Endoscopic vacuum therapy significantly improves clinical outcomes of anastomotic leakages after 2-stage, 3-stage, and transhiatal esophagectomies.

BACKGROUND: Anastomotic leakages after esophagectomies continue to constitute significant morbidity and mortality. Intrathoracic anastomoses pose a high risk for mediastinitis, sepsis, and death, if a leak is not addressed timely and appropriately. However, there are no standardized treatment recommendations or algorithms as for how to treat these leakages. METHODS: The study included all patients at the University Hospital Regensburg, who developed an anastomotic leakage after esophagectomy with gastric pull-up reconstruction from 2007 to 2022. Patients receiving conventional treatment options for an anastomotic leakage (stents, drainage tubes, clips, etc.) were compared to patients receiving endoscopic vacuum-assisted closure (eVAC) therapy as their mainstay of treatment. Treatment failure was defined as cervical esophagostomy formation or death. RESULTS: In total, 37 patients developed an anastomotic leakage after esophagectomy with a gastric pull-up reconstruction. Twenty patients were included into the non-eVAC cohort, whereas 17 patients were treated with eVAC. Treatment failure was observed in 50% of patients (n = 10) in the non-eVAC cohort and in 6% of patients (n = 1) in the eVAC cohort (p < 0.05). The 90-day mortality in the non-eVAC cohort was 15% (n = 3) compared to 6% (n = 1) in the eVAC cohort. Cervical esophagostomy formation was required in 40% of cases (n = 8) in the non-eVAC cohort, whereas no patient in the eVAC cohort underwent cervical esophagostomy formation. CONCLUSION: eVAC therapy for leaking esophagogastric anastomoses appears to be superior to other treatment strategies as it significantly reduces morbidity and mortality. Therefore, we suggest eVAC as an essential component in the treatment algorithm for anastomotic leakages following esophagectomies, especially in patients with intrathoracic anastomoses.

Influence of hepatic fibrosis and inflammation: Correlation between histopathological changes and Gd-EOB-DTPA-enhanced MR imaging.

OBJECTIVE: To evaluate the influence of an active inflammatory process in the liver on Gd-EOB-DTPA-enhanced MR imaging in patients with different degrees of fibrosis/cirrhosis. MATERIAL AND METHODS: Overall, a number of 91 patients (61 men and 30 women; mean age 58 years) were included in this retrospective study. The inclusion criteria for this study were Gd-EOB-DTPA-enhanced MRI of the liver and histopathological evaluation of fibrotic and inflammatory changes. T1-weighted VIBE sequences of the liver with fat suppression were evaluated to determine the relative signal change (RE) between native and hepatobiliary phase (20min). In simple and multiple linear regression analyses, the influence of liver fibrosis/cirrhosis (Ishak score) and the histopathological degree of hepatitis (Modified Hepatic Activity Index, mHAI) on RE were evaluated. RESULTS: RE decreased significantly with increasing liver fibrosis/cirrhosis (p < 0.001) and inflammation (mHAI, p = 0.004). In particular, a correlation between RE and periportal or periseptal boundary zone hepatitis (moth feeding necrosis, mHAI A, p = 0.001) and portal inflammation (mHAI D, p < 0.001) was observed. In multiple linear regression analysis, both the degree of inflammation and the degree of fibrosis were significant predictors for RE (p < 0.01). CONCLUSION: The results of this study suggest that the MR-based hepatic enhancement index RE is not only influenced by the degree of fibrosis, but also by the degree of inflammation.

X-ray, spectroscopic and normal-mode dynamics of calexcitin: structure-function studies of a neuronal calcium-signalling protein.

