The phosphatidylinositol 3‑kinase/mammalian target of rapamycin (PI3K/mTOR) signaling pathway is a therapeutic target for various types of human tumors, and dual PI3K/mTOR inhibitors demonstrate ...antitumor activities in both preclinical and clinical studies. However, resistance mechanisms limit their abilities. As the molecular mechanisms involved in the cellular resistance are not clear in any canine tumors, an understanding of resistance mechanisms would support the potential use of dual PI3K/mTOR inhibitors in canine tumors. The antitumor activity of gedatolisib on cell viability, protein phosphorylation, and cell cycle distribution was assessed using 12 canine tumor cell lines from 6 types of tumors. In addition, the molecular determinants involved in the cellular sensitivity to gedatolisib were explored by investigating the involvement of serum‑and‑glucocorticoid‑induced kinase 1 (SGK1),
, and ATP‑binding cassette, subfamily B, member 1 (ABCB1). The results demonstrated that gedatolisib decreased cell viability in all cell lines, with IC
values <1 µM in 10 of the 12 lines. Gedatolisib inhibited Akt and mTOR complex 1 substrate phosphorylation and induced G
/G
cell cycle arrest. However, certain cell lines with higher IC
values were more resistant to these effects. These cell lines exhibited higher ABCB1 activity and the ABCB1 inhibitor cyclosporin A enhanced the decrease of cell viability caused by gedatolisib. SGK1 overexpression did not confer resistance to gedatolisib. The mutations of E545K and H1047R in
were not observed. The present results indicated that gedatolisib decreased cell viability in canine tumor cell lines and ABCB1 played an important role in gedatolisib resistance, supporting the potential use of gedatolisib for canine tumors.
Infection prevention and control (IPC) in veterinary medicine is crucial to protect patients, owners, staff, and the public. An IPC programme is recommended for every animal hospital. The objective ...of this retrospective longitudinal study was to describe the changes in bacterial and multidrug-resistant (MDR) bacterial isolates and self-reported hand hygiene awareness and practices after an IPC programme to assess the long-term effect of this programme in small animal veterinary medicine. The IPC programme was implemented at our veterinary teaching hospital in April 2018, which included the establishment of an infection control task force, regular IPC lectures and poster campaigns, infrastructure improvement, and manual refinement. Laboratory-based surveillance was retrospectively conducted before and after the programme (January 2016–December 2022). Level and slope changes in bacterial isolates were evaluated using interrupted time-series analysis. Self-reported hand hygiene awareness and practices were assessed using an annual questionnaire. Additionally, hygiene product purchases during the study period were investigated.
The monthly number of total and MDR bacterial isolates decreased significantly after the programme (MDR level change: −0.426; 95% confidence interval: −0.744, −0.109; P = 0.009; and MDR slope change: −0.035; 95% confidence interval: −0.058, −0.011; P = 0.003). Additionally, awareness of hand hygiene before touching animals improved after the programme. Overall self-reported hand hygiene practices improved, and hygiene product purchases significantly increased. These results suggested that the IPC programme may have long-term effects regarding reducing total and MDR bacterial isolates and improving hand hygiene awareness in veterinary medicine.
•Infection prevention and control (IPC) programmes promote IPC like hand hygiene.•The number of bacterial isolates decreased after the IPC programme.•The number of multidrug-resistant bacterial isolates also decreased.•The changes of isolates were minimal after the coronavirus disease 2019 pandemic.•Overall hand hygiene awareness was improved after the IPC programme.
We present the transfer of the spatially variant polarization of topologically structured light to the spatial spin texture in a semiconductor quantum well. The electron spin texture, which is a ...circular pattern with repeating spin-up and spin-down states whose repetition rate is determined by the topological charge, is directly excited by a vector vortex beam with a spatial helicity structure. The generated spin texture efficiently evolves into a helical spin wave pattern owing to the spin-orbit effective magnetic fields in the persistent spin helix state by controlling the spatial wave number of the excited spin mode. By tuning the repetition length and azimuthal angle, we simultaneously generate helical spin waves with opposite phases by a single beam.
•We analyzed effects of somatic mutation on antigen binding and stability using scFvs.•Thermal stability of Tyr95-type is higher than that of Gly95-type.•Gly95-type greatly gains thermal stability ...upon hapten binding.•BCR stability would correlate with B-cell population.
Immune response to T-cell-dependent antigens is highly dynamic; several B-cell clones responsible for antibody production appear alternately during immunization. It was previously shown that at least two-types of antibodies are secreted after immunization with (4-hydroxy-3-nitrophenyl)acetyl (NP); one has Tyr and another has Gly at position 95 of the heavy chain (referred to as Tyr95- and Gly95-type). The former appeared at an early stage, while the latter appeared at a late stage, i.e., after secondary immunization, although Fv domains of these antibodies were encoded by same genes of variable heavy and light chains. We examined whether any biophysical properties of antigen-combing sites relate to this shift in B-cell clones by preparing single-chain Fv (scFv). Thermodynamic and kinetic parameters of the interaction of scFv with various haptens are in accordance with those of intact antibodies, indicating that scFvs are appropriate models for the study on structure and function of antibodies. Next, we measured thermal stability of scFvs using differential scanning calorimetry and found that the apparent melting temperature of free Tyr95-type was 64–66°C,while that of Gly95-type was 47–48°C, indicating that the latter was highly unstable. However, Gly95-type greatly gained thermal stability because of hapten binding. We discussed the relationship between thermal stability resulted by hapten binding and dynamism of antibody response during immunization.
