Restoring Ureagenesis in Hepatocytes by CRISPR/Cas9-mediated Genomic Addition to Arginase-deficient Induced Pluripotent Stem Cells.

Urea cycle disorders are incurable enzymopathies that affect nitrogen metabolism and typically lead to hyperammonemia. Arginase deficiency results from a mutation in Arg1, the enzyme regulating the final step of ureagenesis and typically results in developmental disabilities, seizures, spastic diplegia, and sometimes death. Current medical treatments for urea cycle disorders are only marginally effective, and for proximal disorders, liver transplantation is effective but limited by graft availability. Advances in human induced pluripotent stem cell research has allowed for the genetic modification of stem cells for potential cellular replacement therapies. In this study, we demonstrate a universally-applicable CRISPR/Cas9-based strategy utilizing exon 1 of the hypoxanthine-guanine phosphoribosyltransferase locus to genetically modify and restore arginase activity, and thus ureagenesis, in genetically distinct patient-specific human induced pluripotent stem cells and hepatocyte-like derivatives. Successful strategies restoring gene function in patient-specific human induced pluripotent stem cells may advance applications of genetically modified cell therapy to treat urea cycle and other inborn errors of metabolism

iPSC-Derived Vascular Cell Spheroids as Building Blocks for Scaffold-Free Biofabrication

Recently a protocol is established to obtain large quantities of human induced pluripotent stem cells (iPSC)-derived endothelial progenitors, called endothelial colony forming cells (ECFC), and of candidate smooth-muscle forming cells (SMFC). Here, the suitability for assembling in spheroids, and in larger 3D cell constructs is tested. iPSC-derived ECFC and SMFC are labeled with tdTomato and eGFP, respectively. Spheroids are formed in ultra-low adhesive wells, and their dynamic proprieties are studied by time-lapse microscopy, or by confocal microscopy. Spheroids are also tested for fusion ability either in the wells, or assembled on the Regenova 3D bioprinter which laces them in stainless steel micro-needles (the “Kenzan” method). It is found that both ECFC and SMFC formed spheroids in about 24 h. Fluorescence monitoring indicated a continuous compaction of ECFC spheroids, but stabilization in those prepared from SMFC. In mixed spheroids, the cell distribution changed continuously, with ECFC relocating to the core, and showing pre-vascular organization. All spheroids have the ability of in-well fusion, but only those containing SMFC are robust enough to sustain assembling in tubular structures. In these constructs a layered distribution of alpha smooth muscle actin-positive cells and extracellular matrix deposition is found. In conclusion, iPSC-derived vascular cell spheroids represent a promising new cellular material for scaffold-free biofabrication

Low-frequency cortical activity is a neuromodulatory target that tracks recovery after stroke.

Recent work has highlighted the importance of transient low-frequency oscillatory (LFO; <4 Hz) activity in the healthy primary motor cortex during skilled upper-limb tasks. These brief bouts of oscillatory activity may establish the timing or sequencing of motor actions. Here, we show that LFOs track motor recovery post-stroke and can be a physiological target for neuromodulation. In rodents, we found that reach-related LFOs, as measured in both the local field potential and the related spiking activity, were diminished after stroke and that spontaneous recovery was closely correlated with their restoration in the perilesional cortex. Sensorimotor LFOs were also diminished in a human subject with chronic disability after stroke in contrast to two non-stroke subjects who demonstrated robust LFOs. Therapeutic delivery of electrical stimulation time-locked to the expected onset of LFOs was found to significantly improve skilled reaching in stroke animals. Together, our results suggest that restoration or modulation of cortical oscillatory dynamics is important for the recovery of upper-limb function and that they may serve as a novel target for clinical neuromodulation

Gene expression profiling of tumour epithelial and stromal compartments during breast cancer progression

