Aggregation of recount3 RNA-seq data improves inference of consensus and tissue-specific gene co-expression networks.

Background: Gene co-expression networks (GCNs) describe relationships among expressed genes key to maintaining cellular identity and homeostasis. However, the small sample size of typical RNA-seq experiments which is several orders of magnitude fewer than the number of genes is too low to infer GCNs reliably. recount3, a publicly available dataset comprised of 316,443 uniformly processed human RNA-seq samples, provides an opportunity to improve power for accurate network reconstruction and obtain biological insight from the resulting networks. Results: We compared alternate aggregation strategies to identify an optimal workflow for GCN inference by data aggregation and inferred three consensus networks: a universal network, a non-cancer network, and a cancer network in addition to 27 tissue context-specific networks. Central network genes from our consensus networks were enriched for evolutionarily constrained genes and ubiquitous biological pathways, whereas central context-specific network genes included tissue-specific transcription factors and factorization based on the hubs led to clustering of related tissue contexts. We discovered that annotations corresponding to context-specific networks inferred from aggregated data were enriched for trait heritability beyond known functional genomic annotations and were significantly more enriched when we aggregated over a larger number of samples. Conclusion: This study outlines best practices for network GCN inference and evaluation by data aggregation. We recommend estimating and regressing confounders in each data set before aggregation and prioritizing large sample size studies for GCN reconstruction. Increased statistical power in inferring context-specific networks enabled the derivation of variant annotations that were enriched for concordant trait heritability independent of functional genomic annotations that are context-agnostic. While we observed strictly increasing held-out log-likelihood with data aggregation, we noted diminishing marginal improvements. Future directions aimed at alternate methods for estimating confounders and integrating orthogonal information from modalities such as Hi-C and ChIP-seq can further improve GCN inference.

Complications and pitfalls of endovenous laser therapy for varicose veins of lower extremities.

Varicose veins of lower extremities are a common disorder affecting a significant proportion of adult population. Endovenous techniques have been used in clinical practice for more than 20 years for their radical treatment; of these, laser therapy is one of the most widely utilized options. However, this treatment modality, too, has its specific side effects and complications which can be divided into perioperative (difficult venous access, fiber or sheath damage, vasovagal reaction with a drop in blood pressure and bradycardia, cutaneous thermal injury), and postoperative (bruising and hematomas, exceptionally pain, superficial venous thrombosis (phlebitis) and fibrous cord formation or pigmentations). Paresthesias, infections, arteriovenous fistulas, deep venous thrombosis and pulmonary embolism are very rare. The introduction of new generators with higher wavelengths and fibers with radial emission of laser energy has substantially reduced the side effects and complications. Performing the surgery in the outpatient setting using tumescent local anesthesia and under perioperative ultrasound control is an essential condition of the procedure.

Lightning rod resonance of a plasmonic near-field transducer.

We demonstrate the lightning-rod resonance of a lollipop near-field transducer integrated in magnetic writer for heat-assisted magnetic recording by collecting the two-photon excited photoluminescence (TPL) signal when excited by a pulsed femto-second fiber laser tuned to the desired mode resonance. The lollipop transducer consists of a round disk and a protruding peg to take advantage of the lightning-rod effect. It is found that the TPL signal is extremely sensitive to the peg length where even a 3-5 nm deviation from the optimal peg length halves the TPL signal. This method conveniently quantifies the optical performance of an NFT device in situ as a function of geometry with a resolution of better than the light wavelength (λ) divided by 200.

Protease-modulating polyacrylate-based hydrogel stimulates wound bed preparation in venous leg ulcers--a randomized controlled trial.

BACKGROUND: Stringent control of proteolytic activity represents a major therapeutic approach for wound-bed preparation. OBJECTIVES: We tested whether a protease-modulating polyacrylate- (PA-) containing hydrogel resulted in a more efficient wound-bed preparation of venous leg ulcers when compared to an amorphous hydrogel without known protease-modulating properties. METHODS: Patients were randomized to the polyacrylate-based hydrogel (n = 34) or to an amorphous hydrogel (n = 41). Wound beds were evaluated by three blinded experts using photographs taken on days 0, 7 and 14. RESULTS: After 14 days of treatment there was an absolute decrease in fibrin and necrotic tissue of 37.6 ± 29.9 percentage points in the PA-based hydrogel group and by 16.8 ± 23.0 percentage points in the amorphous hydrogel group. The absolute increase in the proportion of ulcer area covered by granulation tissue was 36.0 ± 27.4 percentage points in the PA-based hydrogel group and 14.5 ± 22.0 percentage points in the control group. The differences between the groups were significant (decrease in fibrin and necrotic tissue P = 0.004 and increase in granulation tissue P = 0.0005, respectively). CONCLUSION: In particular, long-standing wounds profited from the treatment with the PA-based hydrogel. These data suggest that PA-based hydrogel dressings can stimulate normalization of the wound environment, particularly in hard-to-heal ulcers.

