0.33,0.71).

Conclusions

The cumulative 10-year incidence (mean: 4.2%) of DR in a North Chinese population was significantly associated with a higher HbA1c value, longer known duration of diabetes mellitus, higher estimated CSFP and shorter axial length (P<0.001). Shorter axial length (or hyperopia) and, potentially, higher CSFP may be additional risk factors to be taken into account when counseling and treating patients with diabetes mellitus.     

Overexpression of JAM-A in Non-Small Cell Lung Cancer Correlates with Tumor Progression

The objective of the current study was to determine the clinical significance of junctional adhesion molecule A (JAM-A) in patients with non-small cell lung cancer (NSCLC) and the biological function of JAM-A in NSCLC cell lines. We showed that JAM-A is predominantly expressed in cell membranes and high expression of JAM-A occurred in 37% of lung tumor specimens compared to corresponding normal tissues. High expression of JAM-A was significantly correlated with TNM stage (P = 0.021), lymph node metastasis (P = 0.007), and decreased overall survival (P = 0.02), In addition, we observed that silencing JAM-A by small interfering RNA inhibited tumor cell proliferation and induced cell cycle arrest at the G1/S boundary. Western blotting analysis revealed that knockdown of JAM-A decreased the protein levels of cyclin D1, CDK4, 6, and P-Rb. Thus, JAM-A plays an important role in NSCLC progression.     

Relationship of Circulating Soluble Urokinase Plasminogen Activator Receptor (suPAR) Levels to Disease Control in Asthma and Asthmatic Pregnancy

Asthma has a high burden of morbidity if not controlled and may frequently complicate pregnancy, posing a risk for pregnancy outcomes. Elevated plasma level of the inflammatory biomarker soluble urokinase plasminogen activator receptor (suPAR) is related to a worse prognosis in many conditions such as infectious, autoimmune, or pregnancy-related diseases; however the value of suPAR in asthma and asthmatic pregnancy is unknown. The present study aimed to investigate the suPAR, CRP and IL-6 levels in asthma (asthmatic non-pregnant, ANP; N = 38; female N = 27) and asthmatic pregnancy (AP; N = 15), compared to healthy non-pregnant controls (HNP; N = 29; female N = 19) and to healthy pregnant women (HP; N = 58). The relationship between suPAR levels and asthma control was also evaluated. The diagnostic efficacy of suPAR in asthma control was analyzed using ROC analysis. IL-6 and CRP levels were comparable in all study groups. Circulating suPAR levels were lower in HP and AP than in HNP and ANP subjects, respectively (2.01 [1.81–2.38] and 2.39 [2.07–2.69] vs. 2.60 [1.82–3.49] and 2.84 [2.33–3.72] ng/mL, respectively, p = 0.0001). suPAR and airway resistance correlated in ANP (r = 0.47, p = 0.004). ROC analysis of suPAR values in ANP patients with PEF above and below 80% yielded an AUC of 0.75 (95% CI: 0.57–0.92, p = 0.023) and with ACT total score above and below 20 an AUC of 0.80 (95% CI: 0.64–0.95, p = 0.006). The cut-off value of suPAR to discriminate between controlled and not controlled AP and ANP was 4.04 ng/mL. In conclusion, suPAR may help the objective assessment of asthma control, since it correlates with airway resistance and has good sensitivity in the detection of impaired asthma control. Decrease in circulating suPAR levels detected both in healthy and asthmatic pregnant women presumably represents pregnancy induced immune tolerance.     

The Value of Source Data Verification in a Cancer Clinical Trial

Background

Source data verification (SDV) is a resource intensive method of quality assurance frequently used in clinical trials. There is no empirical evidence to suggest that SDV would impact on comparative treatment effect results from a clinical trial.

Methods

Data discrepancies and comparative treatment effects obtained following 100% SDV were compared to those based on data without SDV. Overall survival (OS) and Progression-free survival (PFS) were compared using Kaplan-Meier curves, log-rank tests and Cox models. Tumour response classifications and comparative treatment Odds Ratios (ORs) for the outcome objective response rate, and number of Serious Adverse Events (SAEs) were compared. OS estimates based on SDV data were compared against estimates obtained from centrally monitored data.

Findings

Data discrepancies were identified between different monitoring procedures for the majority of variables examined, with some variation in discrepancy rates. There were no systematic patterns to discrepancies and their impact was negligible on OS, the primary outcome of the trial (HR (95% CI): 1.18(0.99 to 1.41), p = 0.064 with 100% SDV; 1.18(0.99 to 1.42), p = 0.068 without SDV; 1.18(0.99 to 1.40), p = 0.073 with central monitoring). Results were similar for PFS. More extreme discrepancies were found for the subjective outcome overall objective response (OR (95% CI): 1.67(1.04 to 2.68), p = 0.03 with 100% SDV; 2.45(1.49 to 4.04), p = 0.0003 without any SDV) which was mostly due to differing CT scans.

Interpretation

Quality assurance methods used in clinical trials should be informed by empirical evidence. In this empirical comparison, SDV was expensive and identified random errors that made little impact on results and clinical conclusions of the trial. Central monitoring using an external data source was a more efficient approach for the primary outcome of OS. For the subjective outcome objective response, an independent blinded review committee and tracking system to monitor missing scan data could be more efficient than SDV.     

Can Experience Improve Hospital Management?

Background

Experience curve effects were first observed in the industrial arena as demonstrations of the relationship between experience and efficiency. These relationships were largely determined by improvements in management efficiency and quality of care. In the health care industry, volume-outcome relationships have been established with respect to quality of care improvement, but little is known about the effects of experience on management efficiency. Here, we examine the relationship between experience and hospital management in Japanese hospitals.

Methods

The study sample comprised individuals who had undergone surgery for unruptured abdominal aortic aneurysms and had been discharged from participant hospitals between April 1, 2006 and December 31, 2008. We analyzed the association between case volume (both at the hospital and surgeon level) and postoperative complications using multilevel logistic regression analysis. Multilevel log-linear regression analyses were performed to investigate the associations between case volume and length of stay (LOS) before and after surgery.

Results

We analyzed 909 patients and 849 patients using the hospital-level and surgeon-level analytical models, respectively. The odds ratio of postoperative complication occurrence for an increase of one surgery annually was 0.981 (P<0.001) at the hospital level and 0.982 (P<0.001) at the surgeon level. The log-linear regression analyses showed that shorter postoperative LOS was significantly associated with high hospital-level case volume (coefficient for an increase of one surgery: −0.006, P = 0.009) and surgeon-level case volume (coefficient for an increase of one surgery: −0.011, P = 0.022). Although an increase of one surgery annually at the hospital level was statistically associated with a reduction of preoperative LOS by 1.1% (P = 0.006), there was no significant association detected between surgeon-level case volume and preoperative LOS (P = 0.504).

Conclusion

Experience at the hospital level may contribute to the improvement of hospital management efficiency.