Testing the procedure of simulated annealing by refining homologous immunoglobulin light-chain dimers

The refinement of antigen-binding fragment structures by the method of simulated annealing was tested. Using the program X-PLOR, we refined the structure of one immunoglobulin light-chain dimer against 2.8 A diffraction data collected for a homologous light-chain dimer. The refinement proceeded smoothly; alpha-carbons of the conserved segments of the domain moved to the positions in the reference structure solved independently. An average movement of approximately 1.5 A for atoms in the variable domains (half of the molecule) was observed. Though the final R-factors and energy terms of the reference and test structures were very similar, some of the chain segments of the hypervariable loops (HVR3s) and the ends of some side chains did not converge to the positions in the reference structure. Therefore, although globally the refinement worked very well, positions of the loops and the side chains that are critical for immunoglobulin function have to be carefully examined by difference Fourier techniques.     

Genetic mapping to 10q23.3-q24.2, in a large Italian pedigree, of a new syndrome showing bilateral cataracts, gastroesophageal reflux, and spastic paraparesis with amyotrophy.

We have recently observed a large pedigree with a new rare autosomal dominant spastic paraparesis. In three subsequent generations, 13 affected individuals presented with bilateral cataracts, gastroesophageal reflux with persistent vomiting, and spastic paraparesis with amyotrophy. Bilateral cataracts occurred in all affected individuals, with the exception of one patient who presented with a chorioretinal dystrophy, whereas clinical signs of spastic paraparesis showed a variable expressivity. Using a genomewide mapping approach, we mapped the disorder to the long arm of chromosome 10 on band q23.3-q24.2, in a 12-cM chromosomal region where additional neurologic disorders have been localized. The spectrum of phenotypic manifestations in this family is reminiscent of a smaller pedigree, reported recently, confirming the possibility of a new syndrome. Finally, the anticipation of symptoms suggests that an unstable trinucleotide repeat may be responsible for the condition.     

Cortical ultrastructure of freeze-substituted protonemata of the mossFunaria hygrometrica

Summary The ultrastructural organization of the cortical cytoplasm has been examined in caulonemata, branches and buds of the mossFunaria hygrometrica, which were prepared by rapid freeze-fixation and freeze-substitution (FS). The same structural components occur in the cortex of all three cell types: microtubules (MTs), endoplasmic reticulum (ER), coated and uncoated vesicles, coated pits, and dictyosomes. However, the configuration and density of the cortical ER varies between the three. Caulonemata have an open, polygonal network of ER associated with long MTs oriented mostly parallel to the length of the cell. Lamellar ER, covered with polysomes, is interspersed in the network. Branches have a more tightly arranged ER network, at places occurring in a thick layer, and occasional polysome-decorated lamellae. MTs, which extend to the tip of the branch, are oriented mainly parallel to the cell's long axis and are associated with the cortical ER. Buds have the tightest ER network, which is frequently arranged in a thick layer. Tubules in the polygonal ER of buds are densely covered with ribosomes, whereas tubules in the ER network of caulonemata and branches range from nearly smooth to moderately rough. Closely-spaced ER lamellae, with many polysomes, occur in some buds. The MTs of buds extend into the apical dome and are associated with the cortical ER, but are more randomly oriented than in caulonemata or branches. Close appositions between the ER and PM are observed in all three cells, but are more frequent in buds.Abbreviations DiOC₆(3) 3,3-dihexyloxacarbocyanine iodide - ER endoplasmic reticulum - FS freeze-substitution - MT microtubule - MF microfilament - PM plasma membrane     

Benzimidazole derivatives endowed with potent antileishmanial activity

Two sets of benzimidazole derivatives were synthesised and tested in vitro for activity against promastigotes of Leishmania tropica and L. infantum. Most of the tested compounds resulted active against both Leishmania species, with IC₅₀ values in the low micromolar/sub-micromolar range. Among the set of 2-(long chain)alkyl benzimidazoles, whose heterocyclic head was quaternised, compound 8 resulted about 100-/200-fold more potent than miltefosine, even if the selectivity index (SI) versus HMEC-1 cells was only moderately improved. In the set of 2-benzyl and 2-phenyl benzimidazoles, bearing a basic side chain in position 1, compound 28 (2-(4-chlorobenzyl)-1-lupinyl-5-trifluoromethylbenzimidazole) was 12-/7-fold more potent than miltefosine, but exhibited a further improved SI. Therefore, compounds 8 and 28 represent interesting hit compounds, susceptible of structural modification to improve their safety profiles.     

Identification and characterization of protein cross-links induced by oxidative reactions

Introduction: Protein cross-links are common in biological systems and are generated both deliberately as a part of normal metabolism and also accidently as a result of exposure to external factors such as oxidation and glycation stresses. These cross-links can be both positive and negative for biological function, with high levels of inappropriate cross-links being associated with multiple human pathologies, as well as loss of protein structure and function in the food, agricultural, and pharmaceutical fields.

