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Iara F Kretzer,1,2 Durvanei A Maria,3 Maria C Guido,1 Thaís C Contente,1 Raul C Maranhão1,2 1Laboratory of Metabolism and Lipids, Heart Institute of the Medical School Hospital, 2Department of Clinical Chemistry, Faculty of Pharmaceutical Sciences, University of São Paulo, 3Biochemistry and Biophysics Laboratories, Butantan Institute, São Paulo, Brazil Purpose: Lipid nanoemulsions (LDEs) that bind to low-density lipoprotein (LDL) receptors used as carriers of paclitaxel (PTX) can decrease toxicity and increase PTX antitumoral action. The administration of simvastatin (Simva), which lowers LDL-cholesterol, was tested as an adjuvant to commercial PTX and to PTX associated with LDE (LDE-PTX). Materials and methods: B16F10 melanoma-bearing mice were treated with saline solution or LDE (controls), Simva, PTX, PTX and Simva, LDE-PTX, and LDE-PTX and Simva: PTX dose 17.5 µmol/kg (three intraperitoneal injections, 3 alternate days): Simva 50 mg/kg/day by gavage, 9 consecutive days. Results: Compared with saline controls, 95% tumor-growth inhibition was achieved by LDE-PTX and Simva, 61% by LDE-PTX, 44% by PTX and Simva, and 43% by PTX. Simva alone had no effect. Metastasis developed in only 37% of the LDE-PTX and Simva, 60% in LDE-PTX, and 90% in PTX and Simva groups. Survival rates were higher in LDE-PTX and Simva and in LDE-PTX groups. The LDE-PTX and Simva group presented tumors with reduced cellular density and increased collagen fibers I and III. Tumors from all groups showed reduction in immunohistochemical expression of ICAM, MCP-1, and MMP-9; LDE-PTX and Simva presented the lowest MMP-9 expression. Expression of p21 was increased in the Simva, LDE-PTX, and LDE-PTX and Simva groups. In the Simva and LDE-PTX and Simva groups, expression of cyclin D1, a proliferation and survival promoter of tumor cells, was decreased. Therapy with LDE-PTX and Simva showed negligible toxicity compared with PTX and Simva, which resulted in weight loss and myelosuppression. Conclusion: Simva increased the antitumor activity of PTX carried in LDE but not of PTX commercial presentation, possibly because statins increase the expression of LDL receptors that internalize LDE-PTX. Keywords: lipid nanoparticles, drug delivery, statins, cancer treatment, B16F10 melanoma

Juliana A Moura1, Claudete J Valduga2, Elaine R Tavares1, Iara F Kretzer1,4, Durvanei A Maria3, Raul C Maranhão1,4 1Heart Institute of the Medical School Hospital, University of São Paulo; 2Bandeirante University of São Paulo; 3Butantan Institute, 4Faculty of Pharmaceutical Sciences of the University of São Paulo, São Paulo, Brazil Background: Lipid nanoemulsions that bind to low-density lipoprotein receptors can concentrate chemotherapeutic agents in tissues with low-density lipoprotein receptor overexpression and decrease the toxicity of the treatment. The aim of this study was to develop a new formulation using a lipophilic derivative of methotrexate, ie, didodecyl methotrexate (ddMTX), associated with a lipid nanoemulsion (ddMTX-LDE). Methods: ddMTX was synthesized by an esterification reaction between methotrexate and dodecyl bromide. The lipid nanoemulsion was prepared by four hours of ultrasonication of a mixture of phosphatidylcholine, triolein, and cholesteryloleate. Association of ddMTX with the lipid nanoemulsion was performed by additional cosonication of ddMTX with the previously prepared lipid nanoemulsion. Formulation stability was evaluated, and cell uptake, cytotoxicity, and acute animal toxicity studies were performed. Results: The yield of ddMTX incorporation was 98% and the particle size of LDE-ddMTX was 60 nm. After 48 hours of incubation with plasma, approximately 28% ddMTX was released from the lipid nanoemulsion. The formulation remained stable for at least 45 days at 4°C. Cytotoxicity of LDE-ddMTX against K562 and HL60 neoplastic cells was higher than for methotrexate (50% inhibitory concentration [IC50] 1.6 versus 18.2 mM and 0.2 versus 26 mM, respectively), and cellular uptake of LDE-ddMTX was 90-fold higher than that of methotrexate in K562 cells and 75-fold in HL60 cells. Toxicity of LDE-ddMTX, administered at escalating doses, was higher than for methotrexate (LD50 115 mg/kg versus 470 mg/kg; maximum tolerated dose 47 mg/kg versus 94 mg/kg) in mice. However, the hematological toxicity of LDE-ddMTX was lower than for methotrexate. Conclusion: LDE-ddMTX was stable, and uptake of the formulation by neoplastic cells was remarkably greater than of methotrexate, which resulted in markedly greater cytotoxicity. LDE-ddMTX is thus a promising formulation to be tested in future animal models of cancer or rheumatic disease, wherein methotrexate is widely used. Keywords: nanoparticles, methotrexate, didodecyl methotrexate, drug delivery, cholesterol

