Quantitative Evaluation of Noncovalent Interactions between 3,4-Dimethyl-1H-pyrazole and Dissolved Humic Substances by NMR Spectroscopy.

Nitrification inhibitors (NI) represent a valid chemical strategy to retard nitrogen oxidation in soil and limit nitrate leaching or nitrogen oxide emission. We hypothesized that humic substances can complex NI, thus affecting their activity, mobility, and persistence in soil. Therefore, we focused on 3,4-dimethylpyrazole phosphate (DMPP) by placing it in contact with increasing concentrations of model fulvic (FA) and humic (HA) acids. The complex formation was assessed through advanced and composite NMR techniques (chemical shift drift, line-broadening effect, relaxation times, saturation transfer difference (STD), and diffusion ordered spectroscopy (DOSY)). Our results showed that both humic substances interacted with DMPP, with HA exhibiting a significantly greater affinity than FA. STD emphasized the pivotal role of the aromatic signal, for HA-DMPP association, and both alkyl methyl groups, for FA-DMPP association. The fractions of complexed DMPP were determined on the basis of self-diffusion coefficients, which were then exploited to calculate both the humo-complex affinity constants and the free Gibbs energy (Kd and ΔG for HA were 0.5169 M and -1636 kJ mol-1, respectively). We concluded that DMPP-based NI efficiency may be altered by soil organic matter, characterized by a pronounced hydrophobic nature. This is relevant to improve nitrogen management and lower its environmental impact.

Platelet-Rich Fibrin in Combination with Local Antibiotics Optimizes Wound Healing After Deep Sternal Wound Problems and Prevents Reinfection.

OBJECTIVE: Disturbed wound healing is a significant problem in patients after cardiac surgery. Problems with deep sternal wound healing are rare, but can be quite difficult to treat. Furthermore, the therapy is highly expensive and consumes many of the patient's personal resources. Another major obstacle in this patient group is reinfection after secondary wound closure. We examined how to prevent early reinfection through the use of growth factors in combination with local antibiotics. METHODS: Our study included 232 patients with a deep sternal wound healing problem. After initial vacuum therapy, we planned secondary wound closure. During wound closure, we used only platelet-rich fibrin in a PRF group (109 patients). In another group (123 patients), we covered the wounds intraoperatively with a combination of PRF and local antibiotics (PRF CoDelivery). All patients were observed for 30 days for signs of early surgical site infection. RESULTS: After 30 days, 22 patients (20.2%) in the PRF group showed a persistent problem with wound healing with or without reinfection. In contrast, only 12 patients (9.8%) in the PRF CoDelivery group had this problem (p=0.023 PRF vs. PRF CoDelivery). CONCLUSION: The combination of growth factors and antibiotics was associated with a significantly reduced incidence of early reinfection and thus can be expected to have a positive impact on wound healing in complicated scenarios. Furthermore, the combination of PRF and local antibiotics was easy to use. Further studies are needed to verify these initial findings.

Favorable gradients with the mitroflow aortic valve prosthesis in everyday surgery.

BACKGROUND: The supra-annular Mitroflow valve has been implanted for over 20 years, preferably in small aortic roots. Early outcomes and gradients in unselected elderly patients receiving aortic valve surgery are reported in this study. METHODS: Mitroflow valves were implanted in 190 consecutive patients (70% females). Mean age was 75.6 ± 5.5 years, body mass index was 28.2 ± 5.3 kg/m2, and logistic EuroSCORE 17.8 ± 16.5%. There were 170 single, 17 double, and 3 triple valve procedures. Of all, 46.8% of surgeries were performed with coronary artery bypass grafting and 39.5% were performed with isolated aortic valve replacement (AVR). Rates of redo and endocarditic cases were 14.2 and 4.7%. RESULTS: The 19-mm (n = 14), 21-mm (n = 135), and 23-mm (n = 41) valves showed mean gradients of 16.1 ± 7.4 mm Hg, 15.8 ± 7.1 mm Hg, and 11.6 ± 4.1 mm Hg, respectively, before discharge. Maximum gradients were 27.5 ± 13.0, 28.4 ± 12.6, and 21.6 ± 7.6 mm Hg, respectively. Correlation between gradients and sizes was significant. In-hospital mortality was 12.6% overall (6.7% in isolated AVR). The rate of valve-related events was low (mild paravalvular leakage 2.1%, neurologic 3.2%, bleeding 3.2%, atrioventricular block 4.2%, no thrombosis). CONCLUSION: The Mitroflow valve can be safely implanted in elderly patients undergoing advanced surgical procedures. Gradients were acceptably low even in the smallest valves.

