Pyrogen retention: Comparison of the novel medium cut-off (MCO) membrane with other dialyser membranes.

Haemodialysis effectively removes small solutes and smaller-sized middle molecules from the blood; however, the clearance of larger middle molecules, which have been associated with negative effects, is poor. The novel medium cut-off (MCO) dialysis membrane has larger pore sizes and a more open structure than other high-flux membranes, providing improved removal of larger middle molecules while retaining albumin. However, larger pore sizes may potentially increase permeability to pyrogens, including endotoxins and other bacterial contaminants, that could be present in the dialysis fluid. In this study, we tested the capacity of low-flux, high-flux, MCO and high cut-off dialyser membranes with different pore sizes to prevent pyrogens crossing from dialysate to the blood side in a closed-loop test system, differentiating among lipopolysaccharides, peptidoglycans and bacterial DNA using a toll-like receptor assay. Even though the bacterial contamination levels in our test system exceeded the acceptable pyrogen dose for standard dialysis fluid, levels of lipopolysaccharides, peptidoglycans and bacterial DNA in the blood-side samples were too low to identify potential differences in pyrogen permeability among the membranes. Our results suggest that MCO membranes are suitable for haemodialysis using ISO standard dialysis fluid quality, and retain endotoxins at a similar level as other membranes.

Retention of beneficial molecules and coagulation factors during haemodialysis and haemodiafiltration.

Middle molecules (MMs) are associated with the pathology of uraemia, and are not effectively removed by standard extracorporeal treatments. Increased convection used in haemodiafiltration (HDF) can enhance the removal of MMs; however, high-volume HDF is not available to all patients. The new medium cut-off (MCO) membrane has been developed to allow increased removal of MMs using standard haemodialysis (HD). Improved removal of MMs has been shown with the MCO membrane compared with standard high-flux dialysers, but it is not known whether the increased pore size affects the retention of commonly used medications or that of coagulation factors in dialysis patients. Using an in vitro model, the retention of erythropoietin, heparin, insulin, vancomycin and several coagulation factors (Factors II, VII and X, protein C and antithrombin III) was investigated with the MCO membrane dialyser, compared with high-flux dialysers with polysulfone (in HDF) or polyethersulfone membranes (in HD and HDF). The retention of all molecules investigated was comparable between the MCO membrane and the high-flux dialysers. Results from the in vitro studies suggest that switching from a high-flux dialyser to the MCO membrane should not require changes to the medication dosing or anti-coagulation protocols of dialysis patients.

Requirements and Pitfalls of Dialyzer Sieving Coefficients Comparisons.

Sieving coefficients reported in dialyzer data sheets and instructions for use (IFUs) indicate the potential of different solutes to pass across a particular membrane. Despite being measured in vitro, sieving coefficient data are often used as a predictor of the clinical performance of dialyzers. Although standards for the measurement of sieving coefficients exist, the stated methodologies do not offer sufficient guidance to ensure comparability of test results between different dialyzers. The aim of this work was to investigate the relationship between sieving coefficients and published clinical performance indicators for two solutes, albumin loss and beta-2 microglobulin (ß2 M) reduction ratio (RR), and to assess the impact of different in vitro test parameters on sieving coefficient values for albumin, ß2 M, and myoglobin. Clinical albumin loss and ß2 M RR for commercially available dialyzers used in hemodialysis (HD) and post-dilution hemodiafiltration (HDF) were extracted from the literature and plotted against sieving coefficients reported in data sheets and IFUs. Albumin, ß2 M, and myoglobin sieving coefficients of a selection of dialyzers were measured per the ISO 8637 standard. The impact of in vitro testing conditions was assessed by changing blood flow rate, ultrafiltration (UF) rate, sampling time, and origin of test plasma. Results showed variation in albumin loss and ß2 M RR for the same sieving coefficient across different dialyzers in HD and HDF. Changes in blood flow rates, UF rates, sampling time, and test plasma (bovine vs. human) caused marked differences in sieving coefficient values for all investigated solutes. When identical testing conditions were used, sieving coefficient values for the same dialyzer were reproducible. Testing conditions have a marked impact on the measurement of sieving coefficients, and values should not be compared unless identical conditions are used. Further, variability in observed clinical data in part reflects the lack of definition of test conditions.