Application of plasmonic bowtie nanoantenna arrays for optical trapping, stacking, and sorting.

We present the use of Au bowtie nanoantenna arrays (BNAs) for highly efficient, multipurpose particle manipulation with unprecedented low input power and low-numerical aperture (NA) focusing. Optical trapping efficiencies measured are up to 20× the efficiencies of conventional high-NA optical traps and are among the highest reported to date. Empirically obtained plasmonic optical trapping "phase diagrams" are introduced to detail the trapping response of the BNAs as a function of input power, wavelength, polarization, particle diameter, and BNA array spacing (number density). Using these diagrams, parameters are chosen, employing strictly the degrees-of-freedom of the input light, to engineer specific trapping tasks including (1) dexterous, single-particle trapping and manipulation, (2) trapping and manipulation of two- and three-dimensional particle clusters, and (3) particle sorting. The use of low input power densities (power and NA) suggests that this bowtie nanoantenna trapping system will be particularly attractive for lab-on-a-chip technology or biological applications aimed at reducing specimen photodamage.

Nonlinear optical response from arrays of Au bowtie nanoantennas.

We demonstrate that the optical response of a single Au bowtie nanoantenna can be favorably modified to increase the local intensity by a factor of 10(3) in the feed gap region when a periodic array of antennas are used. We find that the array periodicity can be used to modulate and shape the spectral emission. An analysis of the emission confirms the presence of second-harmonic generation and two-photon photoluminescence, typical of gold nanostructures, but also reveals a portion of the emitted spectrum that cannot be attributed to a single multiphoton process. Our investigations have important implications for understanding the role of resonant nanostructures in designing optical antennas for next-generation photonic technologies.

[A six-year experience with endovenous laser in the treatment of lower extremity varices]. / Sest let zkuseností s endovenózním laserem v terapii varixu dolních kon etin.

AIMS: Endovascular techniques of saphenous ablation are the miniinvasive alternatives of the radical surgical treatment. This study summarizes our own clinical experience with endovenous laser. MATERIAL AND METHODS: During 6 year period we performed in total 723 endovenous laser procedures of trunk varicose veins of lower extremities in 630 patients. Every patient was preoperatively examined clinically and with color duplex machine. Primary varicose veins were operated on in 664 cases, in 59 cases the procedure was performed in recurrent varicose veins with reflux in the residual saphenous trunk. Post-operative follow-up (clinical and duplex ultrasound) was performed after 5 days and 1 month, 6 months and yearly thereafter. The results were evaluated by comparison of CEAP clinical class and quality of life (QoL) pre- and post-operatively, by the percentage of recanalizations and also using Kaplan-Meier life-table method. RESULTS: No thrombosis, nor pulmonary embolism were diagnosed in the post-operative period; from the whole cohort of 723 laser procedures, the postoperative data were available during different time periods in 718 cases (99.3%). Saphenous occlusion was verified in 97.3% after 1 month, non-occlusion or early reopening was seen in 2.7%. In total, 44 non-occluded trunk veins were found during the whole follow-up period (1-72 months, mean 15 months) which represents the final occlusion result of 93.9%. With stronger Kaplan-Meier analysis, we reached 88.9% occlusion rate during the follow-up period of 6 years. Cox regression analysis of factors influencing non-occlusion and early or late recanalisation of saphenous vein found 2 factors with statistical importance: energy per centimeter of vein length (p = 0.04) and laser power (p = 0.04). Cumulative rate of occlusions in 72 months horizon is significantly higher (94%) in patients treated with more than 50 J/cm compared to less than 50 J/cm (87%), log-rank test 0.039. When comparing the influence of laser power on the quality of saphenous occlusion, the treshold of 13W was set arbitrary based on median values in occluded and non-occluded cohorts and using the Kaplan-Meier survival method, the results of the treatment with power less than 13W and more than 13W were analysed. Using the power values less than 13W, the results were significantly better (p = 0.031) compared to power values of 13 W or more. Mean clinical CEAP classification improved from 2.22 (before operation) to 0.24 (1 month after) and 0.48 (last visit) and also QoL was significantly better in laser group compared to traditional surgery group (p < 0.001). The sick leave was also significantly shorter in the laser group ( median 0 days) compared to traditional group (median 40 days), p < 0.01. CONCLUSIONS: The results of endovenous laser ablation of varicose veins depend mainly on meticulous pre-operative ultrasound examination and sufficient technical parameters of the therapy. The present study supports the concept of "slow heating" during the endovenous laser treatment of varicose veins to achieve sufficient energy per centimetre of the vein and the optimal clinical outcome.