ABSTRACT: This report described the use of acoustic radiation force impulse (ARFI) elastography in the diagnosis of two cases of canine malignant peripheral nerve sheath tumors. Both patients presented with lameness in the thoracic limb and no response to previous clinical treatment or trauma history. Physical examination revealed muscular atrophy in one of the thoracic limbs, in both dogs. B-mode axillary ultrasound showed the presence of a predominantly hypoechogenic, heterogeneous, irregular, nonencapsulated nodule with adjacent reactivity, measuring approximately 20.1 mm x 15.4 mm in the first case and 18.3 mm x 10.7 mm in the second. ARFI elastography was performed, showing areas of significant stiffness, with an average shear wave velocity of 4.12 m/s (case 1) and 4.35 m/s (case 2), suggesting malignancy. In both cases, the affected limb was amputated, and the tumors sent for histopathological analysis, which were; subsequently, diagnosed as malignant tumors of the peripheral nerve sheath. The ultrasonographic methods used were essential in the diagnosis and therapeutic conduct.

Developing recombinant Saccharomyces cerevisiae strains capable of transporting and fermenting cellobiose directly is a promising strategy for second-generation ethanol production from lignocellulosic biomass. In this study, we cloned and expressed in the S. cerevisiae CEN.PK2-1C strain an intracellular β-glucosidase (SpBGL7) from Spathaspora passalidarum and co-expressed the cellobiose transporter SiHXT2.4 from Scheffersomyces illinoinensis, and two putative transporters, one from Candida tropicalis (CtCBT1 gene), and one from Meyerozyma guilliermondii (MgCBT2 gene). While all three transporters allowed cell growth on cellobiose, only the MgCBT2 permease allowed cellobiose fermentation, although cellobiose consumption was incomplete. The analysis of the β-glucosidase and transport activities revealed that the cells stopped consuming cellobiose due to a drop in the transport activity. Since ubiquitinylation of lysine residues at the N- or C-terminal domains of the permease are involved in the endocytosis and degradation of sugar transporters, we constructed truncated versions of the permease lacking lysine residues at the C-terminal domain (MgCBT2ΔC), and at both the C- and N-terminal domain (MgCBT2ΔNΔC) and co-expressed these permeases with the SpBGL7 β-glucosidase in an industrial strain. While the strain harboring the MgCBT2ΔC transporter continued to produce incomplete cellobiose fermentations as the wild-type MgCBT2 permease, the strain with the MgCBT2ΔNΔC permease was able to consume and ferment all the cellobiose present in the medium. Thus, our results highlight the importance of expressing cellobiose transporters lacking lysine at the N- and C-terminal domains for efficient cellobiose fermentation by recombinant S. cerevisiae.

Abstract Background Canine hip dysplasia is a common orthopedic disease in veterinary practice. The diagnosis is made by radiographic examinations that evaluate bone alterations associated with hip dysplasia. Although radiographic examination is the gold standard for diagnosis, it does not allow a detailed evaluation of soft tissues such as the joint capsule and periarticular muscles. This study aimed to evaluate the accuracy of B-mode ultrasonography and acoustic radiation force impulse (ARFI) elastography in assessing the joint capsule and periarticular muscles of dogs using the Orthopedic Foundation of Animals (OFA) classification and the distraction index (DI) in the early and late diagnosis of hip dysplasia. This study sought to propose a protocol for the ultrasonographic evaluation of the structures involved in canine hip dysplasia. Methods Radiographic and ultrasonographic evaluations were performed on 108 hip joints of 54 dogs. Thirty dogs were older than 2 years and 24 were aged between 4 and 10 months. Results It was verified that an increase in pectineus muscle stiffness (cutoff value > 2.77 m/s) by elastography in some dysplastic dogs and an increase in the thickness of the joint capsule (cutoff value > 0.9 mm) in B-mode ultrasonography, were associated with a distraction index > 0.5, with both having a positive correlation. In B-mode ultrasonographic evaluation, the presence of signs of degenerative joint disease, such as irregularities of the cranial edge of the acetabulum and femoral head, were associated with a distraction index > 0.5 in canines, with a specificity of 94%. In adult dogs, the findings of degenerative joint disease on ultrasound were associated with a diseased OFA classification (P 1.10 mm was diagnostic for dysplasia in unhealthy dogs by OFA. Conclusions ARFI elastography has shown that the pectineus muscle may experience changes in stiffness in dysplastic animals. Additionally, changes in joint capsule thickness can be identified in B-mode in young and adult dogs with dysplastic joints, which contributes to the diagnosis of hip dysplasia.

