Robust risk aggregation with neural networks.

We consider settings in which the distribution of a multivariate random variable is partly ambiguous. We assume the ambiguity lies on the level of the dependence structure, and that the marginal distributions are known. Furthermore, a current best guess for the distribution, called reference measure, is available. We work with the set of distributions that are both close to the given reference measure in a transportation distance (e.g., the Wasserstein distance), and additionally have the correct marginal structure. The goal is to find upper and lower bounds for integrals of interest with respect to distributions in this set. The described problem appears naturally in the context of risk aggregation. When aggregating different risks, the marginal distributions of these risks are known and the task is to quantify their joint effect on a given system. This is typically done by applying a meaningful risk measure to the sum of the individual risks. For this purpose, the stochastic interdependencies between the risks need to be specified. In practice, the models of this dependence structure are however subject to relatively high model ambiguity. The contribution of this paper is twofold: First, we derive a dual representation of the considered problem and prove that strong duality holds. Second, we propose a generally applicable and computationally feasible method, which relies on neural networks, in order to numerically solve the derived dual problem. The latter method is tested on a number of toy examples, before it is finally applied to perform robust risk aggregation in a real-world instance.

The three-dimensional structure of VIM-31--a metallo-ß-lactamase from Enterobacter cloacae in its native and oxidized form.

The metallo-ß-lactamase VIM-31 differs from VIM-2 by only two Tyr224His and His252Arg substitutions. Located close to the active site, the Tyr224His substitution is also present in VIM-1, VIM-4, VIM-7 and VIM-12. The VIM-31 variant was reported in 2012 from Enterobacter cloacae and kinetically characterized. It exhibits globally lower catalytic efficiencies than VIM-2. In the present study, we report the three-dimensional structures of VIM-31 in its native (reduced) and oxidized forms. The so-called 'flapping-loop' (loop 1) and loop 3 of VIM-31 were not positioned as in VIM-2 but instead were closer to the active site as in VIM-4, resulting in a narrower active site in VIM-31. Also, the presence of His224 in VIM-31 disrupts hydrogen-bonding networks close to the active site. Moreover, a third zinc-binding site, which also exists in VIM-2 structures, could be identified as a structural explanation for the decreased activity of VIM-MBLs at high zinc concentrations.

GES-18, a new carbapenem-hydrolyzing GES-Type ß-lactamase from Pseudomonas aeruginosa that contains Ile80 and Ser170 residues.

A clinical isolate of Pseudomonas aeruginosa recovered from the lower respiratory tract of an 81-year-old patient hospitalized in Belgium was sent to the national reference center to determine its resistance mechanism. PCR sequencing identified a new GES variant, GES-18, which differs from the carbapenem-hydrolyzing enzyme GES-5 by a single amino acid substitution (Val80Ile, in the numbering according to Ambler) and from GES-1 by two substitutions (Val80Ile and Gly170Ser). Detailed kinetic characterization showed that GES-18 and GES-5 hydrolyze imipenem and cefoxitin with similar kinetic parameters and that GES-18 was less susceptible than GES-1 to classical ß-lactamase inhibitors such as clavulanate and tazobactam. The overall structure of GES-18 is similar to the solved structures of GES-1 and GES-2, the Val80Ile and Gly170Ser substitutions causing only subtle local rearrangements. Notably, the hydrolytic water molecule and the Glu166 residue were slightly displaced compared to their counterparts in GES-1. Our kinetic and crystallographic data for GES-18 highlight the pivotal role of the Gly170Ser substitution which distinguishes GES-5 and GES-18 from GES-1.

Kinetic and crystallographic studies of extended-spectrum GES-11, GES-12, and GES-14 ß-lactamases.

GES-1 is a class A extended-spectrum ß-lactamase conferring resistance to penicillins, narrow- and expanded-spectrum cephalosporins, and ceftazidime. However, GES-1 poorly hydrolyzes aztreonam and cephamycins and exhibits very low k(cat) values for carbapenems. Twenty-two GES variants have been discovered thus far, differing from each other by 1 to 3 amino acid substitutions that affect substrate specificity. GES-11 possesses a Gly243Ala substitution which seems to confer to this variant an increased activity against aztreonam and ceftazidime. GES-12 differs from GES-11 by a single Thr237Ala substitution, while GES-14 differs from GES-11 by the Gly170Ser mutation, which is known to confer increased carbapenemase activity. GES-11 and GES-12 were kinetically characterized and compared to GES-1 and GES-14. Purified GES-11 and GES-12 showed strong activities against most tested ß-lactams, with the exception of temocillin, cefoxitin, and carbapenems. Both variants showed a significantly increased rate of hydrolysis of cefotaxime, ceftazidime, and aztreonam. On the other hand, GES-11 and GES-12 (and GES-14) variants all containing Ala243 exhibited increased susceptibility to classical inhibitors. The crystallographic structures of the GES-11 and GES-14 ß-lactamases were solved. The overall structures of GES-11 and GES-14 are similar to that of GES-1. The Gly243Ala substitution caused only subtle local rearrangements, notably in the typical carbapenemase disulfide bond. The active sites of GES-14 and GES-11 are very similar, with the Gly170Ser substitution leading only to the formation of additional hydrogen bonds of the Ser residue with hydrolytic water and the Glu166 residue.