The protein calexcitin was originally identified in molluscan photoreceptor neurons as a 20 kDa molecule which was up-regulated and phosphorylated following a Pavlovian conditioning protocol. Subsequent studies showed that calexcitin regulates the voltage-dependent potassium channel and the calcium-dependent potassium channel as well as causing the release of calcium ions from the endoplasmic reticulum (ER) by binding to the ryanodine receptor. A crystal structure of calexcitin from the squid Loligo pealei showed that the fold is similar to that of another signalling protein, calmodulin, the N- and C-terminal domains of which are known to separate upon calcium binding, allowing interactions with the target protein. Phosphorylation of calexcitin causes it to translocate to the cell membrane, where its effects on membrane excitability are exerted and, accordingly, L. pealei calexcitin contains two protein kinase C phosphorylation sites (Thr61 and Thr188). Thr-to-Asp mutations which mimic phosphorylation of the protein were introduced and crystal structures of the corresponding single and double mutants were determined, which suggest that the C-terminal phosphorylation site (Thr188) exerts the greatest effects on the protein structure. Extensive NMR studies were also conducted, which demonstrate that the wild-type protein predominantly adopts a more open conformation in solution than the crystallographic studies have indicated and, accordingly, normal-mode dynamic simulations suggest that it has considerably greater capacity for flexible motion than the X-ray studies had suggested. Like calmodulin, calexcitin consists of four EF-hand motifs, although only the first three EF-hands of calexcitin are involved in binding calcium ions; the C-terminal EF-hand lacks the appropriate amino acids. Hence, calexcitin possesses two functional EF-hands in close proximity in its N-terminal domain and one functional calcium site in its C-terminal domain. There is evidence that the protein has two markedly different affinities for calcium ions, the weaker of which is most likely to be associated with binding of calcium ions to the protein during neuronal excitation. In the current study, site-directed mutagenesis has been used to abolish each of the three calcium-binding sites of calexcitin, and these experiments suggest that it is the single calcium-binding site in the C-terminal domain of the protein which is likely to have a sensory role in the neuron.

Structure of the neuronal protein calexcitin suggests a mode of interaction in signalling pathways of learning and memory.

The three-dimensional structure of the neuronal calcium-sensor protein calexcitin from Loligo pealei has been determined by X-ray analysis at a resolution of 1.8A. Calexcitin is up-regulated following Pavlovian conditioning and has been shown to regulate potassium channels and the ryanodine receptor. Thus, calexcitin is implicated in neuronal excitation and plasticity. The overall structure is predominantly helical and compact with a pronounced hydrophobic core between the N and C-terminal domains of the molecule. The structure consists of four EF-hand motifs although only the first three EF hands are involved in binding calcium ions; the C-terminal EF-hand lacks the amino acids required for calcium binding. The overall structure is quite similar to that of the sarcoplasmic calcium-binding protein from Amphioxus although the sequence identity is very low at 31%. The structure shows that the two amino acids of calexcitin phosphorylated by protein kinase C are close to the domain interface in three dimensions and thus phosphorylation is likely to regulate the opening of the domains that is probably required for binding to target proteins. There is evidence that calexcitin is a GTPase and the residues, which have been implicated by mutagenesis in its GTPase activity, are in a short but highly conserved region of 3(10) helix close to the C terminus. This helix resides in a large loop that is partly sandwiched between the N and C-terminal domains suggesting that GTP binding may also require or may cause domain opening. The structure possesses a pronounced electropositive crevice in the vicinity of the 3(10) helix, that might provide an initial docking site for the triphosphate group of GTP. These findings elucidate a number of the reported functions of calexcitin with implications for neuronal signalling.

Normolipemic plane xanthoma associated with adenocarcinoma and severe itch.

Normolipemic plane xanthomas are yellow-red-colored flat patches or plaques with barely palpable borders, under normolipemic conditions usually involving the eyelids, the lateral sides of the neck, the upper aspect of the trunk, or the flexural folds. Histologically the lesions are characterized by an infiltrate consisting of foamy macrophages in the papillary and middermis with a distinct perivascular localization. Plane xanthoma has been associated with monoclonal gammopathy, cryoglobulinemia, and myeloproliferative disorders. We present a patient in whom plane xanthoma developed on the upper aspect of the back, which was accompanied by severe itch in the affected area. These symptoms started 1 month after resection of an adenocarcinoma of the rectum that was complicated by recurrent abdominal abscesses and, currently, by ongoing inflammatory bowel disease. A hypothetic pathophysiologic scheme of events leading to xanthoma formation in this patient is presented.

