Soluble TNF-Alpha-Receptors I Are Prognostic Markers in TIPS-Treated Patients with Cirrhosis and Portal Hypertension

Background

TNFα levels are increased in liver cirrhosis even in the absence of infection, most likely owing to a continuous endotoxin influx into the portal blood. Soluble TNFα receptors (sTNFR type I and II) reflect release of the short-lived TNFα, because they are cleaved from the cells after binding of TNFα. The aims were to investigate the circulating levels of soluble TNFR-I and -II in cirrhotic patients receiving TIPS.

Methods

Forty-nine patients with liver cirrhosis and portal hypertension (12 viral, 37 alcoholic) received TIPS for prevention of re-bleeding (n = 14), therapy-refractory ascites (n = 20), or both (n = 15). Portal and hepatic venous blood was drawn in these patients during the TIPS procedure and during the control catheterization two weeks later. sTNFR-I and sTNFR-II were measured by ELISA, correlated to clinical and biochemical characteristics.

Results

Before TIPS insertion, sTNFR-II levels were lower in portal venous blood than in the hepatic venous blood, as well as in portal venous blood after TIPS insertion. No significant differences were measured in sTNFR-I levels. Hepatic venous levels of sTNFR-I above 4.5 ng/mL (p = 0.036) and sTNFR-II above 7 ng/mL (p = 0.05) after TIPS insertion were associated with decreased survival. A multivariate Cox-regression survival analysis identified the hepatic venous levels of sTNFR-I (p = 0.004) two weeks after TIPS, and Child score (p = 0.002) as independent predictors of mortality, while MELD-score was not.

Conclusion

Hepatic venous levels of sTNFR-I after TIPS insertion may predict mortality in patients with severe portal hypertension.     

Conserved Gating Elements in TRPC4 and TRPC5 Channels

TRPC4 and TRPC5 proteins share 65% amino acid sequence identity and form Ca²⁺-permeable nonselective cation channels. They are activated by stimulation of receptors coupled to the phosphoinositide signaling cascade. Replacing a conserved glycine residue within the cytosolic S4–S5 linker of both proteins by a serine residue forces the channels into an open conformation. Expression of the TRPC4_G503S and TRPC5_G504S mutants causes cell death, which could be prevented by buffering the Ca²⁺ of the culture medium. Current-voltage relationships of the TRPC4_G503S and TRPC5_G504S mutant ion channels resemble that of fully activated TRPC4 and TRPC5 wild-type channels, respectively. Modeling the structure of the transmembrane domains and the pore region (S4-S6) of TRPC4 predicts a conserved serine residue within the C-terminal sequence of the predicted S6 helix as a potential interaction site. Introduction of a second mutation (S623A) into TRPC4_G503S suppressed the constitutive activation and partially rescued its function. These results indicate that the S4–S5 linker is a critical constituent of TRPC4/C5 channel gating and that disturbance of its sequence allows channel opening independent of any sensor domain.     

Electronic transitions and heterogeneity of the bacteriophytochrome Pr absorption band: An angle balanced polarization resolved femtosecond VIS pump–IR probe study

Photoisomerization of biliverdin (BV) chromophore triggers the photoresponse in native Agp1 bacteriophytochrome. We discuss heterogeneity in phytochrome Pr form to account for the shape of the absorption profile. We investigated different regions of the absorption profile by angle balanced polarization resolved femtosecond VIS pump–IR probe spectroscopy. We studied the Pr form of Agp1 with its natural chromophore and with a sterically locked 18Et-BV (locked Agp1). We followed the dynamics and orientations of the carbonyl stretching vibrations of ring D and ring A in their ground and electronically excited states. Photoisomerization of ring D is reflected by strong signals of the ring D carbonyl vibration. In contrast, orientational data on ring A show no rotation of ring A upon photoexcitation. Orientational data allow excluding a ZZZasa geometry and corroborates a nontwisted ZZZssa geometry of the chromophore. We found no proof for heterogeneity but identified a new, to our knowledge, electronic transition in the absorption profile at 644 nm (S₀→S₂). Excitation of the S₀→S₂ transition will introduce a more complex photodynamics compared with S₀→S₁ transition. Our approach provides fundamental information on disentanglement of absorption profiles, identification of chromophore structures, and determination of molecular groups involved in the photoisomerization process of photoreceptors.     