Abstract Background Sepsis is a condition characterized by organic dysfunction, leading to hemodynamic instability and high morbidity and mortality rates in humans and animals. Early identification of perfusion changes and appropriate management of sepsis are crucial for improving patient prognosis. Currently, the Systemic Inflammatory Response Syndrome (SIRS) and Sequential Organ Failure Assessment (SOFA) scores are widely studied for sepsis identification and evaluation of organ dysfunction. However, these scores do not assess gastrointestinal involvement, which is common in this condition. Contrast-enhanced ultrasound (CEUS) and Doppler have been considered promising diagnostic techniques for detecting changes in vascularization and microcirculation in a non-invasive and safe manner, particularly in the gastrointestinal system. This study aimed to evaluate duodenal perfusion using CEUS, as well as abdominal aortic and cranial mesenteric artery blood flow using Doppler ultrasound, and systolic arterial pressure (SAP) in 17 bitches with pyometra and in 10 healthy animals. Results The variables were compared between the pyometra and control groups, as well between patients with and without sepsis determined by the SOFA or SIRS scores. Pyometra was found to cause a reduction in abdominal aortic blood flow volume, aortic peak systolic velocity, and resistivity index as evaluated by Doppler ultrasound. Patients with sepsis according to the SOFA criteria only presented lower SAP. In contrast, sepsis animals identified by the SIRS score exhibited lower SAP, aortic peak systolic velocity, aortic blood flow volume, and aortic resistivity index and additionally, higher peak intensity of contrast in the duodenal wall. Conclusions Pyometra causes a reduction in abdominal aortic blood flow, which is more pronounced in animals with sepsis identified by the SIRS criteria. These animals also exhibited a decrease in systolic blood pressure and an increase in duodenal perfusion, as evident by CEUS. However, these changes were not observed in patients with sepsis identified by the SOFA criteria. The alterations in intestinal perfusion observed in animals with sepsis indicate the presence of inflammation or dysfunction. In this regard, CEUS proves to be a valuable technique for detecting subtle changes in tissue hemodynamics that may not be apparent in conventional exams.

Sparse-angle X-ray Computed Tomography (CT) plays a vital role in industrial quality control but leads to an inherent trade-off between scan time and reconstruction quality. Adaptive angle selection strategies try to improve upon this based on the idea that the geometry of the object under investigation leads to an uneven distribution of the information content over the projection angles. Deep Reinforcement Learning (DRL) has emerged as an effective approach for adaptive angle selection in X-ray CT. While previous studies focused on optimizing generic image quality measures using a fixed number of angles, our work extends them by considering a specific downstream task, namely image-based defect detection, and introducing flexibility in the number of angles used. By leveraging prior knowledge about typical defect characteristics, our task-adaptive angle selection method, adaptable in terms of angle count, enables easy detection of defects in the reconstructed images.

For the last 40 years, special education and applied behavior analysis researchers have discussed the notion of social validity, particularly those researchers who conduct single-case research. A substantial part of this discussion has involved critiquing the rigor of methods used to assess social validity. We conducted a scoping literature review focused on describing and integrating different critiques and recommendations about social validity assessment. In a multi-pronged search, we identified 47 articles addressing rigor in social validity assessment. We used inductive content analysis to chart data about specific critiques and identify a framework of seven key questions that organize these critiques across theoretical and methodological dimensions. We describe the key questions, including examples from the reviewed literature that led to their development. The framework may have practical utility for researchers and reviewers in guiding rigorous social validity assessment in intervention research. Implications are discussed for researchers, reviewers, and journal editors.

Abstract Background The majority of knee endoprostheses are cemented. In an earlier study the effects of different cementing techniques on cement penetration were evaluated using a Sawbone model. In this study we used a human cadaver model to study the effect of different cementing techniques on relative motion between the implant and the femoral shaft component under dynamic loading. Methods Two different cementing techniques were tested in a group of 15 pairs of human fresh frozen legs. In one group a conventional cementation technique was used and, in another group, cementation was done using a pressurizing technique. Under dynamic loading that simulated real life conditions relative motion at the bone-implant interface were studied at 20 degrees and 50 degrees flexion. Results In both scenarios, the relative motion anterior was significantly increased by pressure application. Distally, it was the same with higher loads. No significant difference could be measured posteriorly at 20°. At 50° flexion, however, pressurization reduced the posterior relative motion significantly at each load level. Conclusion The use of the pressurizer does not improve the overall fixation compared to an adequate manual cement application. The change depends on the loading, flexion angle and varies in its proportion in between the interface zones.