Assessment of the association between increasing membrane pore size and endotoxin permeability using a novel experimental dialysis simulation set-up.

BACKGROUND: Membranes with increasing pore size are introduced to enhance removal of large uremic toxins with regular hemodialysis. These membranes might theoretically have higher permeability for bacterial degradation products. In this paper, permeability for bacterial degradation products of membranes of comparable composition with different pore size was investigated with a new in vitro set-up that represents clinical flow and pressure conditions. METHODS: Dialysis was simulated with an AK200 machine using a low-flux, high-flux, medium cut-off (MCO) or high cut-off (HCO) device (n = 6/type). A polyvinylpyrrolidone-solution (PVP) was recirculated at blood side. At dialysate side, a challenge solution containing a filtrated lysate of two water-borne bacteria (Pseudomonas aeruginosa and Pelomononas saccharophila) was infused in the dialysate flow (endotoxin ≥ 4EU/ml). Blood and dialysate flow were set at 400 and 500 ml/min for 60 min. PVP was sampled before (PVPpre) and after (PVPpost) the experiment and dialysate after 5 and 55 min. Limulus Amebocyte Lysate (LAL) test was performed. Additionally, samples were incubated with a THP-1 cell line (24 h) and IL-1ß levels were measured evaluating biological activity. RESULTS: The LAL-assay confirmed presence of 9.5 ± 7.4 EU/ml at dialysate side. For none of the devices the LAL activity in PVPpre vs. PVPpost was significantly different. Although more blood side PVP solutions had a detectable amount of endotoxin using a highly sensitive LAL assay in the more open vs traditional membranes, the permeability for endotoxins of the 4 tested dialysis membranes was not significantly different but the number of repeats is small. None of the PVP solutions induced IL-1ß in the THP-1 assay. CONCLUSIONS: A realisitic in vitro dialysis was developed to assess membrane translocation of bacterial products. LAL activity on the blood side after endotoxin exposure did not change for all membranes. Also, none of the PVPpost solutions induced IL-1ß in the THP-1 bio-assay.

Modulation of leucocytic angiotensin-converting enzymes expression in patients maintained on high-permeable haemodialysis.

BACKGROUND: High mortality of haemodialysis patients is associated with systemic chronic inflammation and overactivation of the renin-angiotensin system (RAS). Insufficient elimination of pro-inflammatory immune mediators, especially in the molecular weight range of 15-45 kDa, may be one of the reasons for this. Employment of haemodialysis membranes with increased permeability was shown to ameliorate the inflammatory response and might modulate the effects of local RAS. In this study, we tested the impact of high cut-off (HCO), medium cut-off (MCO) and high-flux (HF) dialysis on leucocytic transcripts of angiotensin-converting enzymes (ACE and ACE2). Additionally, the impact of HCO, MCO and HF sera and dialysates on local ACEs and inflammation markers was tested in THP-1 monocytes. METHODS: Patients' leucocytes were obtained from our recent clinical studies comparing HCO and MCO dialysers with HF. The cells were subjected to quantitaive polymerase chain reaction (qPCR) analyses with TaqMan probes specific for ACE, ACE2 and angiotensin II (AngII) and Ang1-7 receptors. Sera and dialysates from the clinical trials as well as samples from in vitro dialysis were tested on THP-1 monocytic cells. The cells were subjected to qPCR analyses with TaqMan probes specific for ACE, ACE2, interleukin-6 and tumour necrosis factor α and immunocytochemistry with ACE and ACE2 antibodies. RESULTS: Leucocytes obtained from patients treated with HCO or MCO demonstrated decreased transcript expression of ACE, while ACE2 was significantly upregulated as compared with HF. Receptors for AngII and Ang1-7 remained unchanged. THP-1 monocytes preconditioned with HCO and MCO patients' or in vitro dialysis sera reflected the same expressional regulation of ACE and ACE2 as those observed in HCO and MCO leucocytes. As a complementary finding, treatment with HCO and MCO in vitro dialysates induced a pro-inflammatory response of the cells as demonstrated by elevated messenger RNA expression of tumour necrosis factor α and interleukin-6, as well as upregulation of ACE and decreased levels of ACE2. CONCLUSIONS: Taken together, these data demonstrate that employment of membranes with high permeability eliminates a spectrum of mediators from circulation that affect the RAS components in leucocytes, especially ACE/ACE2.