Solution structure of the LDL receptor EGF-AB pair: a paradigm for the assembly of tandem calcium binding EGF domains.

BACKGROUND: From the observed structure and sequence of a pair of calcium binding (cb) epidermal growth factor-like (EGF) domains from human fibrillin-1, we proposed that many tandem cbEGF domains adopt a conserved relative conformation. The low-density lipoprotein receptor (LDLR), which is functionally unrelated to fibrillin-1, contains a single pair of EGF domains that was chosen for study in the validation of this hypothesis. The LDLR is the protein that is defective in familial hypercholesterolaemia, a common genetic disorder that predisposes individuals to cardiovascular complications and premature death. RESULTS: Here, we present the solution structure of the first two EGF domains from the LDL receptor, determined using conventional NMR restraints and residual dipolar couplings. The cbEGF domains have an elongated, rod-like arrangement, as predicted. The new structure allows a detailed assessment of the consequences of mutations associated with familial hypercholesterolaemia to be made. CONCLUSIONS: The validation of the conserved arrangement of EGF domains in functionally distinct proteins has important implications for structural genomics, since multiple tandem cbEGF pairs have been identified in many essential proteins that are implicated in human disease. Our results provide the means to use homology modeling to probe structure-function relationships in this diverse family of proteins and may hold the potential for the design of novel diagnostics and therapies in the future.

The role of the Src homology 3-Src homology 2 interface in the regulation of Src kinases.

The regulatory fragment of Src kinases, comprising Src homology (SH) 3 and SH2 domains, is responsible for controlled repression of kinase activity. We have used a multidisciplinary approach involving crystallography, NMR, and isothermal titration calorimetry to study the regulatory fragment of Fyn (FynSH32) and its interaction with a physiological activator: a fragment of focal adhesion kinase that contains both phosphotyrosine and polyproline motifs. Although flexible, the preferred disposition of SH3 and SH2 domains in FynSH32 resembles the inactive forms of Hck and Src, differing significantly from LckSH32. This difference, which results from variation in the SH3-SH2 linker sequences, will affect the potential of the regulatory fragments to repress kinase activity. This surprising result implies that the mechanism of repression of Src family members may vary, explaining functional distinctions between Fyn and Lck. The interaction between FynSH32 and focal adhesion kinase is restricted to the canonical SH3 and SH2 binding sites and does not affect the dynamic independence of the two domains. Consequently, the interaction shows no enhancement by an avidity effect. Such an interaction may have evolved to gain specificity through an extended recognition site while maintaining rapid dissociation after signaling.

The relative orientation of the fibronectin 6F1(1)F2 module pair: a 15N NMR relaxation study.

The structure of a pair of modules (6F1(1)F2), that forms part of the collagen-binding region of fibronectin, is refined using heteronuclear relaxation data. A structure of the pair was previously derived from 1H-1H NOE and 3J(HalphaHN) data [Bocquier et al. (1999) Structure, 7, 1451-1460] and a weak module-module interface, comprising Leu19 and Leu28, in 6F1, and Tyr68 in 2F1, was identified. In this study, the definition of the average relative orientation of the two modules is improved using the dependence of 15N relaxation on rotational diffusion anisotropy. This structure refinement is based on the selection of a subset of structures from sets calculated with NOE and 3J(HalphaHN) data alone, using the quality of the fits to the relaxation data as the selection criterion. This simple approach is compared to a refinement strategy where 15N relaxation data are included in the force field as additional restraints [Tjandra et al. (1997) Nat. Struct. Biol., 4, 443-449].

Localization and characterization of the hyaluronan-binding site on the link module from human TSG-6.

