Protocol to generate traceable CAR T cells for syngeneic mouse cancer models.

The efficacy of chimeric antigen receptor (CAR) T cell immunotherapy is limited by insufficient infiltration and activation of T cells due to the immunosuppressive tumor microenvironment. Preclinical studies with optimized mouse CAR T cells in immunocompetent mouse cancer models will help define the mechanisms underlying immunotherapy resistance. Here, we present a protocol for preparing mouse T cells and generating CAR T cells. We then detail procedures for testing their therapeutic efficacy and tracking them in a syngeneic mouse glioma model. For complete details on the use and execution of this protocol, please refer to Zhang et al.1.

Spatial analysis of tissue immunity and vascularity by light sheet fluorescence microscopy.

The pathogenesis of cancer and cardiovascular diseases is subjected to spatiotemporal regulation by the tissue microenvironment. Multiplex visualization of the microenvironmental components, including immune cells, vasculature and tissue hypoxia, provides critical information underlying the disease progression and therapy resistance, which is often limited by imaging depth and resolution in large-volume tissues. To this end, light sheet fluorescence microscopy, following tissue clarification and immunostaining, may generate three-dimensional high-resolution images at a whole-organ level. Here we provide a detailed description of light sheet fluorescence microscopy imaging analysis of immune cell composition, vascularization, tissue perfusion and hypoxia in mouse normal brains and hearts, as well as brain tumors. We describe a procedure for visualizing tissue vascularization, perfusion and hypoxia with a transgenic vascular labeling system. We provide the procedures for tissue collection, tissue semi-clearing and immunostaining. We further describe standard methods for analyzing tissue immunity and vascularity. We anticipate that this method will facilitate the spatial illustration of structure and function of the tissue microenvironmental components in cancer and cardiovascular diseases. The procedure requires 1-2 weeks and can be performed by users with expertise in general molecular biology.

Impact of Baseline Expectancy on Outcome Prediction of Real and Sham Acupuncture for Persistent Chemotherapy-Induced Peripheral Neuropathy Pain in Solid Tumor Survivors: A Secondary Analysis of a Randomized Clinical Trial.

BACKGROUND: Chemotherapy-induced peripheral neuropathy (CIPN) pain significantly worsens cancer survivors' quality of life. Expectancy may play an important role in acupuncture response. We sought to explore whether expectancy predicts pain outcome in real acupuncture (RA) and sham acupuncture (SA) in cancer survivors. METHODS: We analyzed data from a randomized clinical trial that evaluated the effect of RA on CIPN symptoms compared to SA and wait list control (WLC) in 75 cancer survivors. This secondary analysis was limited to CIPN pain measured by the Numeric Rating Scale (NRS), graded from 0 to 10. Interventions were delivered over 8 weeks. SA was implemented using a combination of non-acupuncture points and a non-insertion procedure. Patient expectancy was measured by the Acupuncture Expectancy Scale (AES) 3 times during the study. We used a linear regression model to evaluate if the NRS score was associated with the baseline AES score at the end of treatment (week 8), adjusting for baseline NRS score. RESULTS: AES was similar among 3 groups at baseline (RA: 11.8 ± 2.7; SA: 12.1 ± 3.8.; WLC: 14.6 ± 4.2; P = .062). Baseline AES was not found to be significantly associated with the week 8 NRS score among patients in all RA, SA, and WLC groups (all P > .05). However, we found a trend that higher baseline AES predicted lower NRS score at week 8 in the SA group: a one-point higher score on baseline expectancy was associated with a 0.3-point reduction in NRS pain score (P = .059) at week 8. CONCLUSIONS: The association of baseline expectancy and acupuncture response was similar between RA and SA. However, SA seemed to rely more on expectancy than RA. Further studies with larger sample sizes are needed to confirm this finding.

Clinical Outcomes of Micropulse Transscleral Cyclophotocoagulation in Patients with a History of Keratoplasty.