Removal of Cell-Activating Substances Using Dialyzers With Various Permeability Profiles.

Despite multiple efforts to target an improvement in clinical outcomes of patients with end-stage renal disease, several challenges must still be addressed. Dialysis patients are at a high risk for complications, as reflected by increasing mortality rates. The objective of this study is to assess the impact of the application of dialyzers with varying permeability profiles on the removal of cell-activating substances from the blood of hemodialysis (HD) patients. Dialysate samples were collected using Revaclear 400 (RC) and MCO-Ci400 (MCO-CI). Total protein and solute marker concentrations were determined for the concentrated sample. The response of tubular epithelial cells (TECs) to the dialysate samples was assessed via measurement of interleukin 6, cell viability, and morphology. Proteomic analysis of the dialysate samples was performed using liquid chromatography coupled to tandem mass spectrometry. Treatment of TECs with the MCO-CI dialysate resulted in significantly decreased cell viability compared with the RC dialysate. TECs incubated with samples from MCO-CI lost their typical brick-like shape and cell-cell connections. Proteomic analysis of dialysate samples indicated multiple pro-apoptotic and pro-inflammatory proteins, supporting the observed phenotype. Additionally, application of the MCO-CI dialyzer allowed for more efficient removal of proteins associated with advanced chronic kidney disease stages. Collectively, the use of dialyzer with a higher permeability profile enabled more efficient removal of cell-activating and toxic substances from the blood of HD patients. However, a further large-scale study is needed to address benefits and associated risks for patients.

In vitro benchmark of cytokine removal by dialyzers with various permeability profiles.

PURPOSE: Removal of cytokines is relevant for dialysis patients as they are suspected to promote cardiovascular complications. The objective of this study was to benchmark membranes with different permeability profiles under standardized in vitro test conditions using miniaturized devices with respect to their ability to remove cytokines from human serum and to lower cell activating potential. METHODS: In vitro dialysis was used to dialyze cytokine enriched serum in 3 independent experiments per tested membrane. IL-6 in the serum and dialysate was measured at defined times by enzyme-linked immunosorbent assay. IL-8, IL-1ß, IL-6 and TNF-α in dialysate were measured by immunoassay. Dialysate samples were subjected to cultured tubular epithelial cells or human fibroblasts to study cell activation via IL-6 generation. Dialysate samples were added to human whole blood with subsequent analysis of granulocyte and monocyte activation by detection of CD11b. RESULTS: IL-6 decreased in serum and increased in dialysate during in vitro dialysis. IL-8, IL-1ß, and TNF-α were identified in dialysate. Dialysate added to cell cultures increased IL-6 concentration in culture medium or increased expression of CD11b. High cut-off membranes showed the strongest transfer of cytokines, albumin and total proteins from serum to dialysate and led to strongest cell activation. This effect was lower for medium cut-off membranes and lowest for conventional high-flux membranes. CONCLUSIONS: This study demonstrated an in vitro test by which membranes were benchmarked with respect to cytokine and cell activation removal capacity. Cell activation levels could be influenced by the choice of membrane by altering cytokine concentration levels.