Areas covered: This review covers recent advances in the detection and identification of protein cross-links arising from oxidation reactions, mediated by both radicals and two-electron oxidants. Information on both enzymatic and nonenzymatic cross-linking is reviewed, both where this is intentional, as part of normal metabolism, and accidental and a potential cause of disease.

Expert commentary: The advantages and drawbacks of various methods available for the detection, identification, and quantification of these species are discussed, as well as some of the mechanisms and processes known to give rise to these species.     

Effect of aging on mechanical stresses,deformations, and hemodynamics in human femoropopliteal artery due to limb flexion

Femoropopliteal artery (FPA) reconstructions are notorious for poor clinical outcomes. Mechanical and flow conditions that occur in the FPA with limb flexion are thought to play a significant role, but are poorly characterized. FPA deformations due to acute limb flexion were quantified using a human cadaver model and used to build a finite element model that simulated surrounding tissue forces associated with limb flexion-induced deformations. Strains and intramural principal mechanical stresses were determined for seven age groups. Computational fluid dynamics analysis was performed to assess hemodynamic variables. FPA shape, stresses, and hemodynamics significantly changed with age. Younger arteries assumed straighter positions in the flexed limb with less pronounced bends and more uniform stress distribution along the length of the artery. Even in the flexed limb posture, FPAs younger than 50 years of age experienced tension, while older FPAs experienced compression. Aging resulted in localization of principal mechanical stresses to the adductor hiatus and popliteal artery below the knee that are typically prone to developing vascular pathology. Maximum principal stresses in these areas increased threefold to fivefold with age with largest increase observed at the adductor hiatus. Atheroprotective wall shear stress reduced after 35 years of age, and atheroprone and oscillatory shear stresses increased after the age of 50. These data can help better understand FPA pathophysiology and can inform the design of targeted materials and devices for peripheral arterial disease treatments.     

Optimising the clinical strategy for autoimmune liver diseases: Principles of value-based medicine

Background

Autoimmune hepatitis, primary biliary cholangitis, and primary sclerosing cholangitis represent the three major autoimmune liver diseases (AILDs). Their management is highly specialized, requires a multidisciplinary approach and often relies on expensive, orphan drugs. Unfortunately, their treatment is often unsatisfactory, and the care pathway heterogeneous across different centers. Disease-specific clinical outcome indicators (COIs) able to evaluate the whole cycle of care are needed to assist both clinicians and administrators in improving quality and value of care. Aim of our study was to generate a set of COIs for the three AILDs. We then prospectively validated these indicators based on a series of consecutive patients recruited at three tertiary clinical centers in Lombardy, Italy.

Discovery of morpholine-based aryl sulfonamides as Nav1.7 inhibitors

Replacement of the piperidine ring in the lead benzenesulfonamide Na_v1.7 inhibitor 1 with a weakly basic morpholine core resulted in a significant reduction in Na_v1.7 inhibitory activity, but the activity was restored by shortening the linkage from methyleneoxy to oxygen. These efforts led to a series of morpholine-based aryl sulfonamides as isoform-selective Na_v1.7 inhibitors. This report describes the synthesis and SAR of these analogs.     

Exploiting a water network to achieve enthalpy-driven,bromodomain-selective BET inhibitors

Within the last decade, the Bromodomain and Extra-Terminal domain family (BET) of proteins have emerged as promising drug targets in diverse clinical indications including oncology, auto-immune disease, heart failure, and male contraception. The BET family consists of four isoforms (BRD2, BRD3, BRD4, and BRDT/BRDT6) which are distinguished by the presence of two tandem bromodomains (BD1 and BD2) that independently recognize acetylated-lysine (KAc) residues and appear to have distinct biological roles. BET BD1 and BD2 bromodomains differ at five positions near the substrate binding pocket: the variation in the ZA channel induces different water networks nearby. We designed a set of congeneric 2- and 3-heteroaryl substituted tetrahydroquinolines (THQ) to differentially engage bound waters in the ZA channel with the goal of achieving bromodomain selectivity. SJ830599 (9) showed modest, but consistent, selectivity for BRD2-BD2. Using isothermal titration calorimetry, we showed that the binding of all THQ analogs in our study to either of the two bromodomains was enthalpy driven. Remarkably, the binding of 9 to BRD2-BD2 was marked by negative entropy and was entirely driven by enthalpy, consistent with significant restriction of conformational flexibility and/or engagement with bound waters. Co-crystallography studies confirmed that 9 did indeed stabilize a water-mediated hydrogen bond network. Finally, we report that 9 retained cytotoxicity against several pediatric cancer cell lines with EC₅₀ values comparable to BET inhibitor (BETi) clinical candidates.     

Effect of intraoperative local application of 3% hydrogen peroxide on pocket infections following cardiac implantable electronic device implantation: An observational study

Aims

The aim of this study was to analyze whether local application of 3% hydrogen peroxide (H2O2) additionally to standard antibiotic prophylaxis following implantation of cardiac implantable electronic devices (CIED) reduces the incidence of pocket infections (PI).