PURPOSE: To examine the surgical outcomes and graft conditions in patients receiving micropulse transscleral cyclophotocoagulation (MP-TSCPC) to treat post-keratoplasty ocular hypertension. METHODS: This retrospective observational study included 30 eyes of 28 consecutive glaucoma patients with a history of penetrating keratoplasty (PKP) or Descemet's stripping automated endothelial keratoplasty (DSAEK) who underwent MP-TSCPC at the University of California, San Francisco from 09/2015 to 08/2018. Using the Wilcoxon signed-rank test, we compared preoperative and postoperative intraocular pressure (IOP), number of glaucoma medications, visual acuity, and central corneal thickness at 1, 3, 6, and 12 months. Postoperative complications, additional surgeries, and graft failures were also recorded at these follow-up times. Linear regression model was used to study whether PKP vs. DSAEK affects the effectiveness of MP-TSCPC. RESULTS: Thirty eyes from 28 patients were followed for 12 months. IOP was significantly decreased from preop at all follow-up points (P < 0.001). There was no significant change in the number of glaucoma drops, visual acuity, or CCT. At 12 months, 21 of the 30 eyes met the definition of success, and only one underwent repeat PKP due to graft rejection. The type of corneal transplant was not a significant factor for IOP reduction at the last follow-up. CONCLUSIONS: MP-TSCPC achieved desirable IOP control and success rates for postkeratoplasty patients while resulting in minimal complications and graft failure. It appears to be a safe and effective procedure in patients who received corneal transplant with one-year follow-up.

Numerical Modeling of Sub-Wavelength Anti-Reflective Structures for Solar Module Applications.

This paper reviews the current progress in mathematical modeling of anti-reflective subwavelength structures. Methods covered include effective medium theory (EMT), finite-difference time-domain (FDTD), transfer matrix method (TMM), the Fourier modal method (FMM)/rigorous coupled-wave analysis (RCWA) and the finite element method (FEM). Time-based solutions to Maxwell's equations, such as FDTD, have the benefits of calculating reflectance for multiple wavelengths of light per simulation, but are computationally intensive. Space-discretized methods such as FDTD and FEM output field strength results over the whole geometry and are capable of modeling arbitrary shapes. Frequency-based solutions such as RCWA/FMM and FEM model one wavelength per simulation and are thus able to handle dispersion for regular geometries. Analytical approaches such as TMM are appropriate for very simple thin films. Initial disadvantages such as neglect of dispersion (FDTD), inaccuracy in TM polarization (RCWA), inability to model aperiodic gratings (RCWA), and inaccuracy with metallic materials (FDTD) have been overcome by most modern software. All rigorous numerical methods have accurately predicted the broadband reflection of ideal, graded-index anti-reflective subwavelength structures; ideal structures are tapered nanostructures with periods smaller than the wavelengths of light of interest and lengths that are at least a large portion of the wavelengths considered.

Gibberellin-associated cisgenes modify growth, stature and wood properties in Populus.

We studied the effects on plant growth from insertion of five cisgenes that encode proteins involved in gibberellin metabolism or signalling. Intact genomic copies of PtGA20ox7, PtGA2ox2,Pt RGL1_1, PtRGL1_2 and PtGAI1 genes from the genome-sequenced Populus trichocarpa clone Nisqually-1 were transformed into Populus tremula × alba (clone INRA 717-1B4), and growth, morphology and xylem cell size characterized in the greenhouse. Each cisgene encompassed 1-2 kb of 5' and 1 kb of 3' flanking DNA, as well as all native exons and introns. Large numbers of independent insertion events per cisgene (19-38), including empty vector controls, were studied. Three of the cisgenic modifications had significant effects on plant growth rate, morphology or wood properties. The PtGA20ox7 cisgene increased rate of shoot regeneration in vitro, accelerated early growth, and variation in growth rate was correlated with PtGA20ox7 gene expression. PtRGL1_1 and PtGA2ox2 caused reduced growth, while PtRGL1_2 gave rise to plants that grew normally but had significantly longer xylem fibres. RT-PCR studies suggested that the lack of growth inhibition observed in PtRGL1_2 cisgenic plants was a result of co-suppression. PtGAI1 slowed regeneration rate and both PtGAI1 and PtGA20ox7 gave rise to increased variance among events for early diameter and volume index, respectively. Our work suggests that cisgenic insertion of additional copies of native genes involved in growth regulation may provide tools to help modify plant architecture, expand the genetic variance in plant architecture available to breeders and accelerate transfer of alleles between difficult-to-cross species.