In Vitro Dialysis of Cytokine-Rich Plasma With High and Medium Cut-Off Membranes Reduces Its Procalcific Activity.

Recently developed high-flux (HF) dialysis membranes with extended permeability provide better clearance of middle-sized molecules such as interleukins (ILs). Whether this modulation of inflammation influences the procalcific effects of septic plasma on vascular smooth muscle cells (VSMCs) is not known. To assess the effects of high cut-off (HCO) and medium cut-off (MCO) membranes on microinflammation and in vitro vascular calcification we developed a miniature dialysis model. Plasma samples from lipopolysaccharide-spiked blood were dialyzed with HF, HCO, and MCO membranes in an in vitro miniature dialysis model. Afterwards, IL-6 concentrations were determined in dialysate and plasma. Calcifying VSMCs were incubated with dialyzed plasma samples and vascular calcification was assessed. Osteopontin (OPN) and matrix Gla protein (MGP) were measured in VSMC supernatants. IL-6 plasma concentrations were markedly lower with HCO and MCO dialysis. VSMC calcification was significantly lower after incubation with MCO- and HCO-serum compared to HF plasma. MGP and OPN levels in supernatants were significantly lower in the MCO but not in the HCO group compared to HF. In vitro dialysis of cytokine-enriched plasma samples with MCO and HCO membranes reduces IL-6 levels. The induction of vascular calcification by cytokine-enriched plasma is reduced after HCO and MCO dialysis.

Medium Cut-Off (MCO) Membranes Reduce Inflammation in Chronic Dialysis Patients-A Randomized Controlled Clinical Trial.

BACKGROUND: To increase the removal of middle-sized uremic toxins a new membrane with enhanced permeability and selectivity, called Medium Cut-Off membrane (MCO-Ci) has been developed that at the same time ensures the retention of albumin. Because many middle-sized substances may contribute to micro-inflammation we hypothesized that the use of MCO-Ci influences the inflammatory state in hemodialysis patients. METHODS: The randomized crossover trial in 48 patients compared MCO-Ci dialysis to High-flux dialysis of 4 weeks duration each plus 8 weeks extension phase. Primary endpoint was the gene expression of TNF-α and IL-6 in peripheral blood mononuclear cells (PBMCs), secondary endpoints were plasma levels of specified inflammatory mediators and cytokines. RESULTS: After four weeks of MCO-Ci the expression of TNF-α mRNA (Relative quantification (RQ) from 0.92 ± 0.34 to 0.75 ± 0.31, -18.5%, p<0.001)-α and IL-6 mRNA (RQ from 0.78 ± 0.80 to 0.60 ± 0.43, -23.1%, p<0.01) was reduced to a significantly greater extent than with High-flux dialyzers (TNF mRNA-RQ: -14.3%; IL-6 mRNA-RQ: -3.5%). After retransformation of logarithmically transformed data, measurements after MCO were reduced to 82% of those after HF (95% CI 74%-91%). 4 weeks use of MCO-Ci resulted in long-lasting change in plasma levels of several cytokines and other substances with a significant decrease for sTNFR1, kappa and lambda free light chains, urea and an increase for Lp-PLA2 (PLA2G7) compared to High-flux. Albumin levels dropped significantly after 4 weeks of MCO dialysis but increased after additional 8 weeks of MCO dialysis. Twelve weeks treatment with MCO-Ci was well tolerated regarding the number of (S)AEs. In the extension period levels of CRP, TNF-α-mRNA and IL-6 mRNA remained stable in High-flux as well as in MCO-Ci. CONCLUSIONS: MCO-Ci dialyzers modulate inflammation in chronic HD patients to a greater extent compared to High-flux dialyzers. Transcription of pro-inflammatory cytokines in peripheral leukocytes is markedly reduced and removal of soluble mediators is enhanced with MCO dialysis. Serum albumin concentrations stabilize after an initial drop. These results encourage further trials with longer treatment periods and clinical endpoints.