T cell receptor microcluster transport through molecular mazes reveals mechanism of translocation.

Recognition of peptide antigen by T cells involves coordinated movement of T cell receptors (TCRs) along with other costimulatory and signaling molecules. The spatially organized configurations that result are collectively referred to as the immunological synapse. Experimental investigation of the role of spatial organization in TCR signaling has been facilitated by the use of nanopatterned-supported membranes to direct TCR into alternative patterns. Here we study the mechanism by which substrate structures redirect TCR transport. Using a flow-tracking algorithm, the ensemble of TCR clusters within each cell was tracked during synapse formation under various constraint geometries. Shortly after initial cluster formation, a coordinated centripetal flow of approximately 20 nm/s develops. Clusters that encounter substrate-imposed constraint are deflected and move parallel to the constraint at speeds that scale with the relative angle of motion to the preferred centripetal direction. TCR transport is driven by actin polymerization, and the distribution of F-actin was imaged at various time points during the synapse formation process. At early time points, there is no significant effect on actin distribution produced by substrate constraints. At later time points, modest differences were observed. These data are consistent with a frictional model of TCR coupling to cytoskeletal flow, which allows slip. Implications of this model regarding spatial sorting of cell-surface molecules are discussed.

Altered TCR signaling from geometrically repatterned immunological synapses.

The immunological synapse is a specialized cell-cell junction that is defined by large-scale spatial patterns of receptors and signaling molecules yet remains largely enigmatic in terms of formation and function. We used supported bilayer membranes and nanometer-scale structures fabricated onto the underlying substrate to impose geometric constraints on immunological synapse formation. Analysis of the resulting alternatively patterned synapses revealed a causal relation between the radial position of T cell receptors (TCRs) and signaling activity, with prolonged signaling from TCR microclusters that had been mechanically trapped in the peripheral regions of the synapse. These results are consistent with a model of the synapse in which spatial translocation of TCRs represents a direct mechanism of signal regulation.

[Diclofenac inhibits proliferation and matrix formation of osteoblast cells]. / Diclofenac hemmt die Proliferation und Matrixbildung osteoblastärer Zellen.

Cell culture studies have shown that NSAID may influence osteogenic activities of osteoblast cultures. However, these studies did not consider long-term effects on differentiating cells. The influence of Voltaren with the non-steroidal agent diclofenac on proliferation and gene expression of the osteoblast-like cell line SaOS-2 was investigated 2, 9, and 16 days after incubation. Two days after 24 h of incubation, 50 microg/ml diclofenac reduced the proliferation and collagen type I expression while 9 and 16 days later no effect was found on either of the parameters. In contrast, 50 microg/ml NSAID has no effect on alkaline phosphatase expression 2 days after incubation while 9 and 16 days later expression had been reduced. Lower concentrations (1.56 and 0.19 microg/ml) had no effects on the studied parameters. BrdU and MTT test showed that 50 microg/ml diclofenac reduced proliferative and metabolic activity. Lower concentrations (< or =25 microg/ml) had a lower or no influence. The findings indicate that the NSAID impairment depends on cellular differentiation stage and is not confined to the time during or immediately after NSAID incubation. According to these results in vitro testing of drugs should be performed over a longer time period to detect possible long-term impacts.

Tissue engineering of bone: effects of mechanical strain on osteoblastic cells in type I collagen matrices.