The CphAII protein from Aquifex aeolicus exhibits a metal-dependent phosphodiesterase activity.

The CphAII protein from the hyperthermophile Aquifex aeolicus shows the five conserved motifs of the metallo-ß-lactamase (MBL) superfamily and presents 28% identity with the Aeromonas hydrophila subclass B2 CphA MBL. The gene encoding CphAII was amplified by PCR from the A. aeolicus genomic DNA and overexpressed in Escherichia coli using a pLex-based expression system. The recombinant CphAII protein was purified by a combination of heating (to denature E. coli proteins) and two steps of immobilized metal affinity chromatography. The purified enzyme preparation did not exhibit a ß-lactamase activity but showed a metal-dependent phosphodiesterase activity versus bis-p-nitrophenyl phosphate and thymidine 5'-monophosphate p-nitrophenyl ester, with an optimum at 85°C. The circular dichroism spectrum was in agreement with the percentage of secondary structures characteristic of the MBL αßßα fold.

Effects and mechanisms of angiotensin II receptor blockade with telmisartan in a normotensive model of mesangioproliferative nephritis.

BACKGROUND: Renoprotective actions of angiotensin receptor blockers are not well established in normotensive, low-grade proteinuric glomerular diseases. We examined the effect of low-dose telmisartan (LT) and high-dose telmisartan (HT) versus conventional antihypertensive therapy in the rat anti-Thy1.1 model of glomerulonephritis. METHODS: Rats were randomized on Day 4 after disease induction to no treatment (CT, control), LT or HT or hydrochlorothiazide + hydralazine (HCT + H). RESULTS: All rats remained normotensive: HT and HCT + H reduced blood pressure by 15-20%. LT, HT and HCT + H reduced glomerular endothelial cell proliferation and glomerular and interstitial matrix deposition on Day 14. Only HT reduced podocyte damage and tubular cell dedifferentiation on Day 9 and mesangial cell activation on Day 14. By gene expression analysis arrays, we identified discs-large homolog 1 and angiopoietin-like 4 as potential mediators of the HT effects. In addition, we identified several pathways possibly related to the pleiotropic effects of HT, including growth factor signalling, mammalian target of rapamycin signalling, protein ubiquitination, the Wnt-beta catenin pathway and hypoxia signaling. CONCLUSIONS: In summary, treatment with HT, initiated after the induction of disease, ameliorates glomerular and tubulointerstitial damage. We provide the first comprehensive insight into the mechanisms underlying the renoprotective effect of high-dose angiotensin II receptor blockers (ARBs). Our study lays the basis for future investigations on novel pathways affected by ARBs in renal disease.

Broad antibiotic resistance profile of the subclass B3 metallo-ß-lactamase GOB-1, a di-zinc enzyme.

The metallo-ß-lactamase (MBL) GOB-1 was expressed via a T7 expression system in Escherichia coli BL21(DE3). The MBL was purified to homogeneity and shown to exhibit a broad substrate profile, hydrolyzing all the tested ß-lactam compounds efficiently. The GOB enzymes are unique among MBLs due to the presence of a glutamine residue at position 116, a zinc-binding residue in all known class B1 and B3 MBL structures. Here we produced and studied the Q116A, Q116N and Q116H mutants. The substrate profiles were similar for each mutant, but with significantly reduced activity compared with that of the wild-type. In contrast to the Q116H enzyme, which bound two zinc ions just like the wild-type, only one zinc ion is present in Q116A and Q116N. These results suggest that the Q116 residue plays a role in the binding of the zinc ion in the QHH site.

Mercaptophosphonate compounds as broad-spectrum inhibitors of the metallo-beta-lactamases.

Although commercialized inhibitors of active site serine beta-lactamases are currently used in coadministration with antibiotic therapy, no clinically useful inhibitors of metallo-beta-lactamases (MBLs) have yet been discovered. In this paper, we investigated the inhibitory effect of mercaptophosphonate derivatives against the three subclasses of MBLs (B1, B2, and B3). All 14 tested mercaptophosphonates, with the exception of 1a, behaved as competitive inhibitors for the three subclasses. Apart from 13 and 21, all the mercaptophosphonates tested exhibit a good inhibitory effect on the subclass B2 MBL CphA with low inhibition constants (K(i) < 15 muM). Interestingly, compound 18 turned out to be a potent broad spectrum MBL inhibitor. The crystallographic structures of the CphA-10a and CphA-18 complexes indicated that the sulfur atom of 10a and the phosphonato group of 18 interact with the Zn(2+) ion, respectively. Molecular modeling studies of the interactions between compounds 10a and 18 and the VIM-4 (B1), CphA (B2), and FEZ-1 (B3) enzymes brought to light different binding modes depending on the enzyme and the inhibitor, consistent with the crystallographic structures.