Detection of Free Advanced Glycation End Products in Vivo during Hemodialysis.

Advanced glycation end products (AGEs) are often regarded as glycotoxins, which are normally removed by the kidney. Patients with end-stage renal failure rely on hemodialysis (HD) treatment to eliminate these compounds. In the present work, a highly selective LC-MS/MS method was used for quantitation of AGE levels in plasma and in dialysis fluids of HD patients, with a focus on AGE-free adducts. A broad range of 19 amino acid modifications was identified and quantitated. It was expected that the AGE-free adducts are successfully eliminated by dialysis treatment. Indeed, with a mean elimination rate of 71%, this assumption proved to be valid for all target analytes with the exception of pyrraline, which showed an opposite behavior. Here, plasma and dialysate levels increased during the treatment by about 59%. The notions that pyrraline was formed in high amounts in the patient's bloodstream (I) after intake of the corresponding precursor compound 3-deoxyglucosone with the dialysis fluid or (II) by catalytic effects on the formation by the dialysis membrane were ruled out. In contrast, in a dietary study, the comparison of pyrraline concentrations in plasma before and after food consumption confirmed that the increase in pyrraline originates solely from digestion of glycated food proteins. Additionally, by detailed analyses of the food consumed during dialysis sessions, bread rolls with a pyrraline content of about 21.7 µmol per serving were identified as the main source.

Cell-free plasma hemoglobin removal by dialyzers with various permeability profiles.

The release of hemoglobin from mechanically stressed erythrocytes into plasma is a general side effect of extracorporeal therapies, such as extracorporeal membrane oxygenation or hemodialysis. In many reported cases dialysis patients showed elevated cell-free plasma hemoglobin (CPH) levels which are associated with pathophysiological effects. In this in vitro study, the CPH clearance capacity of various filters with different permeability profiles was measured. Simulated dialysis treatments were conducted and clearance was calculated from variations in CPH concentrations over time by measuring plasma absorbance at 405 nm. Conventional high-flux filters exhibited no detectable clearance of CPH. High-flux filters with extended permeability exhibited clearances between 5.8 ± 1.2 and 12.7 ± 1.7 ml/min when tested with plasma and between 5.8 ± 1.2 and 11.3 ± 1.6 ml/min when tested with whole blood. septeX high-cutoff filters had clearances between 13.8 ± 1.8 and 15.5 ± 1.7 ml/min when tested with plasma and of 22.6 ± 2.9 ml/min when tested with whole blood. This study demonstrated that filters with extended permeability and the septeX filter enable CPH removal when used as in chronic and acute settings.

Looking beyond endotoxin: a comparative study of pyrogen retention by ultrafilters used for the preparation of sterile dialyis fluid.

Sterile single-use ultrafilters are used in dialysis for the preparation of the substitution fluid given to patients undergoing dialysis treatments with high convective fluid removal. The retention of pyrogenic agents by the ultrafilters is crucial to avoiding inflammatory responses. The performance of a new single-use ultrafilter (NUF) with a positively charged flat sheet membrane of relatively small membrane area and large pore size was compared to a reference ultrafilter (RUF) with a hollow fiber membrane. Filter performance was tested with various pyrogen-contaminated dialysis fluids by direct pyrogen quantification and by measuring inflammatory responses in cell-based bioassays. The NUF completely retained oligodeoxynucleotides (ODN), whereas the RUF was fully permeable. Both filters tended to decrease biological activity of DNA in filtered bacterial lysates. The NUF reduced lipopolysaccharides (LPS) and LPS-induced biological activity by 100%, whereas the RUF produced filtrates with low but detectable levels of LPS in most cases. Peptidoglycans (PGN) were fully retained both by the NUF and the RUF. The new ultrafilter retained biologically active ODN, which has not yet been described for any other device used in dialysis, and it showed better or equal retention of LPS and PGN even with a smaller membrane surface and larger pore size.

