Dominant negative phenotype of Bacillus thuringiensis Cry1Ab, Cry11Aa and Cry4Ba mutants suggest hetero-oligomer formation among different Cry toxins.

Background - Bacillus thuringiensis Cry toxins are used worldwide in the control of different insect pests important in agriculture or in human health. The Cry proteins are pore-forming toxins that affect the midgut cell of target insects. It was shown that non-toxic Cry1Ab helix a-4 mutants had a dominant negative (DN) phenotype inhibiting the toxicity of wildtype Cry1Ab when used in equimolar or sub-stoichiometric ratios (1:1, 0.5:1, mutant:wt) indicating that oligomer formation is a key step in toxicity of Cry toxins. Methodology/Principal Findings - The DN Cry1Ab-D136N/T143D mutant that is able to block toxicity of Cry1Ab toxin, was used to analyze its capacity to block the activity against Manduca sexta larvae of other Cry1 toxins, such as Cry1Aa, Cry1Ac, Cry1Ca, Cry1Da, Cry1Ea and Cry1Fa. Cry1Ab-DN mutant inhibited toxicity of Cry1Aa, Cry1Ac and Cry1Fa. In addition, we isolated mutants in helix a-4 of Cry4Ba and Cry11Aa, and demonstrate that Cry4Ba-E159K and Cry11Aa-V142D are inactive and completely block the toxicity against Aedes aegypti of both wildtype toxins, when used at sub-stoichiometric ratios, confirming a DN phenotype. As controls we analyzed Cry1Ab-R99A or Cry11Aa-E97A mutants that are located in helix a-3 and are affected in toxin oligomerization. These mutants do not show a DN phenotype but were able to block toxicity when used in 10:1 or 100:1 ratios (mutant:wt) probably by competition of binding with toxin receptors. Conclusions/Significance - We show that DN phenotype can be observed among different Cry toxins suggesting that may interact in vivo forming hetero-oligomers. The DN phenotype cannot be observed in mutants affected in oligomerization, suggesting that this step is important to inhibit toxicity of other toxin

Biofunctionalized Polymer Nanomaterials: Implications on Shapes and Sizes

Nanotechnology has been one of the most widely used tools in various industries such as pharmaceutical, food, and chemistry, among others, for the encapsulation of compounds or even microorganisms. However, an analysis of the methodologies or polymer matrices to be used is rarely generated, and these in turn contribute to the objective of the product that is intended to be designed. In addition to the evaluation of its physicochemical, optical, and rheological characteristics, and others, are a set of technological tools that allow predicting the stability of a colloid, however, some of the factors that have less importance as the effect of the synthesis process on the shape and size that a structure may have, studies have been carried out to evaluate this phenomenon, which has become a determining factor in the design of any nanoscale material

Short-term regulation of peptide YY secretion by a mixed meal or peritoneal glucose-based dialysate in patients with chronic renal failure

This is a pre-copyedited, author-produced version of an article accepted for publication in "Nephrology Dialysis Trasnplantation" following peer review. The version of the record is avaliable online at Oxford Academic website.Instituto de Salud Carlos III, PI051024Instituto de Salud Carlos III, PI070413Xunta de Galicia, PS07/12Xunta de Galicia, 2006/2

Monte Carlo flattening filter design to high energy intraoperative electron beam homogenization

[EN] Intraoperative radiotherapy using mobile linear accelerators is used for a wide variety of malignancies. However, when large fields are used in combination with high energies, a deterioration of the flatness dose profile is measured with respect to smaller fields and lower energies. Indeed, for the LIAC HWL of Sordina, this deterioration is observed for the 12 MeV beam combined with 10 cm (or larger) diameter applicator. Aimed to solve this problem, a flattening filter has been designed and validated evaluating the feasibility of its usage at the upper part of the applicator. The design of the filter was based on Monte Carlo simulations because of its accuracy in modeling components of clinical devices, among other purposes. The LIAC 10 cm diameter applicator was modeled and simulated independently by two different research groups using two different MC codes, reproducing the heterogeneity of the 12 MeV energy beam. Then, an iterative process of filter design was carried out. Finally, the MC designed conical filter with the optimal size and height to obtain the desired flattened beam was built in-house using a 3D printer. During the experimental validation of the applicator-filter, percentage depth dose, beam profiles, absolute and peripheral dose measurements were performed to demonstrate the effectiveness of the filter addition in the applicator. These measurements conclude that the beam has been flattened, from 5.9% with the standard configuration to 1.6% for the configuration with the filter, without significant increase of the peripheral dose. Consequently, the new filter-applicator LIAC configuration can be used also in a conventional surgery room. A reduction of 16% of the output dose and a reduction of 1.1 mm in the D50 of the percentage depth dose was measured with respect to the original configuration. This work is a proof-of-concept that demonstrates that it is possible to add a filter able to flatten the beam delivered by the Sordina LIAC HWL. Future studies will focus on more refined technical solutions fully compatible with the integrity of the applicator, including its sterilization, to be safely introduced in the clinical practice.The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Giuseppe Felici reports a relationship with S.I.T. Sordina IORT Technologies S.p.A. that includes: employment. We thank the professionals and facilities at the 3D printing and metrology department in the Mechanics Unit at IFIC. JV thanks I. Diaz for measuring the PLA density. We thank S.I.T. SORDINA IORT Tech-nologies SpA for providing the applicator used for testing the filter during the measurements. JV, FB, and JP would like to acknowledge the Spanish "Ministerio de Ciencia e Innovacion" (MCIN) grant PID2021-125096NB-I00 funded by MCIN/AEI/10.13039 and the "Generalitat Valenciana" (GVA) grant PROMETEO/2021/064.Oliver-Gil, S.; Vijande, J.; Tejedor-Aguilar, N.; Miró Herrero, R.; Rovira-Escutia, JJ.; Ballester, F.; Juste-Vidal, B.... (2023). Monte Carlo flattening filter design to high energy intraoperative electron beam homogenization. Radiation Physics and Chemistry. 212. https://doi.org/10.1016/j.radphyschem.2023.11110221

Physiological lentiviral vectors for the generation of improved CAR-T cells

Anti-CD19 chimeric antigen receptor (CAR)-T cells have achieved impressive outcomes for the treatment of relapsed and refractory B-lineage neoplasms. However, important limitations still remain due to severe adverse events (i.e., cytokine release syndrome and neuroinflammation) and relapse of 40%-50% of the treated patients. Most CAR-T cells are generated using retroviral vectors with strong promoters that lead to high CAR expression levels, tonic signaling, premature exhaustion, and overstimulation, reducing efficacy and increasing side effects. Here, we show that lentiviral vectors (LVs) expressing the transgene through a WAS gene promoter (AW-LVs) closely mimic the T cell receptor (TCR)/CD3 expression kinetic upon stimulation. These AW-LVs can generate improved CAR-T cells as a consequence of their moderate and TCR-like expression profile. Compared with CAR-T cells generated with human elongation factor alpha (EF1 alpha)-driven-LVs, AW-CAR-T cells exhibited lower tonic signaling, higher proportion of naive and stem cell memory T cells, less exhausted phenotype, and milder secretion of tumor necrosis factor alpha (TNF-alpha) and interferon (IFN)-gamma after efficient destruction of CD19(+) lymphoma cells, both in vitro and in vivo. Moreover, we also showed their improved efficiency using an in vitro CD19(+) pancreatic tumor model. We finally demonstrated the feasibility of large-scale manufacturing of AW-CAR-T cells in guanosine monophosphate (GMP)-like conditions. Based on these data, we propose the use of AWLVs for the generation of improved CAR-T products