Celiac disease (CD) is an autoimmune inflammatory disease with a relatively high prevalence especially in the western hemisphere. A strong genetic component is involved in the pathogenesis of CD with ...virtually all individuals that develop the disease carrying HLA-DQ alleles that encode specific HLA-DQ2 or HLA-DQ8 heterodimers. Consumption of cereals rich in gluten triggers a chronic intestinal inflammation in genetically susceptible individuals leading to the development of CD. Emerging evidence has implicated a central role for IL-15 in the orchestration and perpetuation of inflammation and tissue destruction in CD. Therefore, IL-15 represents an attractive target for development of new therapies for CD. Transgenic mice that express human IL-15 specifically in enterocytes ($T3^b $-hIL-15 Tg mice) develop villous atrophy and severe duodeno-jejunal inflammation with massive accumulation of NKlike CD8⁺ lymphocytes in the affected mucosa. We used these mice to demonstrate that blockade of IL-15 signaling with an antibody (TM-β1) that binds to murine IL-2/IL-15Rbeta (CD122) leads to a reversal of the autoimmune intestinal damage. The present study, along with work of others, provides the rationale to explore IL-15 blockade as a test of the hypothesis that uncontrolled expression of IL-15 is critical in the pathogenesis and maintenance of refractory CD.
Protein structure dynamics are critical for understanding structure-function relationships. An antibody can recognize its antigen, and can evolve toward the immunogen to increase binding strength, in ...a process referred to as affinity maturation. In this study, a single-chain Fv (scFv) antibody against (4-hydroxy-3-nitrophenyl)acetyl, derived from affinity matured type, C6, was designed to comprise the variable regions of light and heavy chains connected by a (GGGGS)3 linker peptide. This scFv was expressed in Escherichia coli in the insoluble fraction, solubilized in the presence of urea, and refolded by stepwise dialysis. The correctly refolded scFv was purified, and its structural, physical, and functional properties were analyzed using analytical ultracentrifugation, circular dichroism spectrometry, differential scanning calorimetry, and surface plasmon resonance biosensor. Thermal stability of C6 scFv increased greatly upon antigen binding, due to favorable enthalpic contributions. Antigen binding kinetics were comparable to those of the intact C6 antibody. Structural dynamics were analyzed using the diffracted X-ray tracking method, showing that fluctuations were suppressed upon antigen binding. The antigen binding energy determined from the angular diffusion coefficients was in good agreement with that calculated from the kinetics analysis, indicating that the fluctuations detected at single-molecule level are well reflected by antigen binding events.
Multicenter, retrospective cohort study.
The aim of this study was to investigate the occurrence and surgical predictors of postoperative shoulder imbalance (PSI) in Lenke type 2A adolescent ...idiopathic scoliosis (AIS).
Although several studies have investigated the factors influencing PSI in Lenke type 2 curves, no studies have analyzed PSI-related factors considering upper instrumented vertebra (UIV) and lumbar modifier type simultaneously.
Patients with Lenke Type 2A AIS treated by spinal fusion were retrospectively identified and their data were extracted from six spine centers in Japan. Inclusion criteria were age between 10 and 20 years at surgery, UIV = T2, major curve 40° to 90°, and follow-up for 24 to 30 months after surgery. We analyzed patient characteristics, surgical characteristics, and preoperative and immediate-postoperative radiographic parameters. We defined patients with lower instrumented vertebra (LIV) equal or proximal to the last touching vertebra (LTV) as selective thoracic fusion (STF-LTV) and patients with LIV distal to the LTV as non-STF-LTV. t Tests, Mann-Whitney U test, χ2 tests, Fisher exact tests, and multivariate logistic regression were used for statistical analyses.
Among the 99 consecutive patients with a mean follow-up of 25.6 months, PSI was seen in 27 (27.3%) patients immediately after and in 17 (17.2%) patients at 24 to 30 months. The univariate analysis revealed that the significant risk factors of PSI were preoperative radiographical shoulder height, non-STF-LTV, and high main thoracic curve (MTC) correction (immediate-postoperative MTC correction rate: ≥70%), with PSI incidence of 40.0%. The multivariate logistic regression analysis indicated that interaction term of non-STF-LTV and high MTC correction was an independent risk factor for PSI (non-STF-LTV and high MTC correction, odds ratio: 5.167, 95% confidence interval: 1.470-18.159, P = 0.010).
To prevent PSI in Lenke Type 2A AIS patients, surgeons should avoid the combination of non-STF-LTV and high MTC correction in those surgeries with UIV as T2.Level of Evidence: 4.
Functional proteins like antibody, cytokine and growth factor have been widely used for basic biological research, diagnosis and cancer therapy. Particularly, antibody drugs as attractive ...biopharmaceuticals will be expected to create an enormous new market. Chinese hamster ovay (CHO) cells are being increasingly used in industry for the production of recombinant therapeutic proteins including antibody drugs. Although three-dimensional culture is preferred to two-dimensional monolayer culture for the efficient large scale culture of CHO cells and subsequent mass production of recombinant proteins, it has the limitation of low protein production. Therefore, a new cell culture em essentially required for an efficient protein production. Here we report on a new three-dimensional cell culture system as a spheroid cell culture on the micropattern array for efficient production of protein in CHO cells. Furthermore, cocultivation of CHO spheroids with feeder cells including bovine aortic endothelial cells (BAEC) and NIH 3T3 was essential to more increase a protein production. The results indicated that CHO heterospheroids cocultured with BAECs were much superior to either CHO monolayers or CHO homospheroids in protein production. Significantly, the above cocultured spheroids in the serum-free medium drastically enhanced protein expression level up to 3-fold compared with CHO spheroids in serum medium, suggesting that a coculture of spheroid system with feeder layer cells is a promising method to enhance protein production under serum-free condition. The spheroid array constructed here is highly usuful as a platform of biopharmaceutical manufacturing as well as tissue and cell based biosensors to detect a wide variety of clinically active compounds through a cellular physiological response.
PDF