Axial helix rotation as a mechanism for signal regulation inferred from the crystallographic analysis of the E. coli serine chemoreceptor.

Bacterial chemotaxis receptors are elongated homodimeric coiled-coil bundles, which transduce signals generated in an N-terminal sensor domain across 15-20nm to a conserved C-terminal signaling subdomain. This signal transduction regulates the activity of associated kinases, altering the behavior of the flagellar motor and hence cell motility. Signaling is in turn modulated by selective methylation and demethylation of specific glutamate and glutamine residues in an adaptation subdomain. We have determined the structure of a chimeric protein, consisting of the HAMP domain from Archaeoglobus fulgidus Af1503 and the methyl-accepting domain of Escherichia coli Tsr. It shows a 21nm coiled coil that alternates between two coiled-coil packing modes: canonical knobs-into-holes and complementary x-da, a variant form related to the canonical one by axial rotation of the helices. Comparison of the obtained structure to the Thermotoga maritima chemoreceptor TM1143 reveals that they adopt different axial rotation states in their adaptation subdomains. This conformational change is presumably induced by the upstream HAMP domain and may modulate the affinity of the chemoreceptor to the methylation-demethylation system. The presented findings extend the cogwheel model for signal transmission to chemoreceptors.

A soluble mutant of the transmembrane receptor Af1503 features strong changes in coiled-coil periodicity.

Structures of full-length, membrane-bound proteins are essential for understanding transmembrane signaling mechanisms. However, in prokaryotic receptors no such structure has been reported, despite active research for many years. Here we present results of an alternative strategy, whereby a transmembrane receptor is made soluble by selective mutations to the membrane-spanning region, chosen by analysis of helix geometry in the transmembrane regions of chemotaxis receptors. We thus converted the receptor Af1503 from Archaeoglobus fulgidus to a soluble form by deleting transmembrane helix 1 and mutating the surface residues of transmembrane helix 2 to hydrophilic amino acids. Crystallization of this protein resulted in the structure of a tetrameric proteolytic fragment representing the modified transmembrane helices plus the cytoplasmic HAMP domain, a ubiquitous domain of prokaryotic signal transducers. The protein forms a tetramer via native parallel dimerization of the HAMP domain and non-native antiparallel dimerization of the modified transmembrane helices. The latter results in a four-helical coiled coil, characterized by unusually large changes in helix periodicity. The structure offers the first view of the junction between the transmembrane region and HAMP and explains the conservation of a key sequence motif in HAMP domains.

Cell-free expression of a mammalian olfactory receptor and unidirectional insertion into small unilamellar vesicles (SUVs).

Although the identification of the multigene family encoding mammalian olfactory receptors were identified more than 20 years ago, we are far from understanding olfactory perception because of the difficulties in functional expression of these receptors in heterologous cell systems. Cell-free (CF) or in vitro expression systems offer an elegant alternative route to cell based protein expression, as the functional expression of membrane proteins can be directly achieved from the genetic template without the need of cell cultivation and protein isolation. Here we investigated in detail the cell-free expression and membrane insertion of the olfactory receptor OR5 in dependence of different experimental conditions like probing different origins of the cell-free expression system (from bacteria, via plants and insects toward mammalian system) and lipid composition of the respective extracts. We provided substantial biochemical indications by radioactive labeling based on [(35)S]-methionine, followed by proteolytic digestion, and we found that the insertion of the olfactory receptor OR5 into liposomes resulted in an unidirectional orientation with the binding side exposed into the aqueous space, resembling the native orientation in the cilia of the olfactory neurons. We report the different results in synthesis capacity for the different in vitro systems employed as we like to demonstrate the first in vitro kit toward and ex situ and ex vivo odorant receptor array.

Real-time PCR as a new in vitro biocompatibility method to measure leukocyte response to surface contact in dialysis filter devices.