The aim of the present study was to investigate the effect of cyclic uniaxial mechanical strain on a human osteoblastic precursor cell line (hFOB 1.19) in three-dimensional type I collagen matrices. Cell seeded collagen constructs were mechanically stretched by a daily application of cyclic uniaxial strain using a special motor-driven apparatus and compared to unstretched controls. Expression of genes involved in cell proliferation and osteoblastic differentiation as well as matrix production were investigated by analyzing the mRNA of histone H4, core binding factor 1, alkaline phosphatase, osteopontin, osteocalcin, and collagen type I (Col I) up to a cultivation period of 3 weeks using real-time PCR. Cyclic stretching of cell seeded Col I matrices at a magnitude occurring in healing bone increased cell proliferation and slightly elevated the expression of nearly all investigated genes over unstrained controls at various time points. It was concluded that mechanical load promotes the proliferation and differentiation of osteoblastic precursor cells in a Col I matrix and that the application of mechanical stimuli may have a beneficial effect on in vitro tissue formation.

[Effects of mechanical strain on human osteoblastic precursor cells in type I collagen matrices]. / Effekte mechanischer Reize auf humane osteoblastäre Zellen in einer dreidimensionalen Kollagen-Typ-I-Matrix.

BACKGROUND: The aim of the present study was to investigate the effect of mechanical strain on human osteoblastic precursor cells in a three-dimensional scaffold. METHODS: Osteoblastic precursor cells were seeded in a collagen type I gel and mechanically stretched by daily application of cyclic uniaxial strain. The expression of histone H4, core binding factor 1, alkaline phosphatase, osteopontin, osteocalcin, and collagen type I was investigated by analysing the mRNA. Cell and matrix orientation were investigated by scanning electron microscopy. RESULTS: Cyclic stretching increased cell proliferation. The expression of osteogenic markers was slightly increased by mechanical strain. The cells and matrix were strictly oriented in the stress direction. CONCLUSION: The application of mechanical load might have a beneficial effect on the quality and quantity of generated bone tissue and might be a important factor in tissue engineering of bone.

In vitro biocompatibility of resorbable experimental glass ceramics for bone substitutes.

Tricalcium phosphate ceramics (TCPs) are increasingly used as bone substitutes. They demonstrate good biocompatibility and degrade relatively slowly. New glass ceramics based on calcium alkali orthophosphates (Ca(2)KNa(PO(4))(2)) were developed that degrade faster than TCP but could have reduced biocompatibility due to their high solubility. Therefore, they were modified by a neutralizing surface treatment. The aim of this study was to evaluate the biocompatibility of some of these ceramics, GB1a, GB9, and GB14, which differ in the amount of added Na, K, Mg, or Si ions, with standard and modified surfaces. The in vitro cytotoxicity of the ceramics GB1a, GB9, and GB14 was determined by the agar diffusion and filter test and the microculture tetrazolium (MTT) assay. In order to investigate the influence of surface modification, these three ceramics were compared to their surface-treated counterparts, GB1aN, GB9N, and GB14N. GB1a, the ceramic with the highest in vitro solubility, showed the strongest toxic influence in all cell culture tests. GB9 and GB14 produced better results. In contrast, the counterparts with modified surfaces exhibited no (GB9N, GB14N) or weak (GB1aN) signs of cytotoxicity. It is concluded that the toxicity of the ceramics GB1a, GB9, and GB14 depends on their solubility. A positive influence of the surface treatment on in vitro biocompatibility was demonstrated. Therefore, the surface-treated glass ceramics could be promising materials for bone replacement.

[In vitro cell behavior of human osteoblasts after physiological dynamic stretching]. / In-vitro-Zellverhalten humaner Osteoblasten nach physiologischen dynamischen Dehnungen.

The cell activity of human bone derived cell cultures was studied after mechanical stimulation by cyclic strain at a magnitude occurring in physiologically loaded bone tissue. Monolayers of subconfluently grown human bone derived cells were stretched in rectangular silicone dishes with cyclic uniaxial movement along their longitudinal axes. Strain was applied over two days for 30 min per day with a frequency of 1 Hz and a strain magnitude of 1000 mustrain. Cyclic stretching of the cells resulted in an increased proliferation (10-48%) and carboxyterminal collagen type I propeptide release (7-49%) of human cancellous bone derived osteoblasts while alkaline phosphatase activity and osteocalcin release were significantly reduced by 9-25% and 5-32% respectively. These results demonstrate that cyclic strain at physiologic magnitude leads to an increase of osteoblast activities related to matrix production while those activities which are characteristic for the differentiated osteoblast and relevant for matrix mineralization are decreased.

