Role of EP3 and EP4 prostaglandin receptors in reorganization of the cytoskeleton in mature human osteoclasts

OBJECTIVE: Osteoclasts are central to the pathophysiology of several bone diseases. Prostaglandin E2 (PGE2) is well known to influence osteoclasts indirectly, but its direct action on osteoclasts is still controversial and the relevant receptors are unknown. We investigated the distribution and function of EP receptors in human mature osteoclasts. METHODS: Osteoclasts were extracted from femurs and tibias of human fetuses obtained from legal abortions. In situ hybridization and immunohistochemistry were used to detect the presence of EP1, EP2, EP3, and EP4 receptors on these cells. Actin staining and fluorescent microscopy were used to detect the effects of receptor activation on the cytoskeleton. RESULTS: Only EP3 and EP4 receptors were detected at the RNA and protein level in osteoclasts. These receptors were functional: PGE2 decreased the number of osteoclasts presenting an actin ring; 11-deoxy-PGE1, an EP2 and EP4 agonist, also decreased the number of tartrate-resistant acid phosphatase-positive cells with an actin ring; sulprostone, an EP3-specific agonist, had no effect on this variable but increased the number of cells with lamellipodia. CONCLUSION: Mature human osteoclasts present 2 subtypes of EP receptors, namely EP3 and EP4, that mediate different actions of PGE2 on these cells: activation of the EP4 receptors inhibits actin ring formation and activation of the EP3 receptors increases the number of lamellipodia. Activation or inhibition of these receptors by specific agents could be used to study and influence osteoclast function.     

Gentamicin affects melanogenesis in normal human melanocytes

Background: Aminoglycoside antibiotics, including gentamicin, despite their ability to induce adverse effects on pigmented tissues, remain valuable and sometimes indispensable for the treatment of various infections. It is known that gentamicin binds to melanin biopolymers, but the relation between this drug affinity to melanin and its toxicity is not well documented. The aim of this work was to examine the impact of gentamicin on viability and melanogenesis in HEMa-LP (light pigmented) and HEMn-DP (dark pigmented) normal human melanocytes.

Methodology/principal findings: The effect of gentamicin on cell viability was determined by 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) assay; melanin content and tyrosinase activity were measured spectrophotometrically. It has been demonstrated that gentamicin induces concentration-dependent loss in melanocytes viability. The application of antibiotic in concentration of 10?mM causes higher reduction in viability of the light pigmented melanocytes (by about 74%) when compared with the dark pigmented ones (by about 62%). The value of the concentration of a drug that produces loss in cell viability by 50% (EC₅₀) for both cell lines was found to be ～7.5?mM. It has been shown that gentamicin causes inhibition of tyrosinase activity and reduces melanin content in light pigmented melanocytes significantly more than in the dark pigmented cells.

Conclusion/significance: We have found that gentamicin modulates melanization process in melanocytes in vitro, what may explain the potential role of melanin biopolymer in the mechanisms of undesirable toxic effects of this drug in vivo, as a result of its accumulation in pigmented tissues. We have also stated that the melanogenesis process in light pigmented melanocytes is more sensitive to the inhibitory effect of gentamicin than in the dark pigmented cells.     

Frequency of HIV-1 dual subtype infections, including intersubtype superinfections, among injection drug users in Bangkok, Thailand

OBJECTIVES: To estimate the frequency and incidence of dual HIV-1 subtype infections, including superinfections, among recent seroconvertors from a cohort of injection drug users (IDUs). METHODS: A total of 1209 HIV-negative IDUs were followed in a prospective cohort study at 15 methadone clinics in Bangkok, Thailand. After 2308 person-years (PY) of follow-up, 133 seroconverted to HIV-1, of which approximately 20% were subtype B and 80% were CRF01_AE (formerly called subtype E). Specimens from 126 individuals were available at time of first seropositive test and specimens from 80 of these 126 individuals were also available more than 12 months later. For each infected participant, we calculated the amount of time to superinfection, loss to follow-up, or to the closest visit more than 12 months after the time of initial seropositivity. RESULTS: Of all 126 seroconverters seen at the time of the first seropositive test result, there was no apparent case of concurrent dual subtype infection detected despite 2301 PY of observation. Overall, the incidence of superinfection was 2.2 per 100 PY [95% confidence interval (CI), 0.3-7.8]. The 1-year incidence of CRF01_AE superinfection following subtype B primary infection was 3.9 per 100 PY (95% CI, 0.1-21.9) and the incidence of subtype B superinfection following CRF01_AE primary infection was 1.5 per 100 PY (95% CI, 0.04-8.3). CONCLUSIONS: Determination of the frequency and incidence of dual HIV-1 subtype infection demonstrates that HIV-1 superinfection is not uncommon in a population with high HIV-1 incidence with more than one circulating strain.     