Effects of plant diversity,N fertilization,and elevated carbon dioxide on grassland soil N cycling in a long‐term experiment

The effects of global environmental changes on soil nitrogen (N) pools and fluxes have consequences for ecosystem functions such as plant productivity and N retention. In a 13‐year grassland experiment, we evaluated how elevated atmospheric carbon dioxide (CO₂), N fertilization, and plant species richness alter soil N cycling. We focused on soil inorganic N pools, including ammonium and nitrate, and two N fluxes, net N mineralization and net nitrification. In contrast with existing hypotheses, such as progressive N limitation, and with observations from other, often shorter, studies, elevated CO₂ had relatively static and small, or insignificant, effects on soil inorganic N pools and fluxes. Nitrogen fertilization had inconsistent effects on soil N transformations, but increased soil nitrate and ammonium concentrations. Plant species richness had increasingly positive effects on soil N transformations over time, likely because in diverse subplots the concentrations of N in roots increased over time. Species richness also had increasingly positive effects on concentrations of ammonium in soil, perhaps because more carbon accumulated in soils of diverse subplots, providing exchange sites for ammonium. By contrast, subplots planted with 16 species had lower soil nitrate concentrations than less diverse subplots, especially when fertilized, probably due to greater N uptake capacity of subplots with 16 species. Monocultures of different plant functional types had distinct effects on N transformations and nitrate concentrations, such that not all monocultures differed from diverse subplots in the same manner. The first few years of data would not have adequately forecast the effects of N fertilization and diversity on soil N cycling in later years; therefore, the dearth of long‐term manipulations of plant species richness and N inputs is a hindrance to forecasting the state of the soil N cycle and ecosystem functions in extant plant communities.     

Volume regulation of murine T lymphocytes relies on voltage-dependent and two-pore domain potassium channels

A variety of ion channels are supposed to orchestrate the homoeostatic volume regulation in T lymphocytes. However, the relative contribution of different potassium channels to the osmotic volume regulation and in particular to the regulatory volume decrease (RVD) in T cells is far from clear. This study explores a putative role of the newly identified K(2P) channels (TASK1, TASK2, TASK3 and TRESK) along with the voltage-gated potassium channel K(V)1.3 and the calcium-activated potassium channel K(Ca)3.1 in the RVD of murine T lymphocytes, using genetic and pharmacological approaches. K(2P) channel knockouts exerted profound effects on the osmotic properties of murine T lymphocytes, as revealed by reduced water and RVD-related solute permeabilities. Moreover, both genetic and pharmacological data proved a key role of K(V)1.3 and TASK2 channels in the RVD of murine T cells exposed to hypotonic saline. Our experiments demonstrate a leading role of potassium channels in the osmoregulation of T lymphocytes under different conditions. In summary, the present study sheds new light on the complex and partially redundant network of potassium channels involved in the basic physiological process of the cellular volume homeostasis and extends the repertoire of potassium channels by the family of K(2P) channels.     

Fluorescence of phytochrome adducts with synthetic locked chromophores

We performed steady state fluorescence measurements with phytochromes Agp1 and Agp2 of Agrobacterium tumefaciens and three mutants in which photoconversion is inhibited. These proteins were assembled with the natural chromophore biliverdin (BV), with phycoerythrobilin (PEB), which lacks a double bond in the ring C-D-connecting methine bridge, and with synthetic bilin derivatives in which the ring C-D-connecting methine bridge is locked. All PEB and locked chromophore adducts are photoinactive. According to fluorescence quantum yields, the adducts may be divided into four different groups: wild type BV adducts exhibiting a weak fluorescence, mutant BV adducts with about 10-fold enhanced fluorescence, adducts with locked chromophores in which the fluorescence quantum yields are around 0.02, and PEB adducts with a high quantum yield of around 0.5. Thus, the strong fluorescence of the PEB adducts is not reached by the locked chromophore adducts, although the photoconversion energy dissipation pathway is blocked. We therefore suggest that ring D of the bilin chromophore, which contributes to the extended π-electron system of the locked chromophores, provides an energy dissipation pathway that is independent on photoconversion.     