In vitro effects of dynamic strain on the proliferative and metabolic activity of human osteoblasts.

AIM OF THE STUDY: It has been well shown by human and animal studies that mechanical load is an important regulator of skeletal mass and architecture. However, cellular reactions which adapt bone tissue to the mechanical environment are not definitively determined. For this purpose we studied the cell activity of human bone derived cell cultures after mechanical stimulation by cyclic, uniaxial strain at a magnitude occurring in normal loaded bone tissue. MATERIALS AND METHODS: Human osteoblasts were isolated from cancellous bone biopsies of 5 different donors. Cell seeding was made in DMEM in a density of 10.000 cells/cm(2) on deformable culture dishes for three days prior to initiating cell stretching at 1000 microstrain, 1Hz for 1800 cycles for two subsequent days with an especially developed cell stretching device. 48h after the second stimulation cells were harvested and cell number was determined with a Coulter Counter. Cell bound alkaline phosphatase activity was analyzed in cell lysates by a colorimetric assay, osteocalcin and CICP (procollagen I propeptide) production were analyzed in cell supernatants with ELISAs. Three parallel cultures were tested. STATISTICS: Wilcoxon. RESULTS: In all experiments mechanical stimulation resulted in a significant increase in cell number (10-48%) and CICP release (7-49%). Simultaneously a significant decrease in alkaline phosphatase activity (9-25%) and osteocalcin release (5-32%) could be observed. CONCLUSIONS: The results demonstrate that cyclic strain at physiologic magnitude leads to an increase of early osteoblast activities related to matrix production while those activities which are characteristic for the differentiated osteoblast and relevant for matrix mineralization are decreased. These new findings confirm in vivo observations about the importance of dynamic strain for bone formation during fracture healing and bone remodeling and could contribute to the optimization of fracture healing.

Dynamic cell stretching increases human osteoblast proliferation and CICP synthesis but decreases osteocalcin synthesis and alkaline phosphatase activity.

The cell activity of human-bone-derived cell cultures was studied after mechanical stimulation by cyclic strain at a magnitude occurring in physiologically loaded bone tissue. Monolayers of subconfluently grown human-bone-derived cells were stretched in rectangular silicone dishes with cyclic predominantly uniaxial movement along their longitudinal axes. Strain was applied over two days for 30 min per day with a frequency of 1 Hz and a strain magnitude of 1000 microstrain. Cyclic stretching of the cells resulted in an increased proliferation (10-48%) and carboxyterminal collagen type I propeptide release (7-49%) of human-cancellous bone-derived osteoblasts while alkaline phosphatase activity and osteocalcin release were significantly reduced by 9-25 and 5-32%, respectively. These results demonstrate that cyclic strain at physiologic magnitude leads to an increase of osteoblast activities related to matrix production while those activities which are characteristic for the differentiated osteoblast and relevant for matrix mineralization are decreased.

Effects of mechanical factors on the fracture healing process.

An interdisciplinary study based on animal experiments, cell culture studies, and finite element models is presented. In a sheep model, the influence of the osteotomy gap size and interfragmentary motion on the healing success was investigated. Increasing gap sizes delayed the healing process. Increasing movement stimulated callus formation but not tissue quality. Typical distributions of intramembranous bone, endochondral ossification, and connective tissue in the fracture gap are quantified. The comparison of the mechanical data determined by a finite element model with the histologic images allowed the attribution of certain mechanical conditions to the type of tissue differentiation. Intramembranous bone formation was found for strains smaller than approximately 5% and small hydrostatic pressure (< 0.15 MPa). Strains less than 15% and hydrostatic pressure more than 0.15 MPa stimulated endochondral ossification. Larger strains led to connective tissue. Cell culture studies on the influence of strain on osteoblasts supported these findings. Proliferation and transforming growth factor beta production was increased for strains up to 5% but decreased for larger strains. Osteoblasts under larger strains (> 4%) turned away from the principal strain axis and avoided larger deformations. It is hypothesized that gap size and the amount of strain and hydrostatic pressure along the calcified surface in the fracture gap are the fundamental mechanical factors involved in bone healing.