The Histidine Phosphocarrier Kinase/Phosphorylase from Bacillus Subtilis Is an Oligomer in Solution with a High Thermal Stability

The histidine phosphocarrier protein (HPr) kinase/phosphorylase (HPrK/P) modulates the phosphorylation state of the HPr protein, and it is involved in the use of carbon sources by Gram-positive bacteria. Its X-ray structure, as concluded from crystals of proteins from several species, is a hexamer; however, there are no studies about its conformational stability, and how its structure is modified by the pH. We have embarked on the conformational characterization of HPrK/P of Bacillus subtilis (bsHPrK/P) in solution by using several spectroscopic (namely, fluorescence and circular dichroism (CD)) and biophysical techniques (namely, small-angle X-ray-scattering (SAXS) and dynamic light-scattering (DLS)). bsHPrK/P was mainly a hexamer in solution at pH 7.0, in the presence of phosphate. The protein had a high conformational stability, with an apparent thermal denaturation midpoint of ~70 °C, at pH 7.0, as monitored by fluorescence and CD. The protein was very pH-sensitive, precipitated between pH 3.5 and 6.5; below pH 3.5, it had a molten-globule-like conformation; and it acquired a native-like structure in a narrow pH range (between pH 7.0 and 8.0). Guanidinium hydrochloride (GdmCl) denaturation occurred through an oligomeric intermediate. On the other hand, urea denaturation occurred as a single transition, in the range of concentrations between 1.8 and 18 µM, as detected by far-UV CD and fluorescence.     

Adenovirus-Mediated Inducible Expression of a PD-L1 Blocking Antibody in Combination with Macrophage Depletion Improves Survival in a Mouse Model of Peritoneal Carcinomatosis

Immune checkpoint inhibitors (ICIs) have demonstrated remarkable efficacy in a growing number of malignancies. However, overcoming primary or secondary resistances is difficult due to pharmacokinetics issues and side effects associated with high systemic exposure. Local or regional expression of monoclonal antibodies (mAbs) using gene therapy vectors can alleviate this problem. In this work, we describe a high-capacity adenoviral vector (HCA-EFZP-aPDL1) equipped with a mifepristone-inducible system for the controlled expression of an anti-programmed death ligand 1 (PD-L1) blocking antibody. The vector was tested in an immune-competent mouse model of colorectal cancer based on implantation of MC38 cells. A single local administration of HCA-EFZP-aPDL1 in subcutaneous lesions led to a significant reduction in tumor growth with minimal release of the antibody in the circulation. When the vector was tested in a more stringent setting (rapidly progressing peritoneal carcinomatosis), the antitumor effect was marginal even in combination with other immune-stimulatory agents such as polyinosinic-polycytidylic acid (pI:C), blocking mAbs for T cell immunoglobulin, mucin-domain containing-3 (TIM-3) or agonistic mAbs for 4-1BB (CD137). In contrast, macrophage depletion by clodronate liposomes enhanced the efficacy of HCA-EFZP-aPDL1. These results highlight the importance of addressing macrophage-associated immunoregulatory mechanisms to overcome resistance to ICIs in the context of colorectal cancer.     

Microencapsulated Bifidobacterium bifidum and Lactobacillus gasseri in Combination with Quercetin Inhibit Colorectal Cancer Development in ApcMin/+ Mice

Recent studies have suggested that flavonoids such as quercetin and probiotics such as Bifidobacterium bifidum (Bf) and Lactobacillus gasseri (Lg) could play a relevant role in inhibiting colon cancer cell growth. Our study investigated the role of dietary supplementation with microencapsulated probiotics (Bf and Lg) along with quercetin in the development of mouse colorectal cancer (CRC). Methods: Adenomatous polyposis coli/multiple intestinal neoplasia (Apc^Min/+) mice were fed a standard diet or the same diet supplemented with microencapsulated probiotics (Bf and Lg strains, 10⁷ CFU/100 g food) or both probiotics strains plus microencapsulated quercetin (15 mg/100 g food) for 73 days. Changes in body and organ weights, energy metabolism, intestinal microbiota, and colon tissue were determined. The expression of genes related to the Wnt pathway was also analyzed in colon samples. Results: Dietary supplementation with microencapsulated probiotics or microencapsulated probiotics plus quercetin reduced body weight loss and intestinal bleeding in Apc^Min/+ mice. An improvement in energy expenditure was observed after 8 weeks but not after 10 weeks of treatment. A supplemented diet with microencapsulated Bf and Lg reduced the number of aberrant crypt foci (ACF) and adenomas by 45% and 60%, respectively, whereas the supplementation with Bf, Lg and quercetin decreased the number of ACF and adenomas by 57% and 80%, respectively. Microencapsulated Bf and Lg in combination with quercetin could exert inhibition of the canonical Wnt/β-catenin signaling pathway in the colon of Apc^Min/+ mice Conclusions: The administration of microencapsulated Bf and Lg, individually or in combination with quercetin, inhibits the CRC development in Apc^Min/+ mice.     