Global and Site-Specific Effect of Phosphorylation on Protein Turnover.

To date, the effects of specific modification types and sites on protein lifetime have not been systematically illustrated. Here, we describe a proteomic method, DeltaSILAC, to quantitatively assess the impact of site-specific phosphorylation on the turnover of thousands of proteins in live cells. Based on the accurate and reproducible mass spectrometry-based method, a pulse labeling approach using stable isotope-labeled amino acids in cells (pSILAC), phosphoproteomics, and a unique peptide-level matching strategy, our DeltaSILAC profiling revealed a global, unexpected delaying effect of many phosphosites on protein turnover. We further found that phosphorylated sites accelerating protein turnover are functionally selected for cell fitness, enriched in Cyclin-dependent kinase substrates, and evolutionarily conserved, whereas the glutamic acids surrounding phosphosites significantly delay protein turnover. Our method represents a generalizable approach and provides a rich resource for prioritizing the effects of phosphorylation sites on protein lifetime in the context of cell signaling and disease biology.

Multi-omic measurements of heterogeneity in HeLa cells across laboratories.

Reproducibility in research can be compromised by both biological and technical variation, but most of the focus is on removing the latter. Here we investigate the effects of biological variation in HeLa cell lines using a systems-wide approach. We determine the degree of molecular and phenotypic variability across 14 stock HeLa samples from 13 international laboratories. We cultured cells in uniform conditions and profiled genome-wide copy numbers, mRNAs, proteins and protein turnover rates in each cell line. We discovered substantial heterogeneity between HeLa variants, especially between lines of the CCL2 and Kyoto varieties, and observed progressive divergence within a specific cell line over 50 successive passages. Genomic variability has a complex, nonlinear effect on transcriptome, proteome and protein turnover profiles, and proteotype patterns explain the varying phenotypic response of different cell lines to Salmonella infection. These findings have implications for the interpretation and reproducibility of research results obtained from human cultured cells.

Gravitation-driven stress-reduced cell handling.

We present a simple lab-on-chip device for handling small samples of delicate cells, e.g. stem cells. It uses a combination of sedimentation and dielectrophoresis. The transport of cells is driven by gravitation. Dielectrophoresis uses radio-frequency electric fields for generating particle-selective forces dependent on size and polarisability. Electrodes along the channels hold particles and/or cells in a defined position and deflect them towards different outlets. The absence of external pumping and the integration of injection and sampling ports allow the processing of tiny sample volumes. Various functions are demonstrated, such as contact-free cell trapping and cell/particle sorting. Pairs of human cells and antibody-coated beads, as they are formed for T cell activation, are separated from unbound beads. The cells experience only low stress levels compared with the stress levels in dielectrophoresis systems, where transport depends on external pumping. Our device is a versatile yet simple tool that finds applications in cellular biotechnology, in particular when an economic solution is required.

Highly controlled electrofusion of individually selected cells in dielectrophoretic field cages.

The prospect of novel therapeutic approaches has renewed the current interest in the fusion of rare cells, like stem cells or primary immune cells. While conventional techniques are only capable of mass fusion, lab-on-a-chip systems often still lack an acceptable method for making the cells available after processing. Here, we present a microfluidic approach for electrofusion on the single-cell level that offers high control over the cells both before and after fusion. For cell pairing and fusion, we employed dielectrophoresis and AC voltage pulses, respectively. Each cell has been characterized and selected before they were paired, fused and released from the fluidic system for subsequent analysis and cultivation. The successful experimental evaluation of our system was further corroborated by numerical simulations. We obtained fusion efficiencies of more than 30% for individual pairs of mouse myeloma and B cell blasts and showed the proliferating ability of the hybrid cells 3 d after fusion. Since aggregates of more than two cells can be fused, the technique could also be developed further for generating giant cells for low-noise electrophysiology in the context of semi-automated pharmaceutical screening procedures.