Catheter Ablation of Inducible Atrial Flutter, in Combination with Atrial Pacing and Antiarrhythmic Drugs (“Hybrid Therapy”) Improves Rhythm Control in Patients with Refractory Atrial Fibrillation

Atrial flutter or tachycardia may coexist with atrial fibrillation [AF] and can be treated with ablation techniques in attempt to reduce the total AF burden. The role of ablation of latent atrial tachyarrhythmias elicited at electrophysiologic study in conjunction with atrial pacing and antiarrhythmic drugs in patients with refractory AF has not been evaluated. We evaluated the efficacy of catheter ablation of electrically induced atrial flutter or atrial tachycardia in improving rhythm control in patients with refractory AF. Methods: Consecutive patients with refractory AF, and spontaneous atrial flutter (Group 1) or without spontaneous atrial flutter (Group 2) underwent programmed stimulation in a baseline drug-free state. All patients had electrically induced atrial flutter or tachycardia. Radiofrequency ablation of the arrhythmia substrate was performed in all patients. Primary endpoints evaluated for patient outcome in both groups included maintenance of rhythm control and freedom from recurrent atrial tachyarrhythmias. Results: Forty-three patients, with a mean age of 66±13 years were studied. Group 1 consisted of 22 patients while Group 2 had 21 patients. Ablation of the tricuspid valve-inferior venacaval isthmus was performed in 41 patients who had common atrial flutter induced at electrophysiologic study. Ablation of other atrial sites was performed in 8 patients with induced atypical flutter and 4 patients with induced atrial tachycardia. Ten of these patients had ablation of more than one arrhythmia. 17 patients (40%) had atrial pacing instituted and 28 patients remained on a class 1/3 antiarrhythmic drug. During a mean follow-up of 26±14 months, 33 patients (82.5%) remained in rhythm control. Actuarial analysis showed 96% of patients in rhythm control at 6 months, 94% at 12 months, and 90% at 24 months. Freedom from symptomatic AF recurrence was 64% at 6 months, 58% at 12 months, and 42% at 24 months. The outcome for both of these endpoints was similar for Group 1 and Group 2 (p = NS). The AF free interval increased significantly from 7±9 days to 172±121 days (p < 0.01) after ablation. This increase was again similar in both the groups. In the 14 patients were who did not receive atrial pacing and who remained on the same class 1/3 antiarrhythmic drug, the AF free interval increased from 18±17 days to 212±102 days (p < 0.01). Conclusions: We conclude that electrophysiologic studies can elicit latent atrial flutter or tachycardia in patients with refractory AF without spontaneous monomorphic atrial tachyarrhythmias. Catheter ablation of electrically induced atrial flutter or tachycardia either alone, or with atrial pacing and with antiarrhythmic drug may improve rhythm control and reduce AF recurrences. This is similar in patients with and without spontaneous atrial flutter and refractory AF.     

Variance stabilization for computing and comparing grand mean waveforms in MEG and EEG

Grand means of time‐varying signals (waveforms) across subjects in magnetoencephalography (MEG) and electroencephalography (EEG) are commonly computed as arithmetic averages and compared between conditions, for example, by subtraction. However, the prerequisite for these operations, homogeneity of the variance of the waveforms in time, and for most common parametric statistical tests also between conditions, is rarely met. We suggest that the heteroscedasticity observed instead results because waveforms may differ by factors and additive terms and follow a mixed model. We propose to apply the asinh‐transformation to stabilize the variance in such cases. We demonstrate the homogeneous variance and the normal distributions of data achieved by this transformation using simulated waveforms, and we apply it to real MEG data and show its benefits. The asinh‐transformation is thus an essential and useful processing step prior to computing and comparing grand mean waveforms in MEG and EEG.     