Virtual Screening of FDA-Approved Drugs against Triose Phosphate Isomerase from Entamoeba histolytica and Giardia lamblia Identifies Inhibitors of Their Trophozoite Growth Phase

Infectious diseases caused by intestinal protozoan, such as Entamoeba histolytica (E. histolytica) and Giardia lamblia (G. lamblia) are a worldwide public health issue. They affect more than 70 million people every year. They colonize intestines causing primarily diarrhea; nevertheless, these infections can lead to more serious complications. The treatment of choice, metronidazole, is in doubt due to adverse effects and resistance. Therefore, there is a need for new compounds against these parasites. In this work, a structure-based virtual screening of FDA-approved drugs was performed to identify compounds with antiprotozoal activity. The glycolytic enzyme triosephosphate isomerase, present in both E. histolytica and G. lamblia, was used as the drug target. The compounds with the best average docking score on both structures were selected for the in vitro evaluation. Three compounds, chlorhexidine, tolcapone, and imatinib, were capable of inhibit growth on G. lamblia trophozoites (0.05–4.935 μg/mL), while folic acid showed activity against E. histolytica (0.186 μg/mL) and G. lamblia (5.342 μg/mL).     

Human-Induced Neural and Mesenchymal Stem Cell Therapy Combined with a Curcumin Nanoconjugate as a Spinal Cord Injury Treatment

We currently lack effective treatments for the devastating loss of neural function associated with spinal cord injury (SCI). In this study, we evaluated a combination therapy comprising human neural stem cells derived from induced pluripotent stem cells (iPSC-NSC), human mesenchymal stem cells (MSC), and a pH-responsive polyacetal–curcumin nanoconjugate (PA-C) that allows the sustained release of curcumin. In vitro analysis demonstrated that PA-C treatment protected iPSC-NSC from oxidative damage in vitro, while MSC co-culture prevented lipopolysaccharide-induced activation of nuclear factor-κB (NF-κB) in iPSC-NSC. Then, we evaluated the combination of PA-C delivery into the intrathecal space in a rat model of contusive SCI with stem cell transplantation. While we failed to observe significant improvements in locomotor function (BBB scale) in treated animals, histological analysis revealed that PA-C-treated or PA-C and iPSC-NSC + MSC-treated animals displayed significantly smaller scars, while PA-C and iPSC-NSC + MSC treatment induced the preservation of β-III Tubulin-positive axons. iPSC-NSC + MSC transplantation fostered the preservation of motoneurons and myelinated tracts, while PA-C treatment polarized microglia into an anti-inflammatory phenotype. Overall, the combination of stem cell transplantation and PA-C treatment confers higher neuroprotective effects compared to individual treatments.     

Curing of mixtures of epoxy resins and 4‐methyl‐1,3‐dioxolan‐2‐one with several initiators

Diglycidyl ether of bisphenol A or 3,4‐epoxycyclohexylmethyl 3,4‐epoxycyclohexane carboxylate were mixed with different proportions of 4‐methyl‐1,3‐dioxolan‐2‐one and cured using lanthanide triflates as initiators. In order to compare the materials obtained, conventional initiators such as boron trifluoride complexes and N,N‐dimethylaminopyridine were also tested. The curing process was followed by differential scanning calorimetry (DSC) and Fourier transform IR in attenuated total reflectance mode. This technique proved that the carbonate accelerates the curing process because it helps to form the active initiating species, although it was not chemically incorporated into the network and remained entrapped in the material. The DSC kinetic study was also reported. © 2006 Wiley Periodicals Inc. J Appl Polym Sci 102: 2086–2093, 2006     

