Shotgun analysis of integral membrane proteins facilitated by elevated temperature

The beneficial effects on peak selectivity and resolution of conducting liquid chromatography (LC) at elevated temperature (e.g., 30-80 degrees C) are generally well-known; however, its importance for peptide recovery is not nearly as well recognized. This report demonstrates that microLC analysis of membrane proteomic samples significantly benefits from the application of heat. Enriched membrane and membrane-embedded peptides (the latter obtained by membrane shaving) were analyzed by microLC-tandem mass spectrometry (MS/MS) from 20 to 60 degrees C using a standard reversed-phase material. Maximal protein and hydrophobic peptide recovery was obtained at 60 degrees C. The membrane-shaving method employed, a recently optimized version of the high pH/proteinase K protocol, provided significant integral membrane protein enrichment: 98% of identified proteins were predicted to have at least one transmembrane domain (87% to have at least three), and 68% of peptides were predicted to contain transmembrane segments. Analysis of this highly enriched sample at elevated temperature increased protein identifications by 400%, and peptide identifications by 500%, as compared to room-temperature separation. Given that most microLC-MS/MS analyses are currently conducted at room temperature, the findings described herein should be of considerable value for improving the comprehensive study of integral membrane proteins.     

A finite element method for 3D hydrostatic water flows

In this paper we propose a numerical method for the solution of the “Quasi-3D” hydrodynamic equations. This approach uses a combination of standard linear finite elements along the vertical direction and non-conforming Raviart-Thomas elements in the horizontal planes. We describe, also, a suitable approximation for the convective terms. Received: 17 March 1999 / Accepted: 17 June 1999     

Tumor-Derived Small Extracellular Vesicles Induce Pro-Inflammatory Cytokine Expression and PD-L1 Regulation in M0 Macrophages via IL-6/STAT3 and TLR4 Signaling Pathways

Tumor-associated macrophages play a key role in promoting tumor progression by exerting an immunosuppressive phenotype associated with the expression of programmed cell death ligand 1 (PD-L1). It is well known that tumor-derived small extracellular vesicles (SEVs) affect the tumor microenvironment, influencing TAM behavior. The present study aimed to examine the effect of SEVs derived from colon cancer and multiple myeloma cells on macrophage functions. Non-polarized macrophages (M0) differentiated from THP-1 cells were co-cultured with SEVs derived from a colorectal cancer (CRC) cell line, SW480, and a multiple myeloma (MM) cell line, MM1.S. The expression of PD-L1, interleukin-6 (IL-6), and other inflammatory cytokines as well as of the underlying molecular mechanisms were evaluated. Our results indicate that SEVs can significantly upregulate the expressions of PD-L1 and IL-6 at both the mRNA and protein levels and can activate the STAT3 signaling pathway. Furthermore, we identified the TLR4/NF-kB pathway as a convergent mechanism for SEV-mediated PD-L1 expression. Overall, these preliminary data suggest that SEVs contribute to the formation of an immunosuppressive microenvironment.     

Hydrogen production by the thermophilic eubacterium Thermotoga neapolitana from storage polysaccharides of the CO2-fixing diatom Thalassiosira weissflogii

Planktic diatoms are the largest primary producers in marine and freshwater habitats. Their dry biomass accumulates up to 50% of lipids and contains water-soluble β-1,3-glucans as major storage products. Because of the world-wide abundance of these photosynthetic protists, β-1,3-glucans may rival cellulose as the polysaccharide with the highest annual production on Earth. Here we show the feasibility of a simple and efficient process leading to bio-hydrogen by dark fermentation of microalgal biomass with the thermophilic bacterium Thermotoga neapolitana. Production of the biogas on minimum medium supplemented only with the extract of the diatom Thalassiosira weissflogii proved that algal biomass per se can serve as substrate for sustaining the biotechnological process with no requirement of any pretreatment and external integration of other nutrients. At the same time, lipids unused for the anaerobic production of the biogas, can be employed for production of bio-diesel, thus considerably increasing the economic potential of these renewable feedstocks.     

Natural convection in a partially open square cavity with internal heat source: An analysis of the opening mass flow

A steady buoyancy-driven flow of air in a partially open square 2D cavity with internal heat source, adiabatic bottom and top walls, and vertical walls maintained at different constant temperatures is investigated numerically in this work. A heat source with 1% of the cavity volume is present in the center of the bottom wall. The cold right wall contains a partial opening occupying 25%, 50% or 75% of the wall. The influence of the temperature gradient between the verticals walls was analyzed for Ra_e = 10³–10⁵, while the influence of the heat source was evaluated through the relation R = Ra_i/Ra_e, investigated at between 400 and 2000. Interesting results were obtained. For a low Rayleigh number, it is found that the isotherm plots are smooth and follow a parabolic shape indicating the dominance of the heat source. But as the Ra_e increases, the flow slowly becomes dominated by the temperature difference between the walls. It is also observed that multiple strong secondary circulations are formed for fluids with a small Ra_e whereas these features are absent at higher Ra_e. The comprehensive analysis is concluded with horizontal air velocity and temperature plots for the opening. The numerical results show a significant influence of the opening on the heat transfer in the cavity.     

The Influence of Baffles on the Natural Convection in Trapezoidal Cavities

This article presents an investigation into natural convection in trapezoidal cavities. It examines a cavity whose floor and upper inclined walls are both adiabatic, while the vertical walls are isothermal. For these isothermal walls, we consider two thermal boundary conditions. Under the first condition, the short wall on the left side is heated as the tall one on the right side is cooled. The second condition is the reverse of the first—the short wall is cooled as the tall one is heated. Considering laminar conditions and a two-dimensional system, steady-state computations are carried out to assess the effects of one and two baffles, the baffle's height (H _b ), Rayleigh number, 10³ ≤ Ra ≤ 10⁶, and three Prandtl number values. To demonstrate the various effects, the results from several designed case studies are shown in terms of isotherms, streamlines, and local and average Nusselt numbers in order. Predictions reveal that the second baffle decreases the cavity's fluid flow and heat transfer. As the height of the baffle rises, the heat transfer drops drastically. Also, two baffles produce more pronounced thermal stratification than only one.