Large-scale characterization of the microvascular geometry in development and disease by tissue clearing and quantitative ultramicroscopy

Three-dimensional assessment of optically cleared, entire organs and organisms has recently become possible by tissue clearing and selective plane illumination microscopy (“ultramicroscopy”). Resulting datasets can be highly complex, encompass over a thousand images with millions of objects and data of several gigabytes per acquisition. This constitutes a major challenge for quantitative analysis. We have developed post-processing tools to quantify millions of microvessels and their distribution in three-dimensional datasets from ultramicroscopy and demonstrate the capabilities of our pipeline within entire mouse brains and embryos. Using our developed acquisition, segmentation, and analysis platform, we quantify physiological vascular networks in development and the healthy brain. We compare various geometric vessel parameters (e.g. vessel density, radius, tortuosity) in the embryonic spinal cord and brain as well as in different brain regions (basal ganglia, corpus callosum, cortex). White matter tract structures (corpus callosum, spinal cord) showed lower microvascular branch densities and longer vessel branch length compared to grey matter (cortex, basal ganglia). Furthermore, we assess tumor neoangiogenesis in a mouse glioma model to compare tumor core and tumor border. The developed methodology allows rapid quantification of three-dimensional datasets by semi-automated segmentation of fluorescently labeled objects with conventional computer hardware. Our approach can aid preclinical investigations and paves the way towards “quantitative ultramicroscopy”.     

Risks of adverse events in patients with orthostatic intolerance undergoing surgery with general anesthesia

Clinical Autonomic Research - Orthostatic intolerance (OI) is a group of disorders characterized by symptoms that occur upon standing and resolve with recumbence. Although well established but not...     

Time efficiency of direct anterior hip arthroplasty compared to postero-lateral approach in elderly patients

IntroductionPrimary arthroplasty of the hip joint is currently one of the most commonly performed procedures in orthopedics. In Poland we are observing significant changes in the age structure. With the prolonged life more and more elderly patients require musculoskeletal surgery to maintain comfortable and painless mobility. Reducing the duration of the procedure reduces the costs of anesthesiology, surgical and instrument teams, as well as the operating room technical team. The aim of the study was to compare the time required to perform hip joint arthroplasty by the direct anterior approach (DAA) with the postero-lateral approach (PLA) in our hospital.Material and methodsA retrospective analysis of 559 total and bipolar cemented and cementless hip replacement procedures based on two operative approaches – the minimally invasive DAA over the course of 2 years, and the standard PLA over the course of 3 years – was performed.ResultsStatistically significant differences were observed between the approaches used for cementless total arthroplasty with regard to the mean treatment times: 51.9 min for the 272 DAA cases, and 78.3 min for the 190 PLA cases (p < 0.0001). For the cementless hemi-arthroplasty procedure, the mean treatment times were 46.9 min in 36 patients for DAA, and 48.2 min for 61 patients for PLA (p = 0.57).ConclusionsMinimally invasive DAA significantly shortens the time of the procedure in elderly patients compared to PLA. Further study is needed to analyze other aspects of those two approaches.     

A novel customizable modular bioreactor system for whole-heart cultivation under controlled 3D biomechanical stimulation

In the last decade, cardiovascular tissue engineering has made great progress developing new strategies for regenerative medicine applications. However, while tissue engineered heart valves are already entering the clinical routine, tissue engineered myocardial substitutes are still restrained to experimental approaches. In contrast to the heart valves, tissue engineered myocardium cannot be repopulated in vivo because of its biological complexity, requiring elaborate cultivation conditions ex vivo. Although new promising approaches—like the whole-heart decellularization concept—have entered the myocardial tissue engineering field, bioreactor technology needed for the generation of functional myocardial tissue still lags behind in the sense of user-friendly, flexible and low cost systems. Here, we present a novel customizable modular bioreactor system that can be used for whole-heart cultivation. Out of a commercially obtainable original equipment manufacturer platform we constructed a modular bioreactor system specifically aimed at the cultivation of decellularized whole-hearts through perfusion and controlled 3D biomechanical stimulation with a simple but highly flexible operation platform based on LabVIEW^®. The modular setup not only allows a wide range of variance regarding medium conditioning under controlled 3D myocardial stretching but can also easily be upgraded for e.g. electrophysiological monitoring or stimulation, allowing for a tailor-made low-cost myocardial bioreactor system.