Fic domain-catalyzed adenylylation: insight provided by the structural analysis of the type IV secretion system effector BepA

Numerous bacterial pathogens subvert cellular functions of eukaryotic host cells by the injection of effector proteins via dedicated secretion systems. The type IV secretion system (T4SS) effector protein BepA from Bartonella henselae is composed of an N‐terminal Fic domain and a C‐terminal Bartonella intracellular delivery domain, the latter being responsible for T4SS‐mediated translocation into host cells. A proteolysis resistant fragment (residues 10–302) that includes the Fic domain shows autoadenylylation activity and adenylyl transfer onto Hela cell extract proteins as demonstrated by autoradiography on incubation with α‐[³²P]‐ATP. Its crystal structure, determined to 2.9‐Å resolution by the SeMet‐SAD method, exhibits the canonical Fic fold including the HPFxxGNGRxxR signature motif with several elaborations in loop regions and an additional β‐rich domain at the C‐terminus. On crystal soaking with ATP/Mg²⁺, additional electron density indicated the presence of a PP_i/Mg²⁺ moiety, the side product of the adenylylation reaction, in the anion binding nest of the signature motif. On the basis of this information and that of the recent structure of IbpA(Fic2) in complex with the eukaryotic target protein Cdc42, we present a detailed model for the ternary complex of Fic with the two substrates, ATP/Mg²⁺ and target tyrosine. The model is consistent with an in‐line nucleophilic attack of the deprotonated side‐chain hydroxyl group onto the α‐phosphorus of the nucleotide to accomplish AMP transfer. Furthermore, a general, sequence‐independent mechanism of target positioning through antiparallel β‐strand interactions between enzyme and target is suggested.     

Distribution of Young's Modulus in Porcine Corneas after Riboflavin/UVA-Induced Collagen Cross-Linking as Measured by Atomic Force Microscopy

Riboflavin/UVA-induced corneal collagen cross-linking has become an effective clinical application to treat keratoconus and other ectatic disorders of the cornea. Its beneficial effects are attributed to a marked stiffening of the unphysiologically weak stroma. Previous studies located the stiffening effect predominantly within the anterior cornea. In this study, we present an atomic force microscopy-derived analysis of the depth-dependent distribution of the Young''s modulus with a depth resolution of 5 µm in 8 cross-linked porcine corneas and 8 contralateral controls. Sagittal cryosections were fabricated from every specimen and subjected to force mapping. The mean stromal depth of the zone with effective cross-linking was found to be 219±67 µm. Within this cross-linked zone, the mean Young''s modulus declined from 49±18 kPa at the corneal surface to 46±17 kPa, 33±11 kPa, 17±5 kPa, 10±4 kPa and 10±4 kPa at stromal depth intervals of 0–50 µm, 50–100 µm, 100–150 µm, 150–200 µm and 200–250 µm, respectively. This corresponded to a stiffening by a factor of 8.1 (corneal surface), 7.6 (0–50 µm), 5.4 (50–100 µm), 3.0 (100–150 µm), 1.6 (150–200 µm), and 1.5 (200–250 µm), when compared to the Young''s modulus of the posterior 100 µm. The mean Young''s modulus within the cross-linked zone was 20±8 kPa (2.9-fold stiffening), while it was 11±4 kPa (1.7-fold stiffening) for the entire stroma. Both values were significantly distinct from the mean Young''s modulus obtained from the posterior 100 µm of the cross-linked corneas and from the contralateral controls. In conclusion, we were able to specify the depth-dependent distribution of the stiffening effect elicited by standard collagen cross-linking in porcine corneas. Apart from determining the depth of the zone with effective corneal cross-linking, we also developed a method that allows for atomic force microscopy-based measurements of gradients of Young''s modulus in soft tissues in general.     

In vivo detection of Staphylococcus aureus endocarditis by targeting pathogen-specific prothrombin activation

Coagulase-positive Staphylococcus aureus (S. aureus) is the major causal pathogen of acute endocarditis, a rapidly progressing, destructive infection of the heart valves. Bacterial colonization occurs at sites of endothelial damage, where, together with fibrin and platelets, the bacteria initiate the formation of abnormal growths known as vegetations. Here we report that an engineered analog of prothrombin could be used to detect S. aureus in endocarditic vegetations via noninvasive fluorescence or positron emission tomography (PET) imaging. These prothrombin derivatives bound staphylocoagulase and intercalated into growing bacterial vegetations. We also present evidence for bacterial quorum sensing in the regulation of staphylocoagulase expression by S. aureus. Staphylocoagulase expression was limited to the growing edge of mature vegetations, where it was exposed to the host and co-localized with the imaging probe. When endocarditis was induced with an S. aureus strain with genetic deletion of coagulases, survival of mice improved, highlighting the role of staphylocoagulase as a virulence factor.