BACKGROUND: The interactions of hyaluronan (HA) with proteins are important in extracellular matrix integrity and leukocyte migration and are usually mediated by a domain termed a Link module. Although the tertiary structure of a Link module has been determined, the molecular basis of HA-protein interactions remains poorly understood. RESULTS: Isothermal titration calorimetry was used to characterize the interaction of the Link module from human TSG-6 (Link_TSG6) with HA oligosaccharides of defined length (HA(4)-HA(16)). All oligomers bound (except HA(4)) with K(d) values ranging from 0.2-0.5 microM at 25 degrees C. The reaction is exothermic with a favourable entropy and the thermodynamic profile is similar to those of other glycosaminoglycan-protein interactions. The HA(8) recognition site on Link_TSG6 was localized by comparing nuclear magnetic resonance (NMR) spectra from a 1:1 complex with free protein. Residues perturbed on HA binding include both amino acids that are likely to be directly involved in the interaction (i.e., Lys11, Tyr59, Asn67, Phe70, Lys72 and Tyr78) and those affected by a ligand-induced conformational change in the beta4/beta5 loop. The sidechain of Asn67 becomes more rigid in the complex suggesting that it is in close proximity to the binding site. CONCLUSIONS: In TSG-6 a single Link module is sufficient for a high-affinity interaction with HA. The HA-binding surface on Link_TSG6 is found in a similar position to that suggested previously for CD44, indicating that its location might be conserved across the Link module superfamily. Here we find no evidence for the involvement of linear sequence motifs in HA binding.

Interface characterization of the type II module pair from fibronectin.

The lone (1)F2(2)F2 modular pair of fibronectin is found in the collagen-binding region. This exclusive localization suggests the (1)F2(2)F2 pair plays an important role in the recognition of collagen. However, no information is currently available about the interaction between the two F2 modules and, thus, the orientation of their putative collagen-binding sites with respect to one another. Comparison of a variety of high-resolution NMR parameters from the F2 modules in isolation and the (1)F2(2)F2 pair was used to establish the extent of interaction between the F2 modules in the pair. Chemical shifts of the F2 modules and the (1)F2(2)F2 pair indicate that the structures of the modules are preserved in the pair and that, with the exception of the covalent linkage, they do not interact. (15)N NMR relaxation data identify significant motion occurring in the linker region of the (1)F2(2)F2 pair, and analyses of the anisotropic diffusion properties of the (1)F2(2)F2 pair are consistent with the modules in the F2 pair tumbling independent of one another.

Backbone dynamics of a cbEGF domain pair in the presence of calcium.

Calcium binding (cb) epidermal growth factor-like (EGF) domains are found in a wide variety of extracellular proteins with diverse functions. In several proteins, including the fibrillins (1 and 2), the low-density lipoprotein receptor, the Notch receptor and related molecules, these domains are organised as multiple tandem repeats. The functional importance of calcium-binding by EGF domains has been underscored by the identification of missense mutations associated with defective calcium-binding, which have been linked to human diseases. Here, we present (15)N backbone relaxation data for a pair of cbEGF domains from fibrillin-1, the defective protein in the Marfan syndrome. The data were best fit using a symmetric top model, confirming the extended conformation of the cbEGF domain pair. Our data demonstrate that calcium plays a key role in stabilising the rigidity of the domain pair on the pico- to millisecond time-scale. Strikingly, the most dynamically stable region of the construct is centred about the domain interface. These results provide important insight into the properties of intact fibrillin-1, the consequences of Marfan syndrome causing mutations, and the ultrastructure of fibrillins and other extracellular matrix proteins.

Effects of proline cis-trans isomerization on TB domain secondary structure.