INTRODUCTION: The mortality risk of dialysis patients is still elevated. Even though there is continuous improvement in the biocompatibility of dialysis devices and treatments, there is clinical evidence of a negative inflammatory impact. One dialysis-related risk factor to be considered in this regard may be the repeated blood exposure to foreign filter surfaces. Standard test methods do not allow differences to be shown between most of the common dialysis devices. METHODS: A new highly sensitive in vitro test system was developed by analyzing the response of leukocytes to surface contact in dialysis filter devices by means of quantitative real time PCR and flow cytometry. Membrane surface studies provided additional physical data. RESULTS: An increase in the transcription level of specific pro-inflammatory genes, particularly IL-1b, TNF alpha, and IL-8, was observed after blood contact to the filter devices. In two sets of pairwise filter comparisons, radiation-sterilized filters showed stronger cell activation, more hydrophilic membranes, and rougher surfaces. CONCLUSIONS: Quantitative real time RT-PCR was shown to be a new in vitro test method with increased sensitivity for detecting differences in activation levels of leukocytes upon membrane contact. Correlating leukocyte activation levels with surface properties opens new opportunities for understanding leukocyte activation upon membrane contact and thus guides further improvements in the biocompatibility of dialysis filter devices.

Mechanism of regulation of receptor histidine kinases.

Bacterial transmembrane receptors regulate an intracellular catalytic output in response to extracellular sensory input. To investigate the conformational changes that relay the regulatory signal, we have studied the HAMP domain, a ubiquitous intracellular module connecting input to output domains. HAMP forms a parallel, dimeric, four-helical coiled coil, and rational substitutions in our model domain (Af1503 HAMP) induce a transition in its interhelical packing, characterized by axial rotation of all four helices (the gearbox signaling model). We now illustrate how these conformational changes are propagated to a downstream domain by fusing Af1503 HAMP variants to the DHp domain of EnvZ, a bacterial histidine kinase. Structures of wild-type and mutant constructs are correlated with ligand response in vivo, clearly associating them with distinct signaling states. We propose that altered recognition of the catalytic domain by DHp, rather than a shift in position of the phospho-accepting histidine, forms the basis for regulation of kinase activity.

Cytochrome c biosensor--a model for gas sensing.

This work is about gas biosensing with a cytochrome c biosensor. Emphasis is put on the analysis of the sensing process and a mathematical model to make predictions about the biosensor response. Reliable predictions about biosensor responses can provide valuable information and facilitate biosensor development, particularly at an early development stage. The sensing process comprises several individual steps, such as phase partition equilibrium, intermediate reactions, mass-transport, and reaction kinetics, which take place in and between the gas and liquid phases. A quantitative description of each step was worked out and finally combined into a mathematical model. The applicability of the model was demonstrated for a particular example of methanethiol gas detection by a cytochrome c biosensor. The model allowed us to predict the optical readout response of the biosensor from tabulated data and data obtained in simple liquid phase experiments. The prediction was experimentally verified with a planar three-electrode electro-optical cytochrome c biosensor in contact with methanethiol gas in a gas tight spectroelectrochemical measurement cell.

The mechanisms of HAMP-mediated signaling in transmembrane receptors.

HAMP domains mediate signal transduction in over 7500 enzyme-coupled receptors represented in all kingdoms of life. The HAMP domain of the putative archaeal receptor Af1503 has a parallel, dimeric, four-helical coiled coil structure, but with unusual core packing, related to canonical packing by concerted axial rotation of the helices. This has led to the gearbox model for signal transduction, whereby the alternate packing modes correspond to signaling states. Here we present structures of a series of Af1503 HAMP variants. We show that substitution of a conserved small side chain within the domain core (A291) for larger residues induces a gradual transition in packing mode, involving both changes in helix rotation and bundle shape, which are most prominent at the C-terminal, output end of the domain. These are correlated with activity and ligand response in vitro and in vivo by incorporating Af1503 HAMP into mycobacterial adenylyl cyclase assay systems.