The progression of ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) marks a critical step in the evolution of breast cancer. There is some evidence to suggest that dynamic interactions between the neoplastic cells and the tumour microenvironment play an important role. Using the whole-genome cDNA-mediated annealing, selection, extension and ligation assay (WG-DASL, Illumina), we performed gene expression profiling on 87 formalin-fixed paraffin-embedded (FFPE) samples from 17 patients consisting of matched IDC, DCIS and three types of stroma: IDC-S ( 10 mm from IDC or DCIS). Differential gene expression analysis was validated by quantitative real time-PCR, immunohistochemistry and immunofluorescence. The expression of several genes was down-regulated in stroma from cancer patients relative to normal stroma from reduction mammoplasties. In contrast, neoplastic epithelium underwent more gene expression changes during progression, including down regulation of SFRP1. In particular, we observed that molecules related to extracellular matrix (ECM) remodelling (e.g. COL11A1, COL5A2 and MMP13) were differentially expressed between DCIS and IDC. COL11A1 was overexpressed in IDC relative to DCIS and was expressed by both the epithelial and stromal compartments but was enriched in invading neoplastic epithelial cells. The contributions of both the epithelial and stromal compartments to the clinically important scenario of progression from DCIS to IDC. Gene expression profiles, we identified differential expression of genes related to ECM remodelling, and specifically the elevated expression of genes such as COL11A1, COL5A2 and MMP13 in epithelial cells of IDC. We propose that these expression changes could be involved in facilitating the transition from in situ disease to invasive cancer and may thus mark a critical point in disease development

Feasibility of interleukin-6 receptor blockade in cardiac antibody-mediated rejection

BACKGROUND: Antibody-mediated rejection (AMR) remains a significant cause of heart transplant mortality with few effective therapies. METHODS: This study aimed to describe initial experience of using interleukin-6 receptor blockade with tocilizumab in the treatment of acute cardiac AMR at Barnes-Jewish Hospital/Washington University Transplant Center from July 2017 to May 2021 (n = 7). Clinical, echocardiographic, and serum alloantibody data were analyzed before and after treatment. RESULTS: All participants demonstrated marked improvement in functional status. Echocardiographic data following 4-6 mo of tocilizumab revealed significant improvements in biventricular systolic function for all participants. Consistent reductions in donor-specific HLA or angiotensin type I receptor antibodies were not observed, suggesting that tocilizumab may act downstream of antibody production. No patient experienced drug-related complications that necessitated discontinuation of therapy. CONCLUSIONS: These findings provide initial insights into the safety and efficacy of interleukin-6 receptor blockade in the treatment of cardiac AMR and support the design of larger prospective studies

Automatic detection and segmentation of multiple brain metastases on magnetic resonance image using asymmetric UNet architecture.

Detection of brain metastases is a paramount task in cancer management due both to the number of high-risk patients and the difficulty of achieving consistent detection. In this study, we aim to improve the accuracy of automated brain metastasis (BM) detection methods using a novel asymmetric UNet (asym-UNet) architecture. An end-to-end asymmetric 3D-UNet architecture, with two down-sampling arms and one up-sampling arm, was constructed to capture the imaging features. The two down-sampling arms were trained using two different kernels (3 × 3 × 3 and 1 × 1 × 3, respectively) with the kernel (1 × 1 × 3) dominating the learning. As a comparison, vanilla single 3D UNets were trained with different kernels and evaluated using the same datasets. Voxel-based Dice similarity coefficient (DSCv), sensitivity (S v), precision (P v), BM-based sensitivity (S BM), and false detection rate (F BM) were used to evaluate model performance. Contrast-enhanced T1 MR images from 195 patients with a total of 1034 BMs were solicited from our institutional stereotactic radiosurgery database. The patient cohort was split into training (160 patients, 809 lesions), validation (20 patients, 136 lesions), and testing (15 patients, 89 lesions) datasets. The lesions in the testing dataset were further divided into two subgroups based on the diameters (small S = 1-10 mm, large L = 11-26 mm). In the testing dataset, there were 72 and 17 BMs in the S and L sub-groups, respectively. Among all trained networks, asym-UNet achieved the highest DSCv of 0.84 and lowest F BM of 0.24. Although vanilla 3D-UNet with a single 1 × 1 × 3 kernel achieved the highest sensitivities for the S group, it resulted in the lowest precision and highest false detection rate. Asym-UNet was shown to balance sensitivity and false detection rate as well as keep the segmentation accuracy high. The novel asym-UNet segmentation network showed overall competitive segmentation performance and more pronounced improvement in hard-to-detect small BMs comparing to the vanilla single 3D UNet