The transforming growth factor beta (TGF-beta) binding protein-like (TB) domain is found principally in proteins localized to extracellular matrix fibrils, including human fibrillin-1, the defective protein in the Marfan syndrome. Analysis of the nuclear magnetic resonance (NMR) data for the sixth TB module from human fibrillin-1 has revealed the existence of two stable conformers that differ in the isomerization states of two proline residues. Unusually, the two isoforms do not readily interconvert and are stable on the time scale of milliseconds. We have computed independent structures of the major and minor conformers of TB6 to assess how the domain fold adjusts to incorporate alternatively cis- or trans-prolines. Based on previous observations, it has been suggested that multiple conformers can only be accommodated in flexible regions of protein structure. In contrast, P22, which exists in trans in the major form and cis in the minor form of TB6, is in a rigid region of the domain, which is confirmed by backbone dynamics measurements. Overall, the structures of the major and minor conformers are similar. However, the secondary structure topologies of the two forms differ as a direct consequence of the changes in proline conformation.

Solution structure of a pair of calcium-binding epidermal growth factor-like domains: implications for the Marfan syndrome and other genetic disorders.

The nuclear magnetic resonance structure of a covalently linked pair of calcium-binding (cb) epidermal growth factor-like (EGF) domains from human fibrillin-1, the protein defective in the Marfan syndrome, is described. The two domains are in a rigid, rod-like arrangement, stabilized by interdomain calcium binding and hydrophobic interactions. We propose a model for the arrangement of fibrillin monomers in microfibrils that reconciles structural and antibody binding data, and we describe a set of disease-causing mutations that provide the first clues to the specificity of cbEFG interactions. The residues involved in stabilizing the domain linkage are highly conserved in fibrillin, fibulin, thrombomodulin, and the low density lipoprotein receptor. We propose that the relative orientation of tandem cbEGF domains in these proteins is similar, but that in others, including Notch, pairs adopt a completely different conformation.

Secondary structure and backbone dynamics of human granulocyte colony-stimulating factor in solution.

The secondary structure and backbone dynamics of the cytokine, human granulocyte colony-stimulating factor (hG-CSF) have been determined by heteronuclear nuclear magnetic resonance (NMR) techniques. Virtually complete NH, C alpha H, C beta H 15N, 13C alpha, and 13C beta assignment of the 175-residue recombinant protein, methionyl-[Cys-17-Ser]-hG-CSF, was achieved by use of three-dimensional (3D) heteronuclear 1H-15N and triple-resonance 1H-15N-13C experiments. Spectra recorded at 750 MHz aided the assignment of severely overlapped regions. The structures of G-CSF from several species have recently been determined by X-ray diffraction [Hill, C. P., Osslund, T. D., & Eisenberg, D. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 5167-5171; Lovejoy, B., Cascio, D., & Eisenberg, D. (1993) J. Mol. Biol. 234, 640-653]. Like several cytokines, hG-CSF has a four-helix topology (A-D) with overhand loop connections, but with an additional helical segment (A') identified in the connection between helix A and helix B. The solution-state determination of the secondary structure is based on short- and medium-range NOEs, backbone J-couplings, and NH exchange data and is corroborated by 13C alpha secondary shifts. The helices are defined as follows: A, 10-38; A',44-53; B, 71-91; C, 102-123; D, 143-172. The dynamics of the amide backbone resonances, investigated using 1H-15N heteronuclear NMR, indicate a rigid protein core with some increased mobility in the AB loop and more pronounced mobility in the CD loop.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of an alcohol education package for non-specialist health care and social workers.

A quasi-experimental non-equivalent control group design was used to assess the influence of a two-day experiential alcohol education package for non-specialist health care and social workers. Four pairs of teams took part in the study: general practice; accident and emergency; medicine for the elderly; and social work. The dual foci of the evaluation were agents' knowledge and attitudes, and these were assessed using a modified version of the Alcohol and Alcohol Problems Perception Questionnaire (AAPPQ). For both variables, the one-month follow-up scores of the education teams were significantly higher than those of the comparisons, although the effect was stronger in the case of therapeutic attitudes than knowledge. There were also significant differences in improvement in attitude scores, with significant effects being observed in the general practice, medicine for the elderly and social work teams but not the accident and emergency. At 6 months, the level of fall-off in improvement varied and this, along with the pattern of change evident in the five components which comprise the AAPPQ attitude scale, was examined and discussed in relation to previous research in this field of inquiry.