Transient measurements of adsorption and diffusion in H-ZSM-5 membranes

A transient permeation method presented here not only determines the adsorption and diffusion properties of the pores that are the transport pathways through zeolite membranes, but nondestructively estimates the effective thickness of the membrane. Transient responses of the permeate concentration to step changes in the feed were measured on two H-ZSM-5 tubular membranes and modeled assuming Maxwell-Stefan diffusion and Langmuir adsorption. The adsorption isotherms determined from these transient measurements at 298 K of N₂ and CO₂ were nearly identical to those measured by calorimetry on H-ZSM-5 powders. The CH₄ isotherm at 298 K was similar to isotherms measured by calorimetry and gravimetric techniques on Na-ZSM-5 and silicalite powders. The similarity of the isotherms indicates that transport of these light gases occurs mainly through zeolite pores. The Maxwell-Stefan diffusion coefficients D_MS depended on concentration and were higher for higher feed partial pressures. Average D_MS values for the two membranes were 7.5, 5 and 1.5×10⁻¹⁰ m²/s for N₂, CH₄, and CO₂, respectively; these are in the same range and order as diffusion coefficients measured in zeolite crystals.     

Test-data generation for control coverage by proof

Many tools can check if a test set provides control coverage; they are, however, of little or no help when coverage is not achieved and the test set needs to be completed. In this paper, we describe how a formal characterisation of a coverage criterion can be used to generate test data; we present a procedure based on traditional programming techniques like normalisation, and weakest precondition calculation. It is a basis for automation using an algebraic theorem prover. In the worst situation, if automation fails to produce a specific test, we are left with a specification of the compliant test sets. Many approaches to model-based testing rely on formal models of a system under test. Our work, on the other hand, is not concerned with the use of abstract models for testing, but with coverage based on the text of programs.     

Effect of cold environment on hepatic microsomal Δ6 and Δ9 desaturase activity of male rats

Male rats maintained at 24 C and then shifted to 5 C for 5 days increased food intake and decreased in growth rate and food conversion. No modification was observed in Δ6 desaturase activity, while Δ9 desaturase activity decreased after this period of time. These results were confirmed by liver microsomal and mitochondrial fatty acid composition. The phospholipid composition of liver microsomes was unaltered, whereas in mitochondria, phosphatidylcholine and sphingomyelin decreased and phosphatidylethanolamine increased due to the cold environment. The influence of food intake and weight changes on fatty acid metabolism was studied using (i) rats maintained at 5 C with restricted food intake to match the food intake of those kept at 24 C with food ad libitum and (ii) rats maintained at 24 C whose food intake was also restricted so that their growth rate would be the same as that of rats maintained at 5 C with food ad libitum, respectively. These results indicate that the negative metabolic balance state of these cold conditions is not an active factor modifier of Δ6 desaturase activity, whereas it decreases Δ9 desaturase activity, reflecting the lipogenic characteristics of the latter enzyme.     

Electrodialytic extraction of Cu,Pb and Cl from municipal solid waste incineration fly ash suspended in water

The possibility of using fly ash from municipal solid waste incineration (MSWI) in, for example, concrete is considered. MSWI fly ash, however, has too high a concentration of heavy metals, which may cause leaching problems during use or problems with waste handling at the end of the lifetime of the concrete. The Cl content in MSWI fly ash is also too high and will cause corrosion problems in reinforced concrete. The possibility of removing some of the unwanted heavy metals (Cu and Pb) together with Cl from an MSWI fly ash suspended in water using an electrodialytic separation method was investigated. The removal of Pb and Cu was found to be highly pH dependent and the highest contents removed were 41 and 90%, respectively. The Cu concentration of the ash decreased from 2200 to 860 mg kg^?1 but the Pb concentration increased from 8560 to 16 800 mg kg^?1, showing that Pb is mainly found in the ash fraction that is least soluble. Hence electrodialytic treatment of the ash suspended in water is not a solution to improve the ash quality in terms of Pb. The water‐soluble Cl content per unit weight of the original ash was 12.4%. The removal of water‐soluble Cl was efficient and >98% of Cl was removed (calculated on the basis of mean initial and final concentrations). This result indicates that electrodialytic extraction may be a method that can be used for the removal of Cl from ash prior to its utilization in concrete. Copyright © 2006 Society of Chemical Industry