Recombinant expression, purification, and functional characterisation of connective tissue growth factor and nephroblastoma-overexpressed protein.

The CCN family of proteins, especially its prominent member, the Connective tissue growth factor (CTGF/CCN2) has been identified as a possible biomarker for the diagnosis of fibrotic diseases. As a downstream mediator of TGF-ß1 signalling, it is involved in tissue scarring, stimulates interstitial deposition of extracellular matrix proteins, and promotes proliferation of several cell types. Another member of this family, the Nephroblastoma-Overexpressed protein (NOV/CCN3), has growth-inhibiting properties. First reports further suggest that these two CCN family members act opposite to each other in regulating extracellular matrix protein expression and reciprocally influence their own expression when over-expressed. We have established stable HEK and Flp-In-293 clones as productive sources for recombinant human CCN2/CTGF. In addition, we generated an adenoviral vector for recombinant expression of rat NOV and established protocols to purify large quantities of these CCN proteins. The identity of purified human CCN2/CTGF and rat CCN3/NOV was proven by In-gel digest followed by ESI-TOF/MS mass spectrometry. The biological activity of purified proteins was demonstrated using a Smad3-sensitive reporter gene and BrdU proliferation assay in permanent cell line EA•hy 926 cells. We further demonstrate for the first time that both recombinant CCN proteins are N-glycosylated.

The structure of the dizinc subclass B2 metallo-beta-lactamase CphA reveals that the second inhibitory zinc ion binds in the histidine site.

Bacteria can defend themselves against beta-lactam antibiotics through the expression of class B beta-lactamases, which cleave the beta-lactam amide bond and render the molecule harmless. There are three subclasses of class B beta-lactamases (B1, B2, and B3), all of which require Zn2+ for activity and can bind either one or two zinc ions. Whereas the B1 and B3 metallo-beta-lactamases are most active as dizinc enzymes, subclass B2 enzymes, such as Aeromonas hydrophila CphA, are inhibited by the binding of a second zinc ion. We crystallized A. hydrophila CphA in order to determine the binding site of the inhibitory zinc ion. X-ray data from zinc-saturated crystals allowed us to solve the crystal structures of the dizinc forms of the wild-type enzyme and N220G mutant. The first zinc ion binds in the cysteine site, as previously determined for the monozinc form of the enzyme. The second zinc ion occupies a slightly modified histidine site, where the conserved His118 and His196 residues act as metal ligands. This atypical coordination sphere probably explains the rather high dissociation constant for the second zinc ion compared to those observed with enzymes of subclasses B1 and B3. Inhibition by the second zinc ion results from immobilization of the catalytically important His118 and His196 residues, as well as the folding of the Gly232-Asn233 loop into a position that covers the active site.

Preliminary X-ray analysis of a human V(H) fragment at 1.8 A resolution.

Small antibody fragments are more useful than full-size antibodies for achieving efficient biodistribution. As a first step towards the design of a clinically desirable antibody fragment, the crystallization of a human V(H) fragment has been achieved. The fragment was derived from the single-chain antibody scFvM12, which recognizes a cancer-specific hypoglycosylated form of mucin. The V(H) fragment was obtained by in-drop digestion of the scFvM12 with a low concentration of the broad-spectrum protease subtilisin Carlsberg. The crystal belongs to the monoclinic space group C2. The crystal diffracted to 1.8 A resolution when analysed at 100 K using a rotating-anode X-ray generator.

The recombinant anti-EGF receptor immunotoxin 425(scFv)-ETA' suppresses growth of a highly metastatic pancreatic carcinoma cell line.

Pancreatic carcinoma still has the highest mortality rate in comparison to any other malignancy. Major reasons are late detection of disease, highly aggressive tumor growth and the early formation of metastases. Thus, novel effective therapies are urgently needed to improve the outcome of the patients. Overexpression of the epidermal growth factor receptor (EGFR) and its ligands has been implicated in the oncogenesis of pancreatic carcinoma and associated with an unfavorable prognosis. Consequently, the EGFR represents a specific target antigen suitable for immunotherapy. We generated a recombinant immunotoxin by fusing the anti-EGFR single chain fragment 425(scFv) to a truncated mutant of Pseudomonas Exotoxin A (ETA'). Using the expression vector pBM1.1, functional 425(scFv)-ETA' was periplasmically expressed under osmotic stress conditions in the presence of compatible solutes. The 72 kDa His10-tagged fusion protein was purified by a combination of metal-ion affinity and molecular size chromatography. Binding activity and specificity of the immunotoxin to the EGFR-positive pancreatic carcinoma cell line L3.6pl was confirmed by flow cytometry and ELISA. Finally, 425(scFv)-ETA' showed significant toxicity toward this cell line reaching 50% inhibition of cell proliferation at a concentration (IC50) of 7.5 ng/ml. This is the first report documenting the specific cytotoxicity of a recombinant immunotoxin towards metastatic pancreatic carcinoma cells, suggesting that EGFR-specific antibody toxins may become valuable therapeutic reagents for the treatment of pancreatic carcinoma.