Cell Cycle-Dependent Induction of Homologous Recombination by a Tightly Regulated I-SceI Fusion Protein

Double-strand break repair is executed by two major repair pathways: non-homologous end joining (NHEJ) and homologous recombination (HR). Whereas NHEJ contributes to the repair of ionizing radiation (IR)-induced double strand breaks (DSBs) throughout the cell cycle, HR acts predominantly during the S and G2 phases of the cell cycle. The rare-cutting restriction endonuclease, I-SceI, is in common use to study the repair of site-specific chromosomal DSBs in vertebrate cells. To facilitate analysis of I-SceI-induced DSB repair, we have developed a stably expressed I-SceI fusion protein that enables precise temporal control of I-SceI activation, and correspondingly tight control of the timing of onset of site-specific chromosome breakage. I-SceI-induced HR showed a strong, positive linear correlation with the percentage of cells in S phase, and was negatively correlated with the G1 fraction. Acute depletion of BRCA1, a key regulator of HR, disrupted the relationship between S phase fraction and I-SceI-induced HR, consistent with the hypothesis that BRCA1 regulates HR during S phase

Tuning the SUPRA CAR to differentiate high/low Her2 levels

On-target, off-tumor toxicity has been a major challenge for chimeric antigen receptor (CAR) T cells to clinically treat solid tumors. This is due to conventional CAR T cells poorly discriminating between high and low tumor associated antigen densities, causing them to attack both cancer and healthy cells. Her2 is a tumor associated antigen where it is minimally expressed in healthy cells and overexpressed in cancer cells of Her2 positive cancer patients. Previous case reports demonstrate that targeting Her2 with conventional ɑ-Her2-CAR could be fatal. To reduce the risk of on-target, off-tumor toxicity, an ultrasensitive CAR T cell platform that highly activates against high antigen density and minimally activates against low antigen density is in need. The Wong lab has developed the split, universal and programmable (SUPRA) CAR system for a safer and more effective CAR. Composed of a universal receptor and an exogenous scFv protein, this split CAR allows safe, effective, and flexible control over T cell activity due to its various tunable components. This thesis aims to demonstrate that the SUPRA CAR can differentiate high and low Her2 densities more than that of a conventional ɑ-Her2-CAR

Enhanced Rg3 negatively regulates Th1 cell responses

Background: Korean Red Ginseng (KRG; Panax ginseng Meyer) is a widely used medicinal herb known to exert various immune modulatory functions. KRG and one of its purified components, ginsenoside Rg3, are known to possess anti-inflammatory activities. How they impact helper T cell-mediated responses is not fully explored. In this study, we attempted to evaluate the effect of KRG extract (KRGE) and ginsenoside Rg3 on Th1 cell responses. Methods: Using well-characterized T cell in vitro differentiation systems, we examined the effects of KRGE or enhanced Rg3 on the Th1-inducing cytokine production from dendritic cells (DC) and the naïve CD4+ T cells differentiation to Th1 cells. Furthermore, we examined the change of Th1 cell population in the intestine after treatment of enhanced Rg3. The influence of KRGE or enhanced Rg3 on Th1 cell differentiation was evaluated by fluorescence-activated cell sorting, enzyme-linked immunosorbent assay, and quantitative real-time polymerase chain reaction. Results: KRGE significantly inhibited the production level of IL-12 from DCs and subsequent Th1 cell differentiation. Similarly, enhanced Rg3 significantly suppressed the expression of interferon gamma (IFNγ) and T-bet in T cells under Th1-skewing condition. Consistent with these effects in vitro, oral administration of enhanced Rg3 suppressed the frequency of Th1 cells in the Peyer's patch and lamina propria cells in vivo. Conclusion: Enhanced Rg3 negatively regulates the differentiation of Th1 cell in vitro and Th1 cell responses in the gut in vivo, providing fundamental basis for the use of this agent to treat Th1-related diseases. Keywords: Enhanced Rg3, IFNγ, IL-12, Korean Red Ginseng extract, Th

Nucleophilic substitution reaction based layer-by-layer growth of superparamagnetic nanocomposite films with high nonvolatile memory performance

Magnetic nanocomposite multilayers including magnetic nanoparticles are prepared using a nucleophilic substitution reaction between 2-bromo-2-methylpropionic acid (-BMPA) and amino (-NH2) groups. The assembly of magnetic nanoparticles in a nonpolar solvent induces densely packed adsorption, which significantly enhances the magnetic and nonvolatile memory properties of nanocomposite films.

Efficacy for Whitlockite for Augmenting Spinal Fusion

Whitlockite (WH) is the second most abundant inorganic component of human bone, accounting for approximately 25% of bone tissue. This study investigated the role of WH in bone remodeling and formation in a mouse spinal fusion model. Specifically, morphology and composition analysis, tests of porosity and surface area, thermogravimetric analysis, an ion-release test, and a cell viability test were conducted to analyze the properties of bone substitutes. The MagOss group received WH, Group A received 100% beta-tricalcium phosphate (β-TCP), Group B received 100% hydroxyapatite (HAp), Group C received 30% HAp/70% β-TCP, and Group D received 60% HAp/40% β-TCP (n = 10 each). All mice were sacrificed 6 weeks after implantation, and micro-CT, hematoxylin and eosin (HE) staining, and Masson trichome (MT) staining and immunohistochemistry were performed. The MagOss group showed more homogeneous and smaller grains, and nanopores (<500 nm) were found in only the MagOss group. On micro-CT, the MagOss group showed larger fusion mass and better graft incorporation into the decorticate mouse spine than other groups. In the in vivo experiment with HE staining, the MagOss group showed the highest new bone area (mean: decortication group, 9.50%; A, 15.08%; B, 15.70%; C, 14.76%; D, 14.70%; MagOss, 22.69%; p < 0.0001). In MT staining, the MagOss group demonstrated the highest new bone area (mean: decortication group, 15.62%; A, 21.41%; B, 22.86%; C, 23.07%; D, 22.47%; MagOss, 26.29%; p < 0.0001). In an immunohistochemical analysis for osteocalcin, osteopontin, and CD31, the MagOss group showed a higher positive area than other groups. WH showed comparable bone conductivity to HAp and β-TCP and increased new bone formation. WH is likely to be used as an improved bone substitute with better bone conductivity than HAp and β-TCP

An Injectable Engineered Cartilage Gel Improves Intervertebral Disc Repair in a Rat Nucleotomy Model

Lower back pain is a major problem caused by intervertebral disc degeneration. A common surgical procedure is lumbar partial discectomy (excision of the herniated disc causing nerve root compression), which results in further disc degeneration, severe lower back pain, and disability after discectomy. Thus, the development of disc regenerative therapies for patients who require lumbar partial discectomy is crucial. Here, we investigated the effectiveness of an engineered cartilage gel utilizing human fetal cartilage-derived progenitor cells (hFCPCs) on intervertebral disc repair in a rat tail nucleotomy model. Eight-week-old female Sprague-Dawley rats were randomized into three groups to undergo intradiscal injection of (1) cartilage gel, (2) hFCPCs, or (3) decellularized extracellular matrix (ECM) (n = 10/each group). The treatment materials were injected immediately after nucleotomy of the coccygeal discs. The coccygeal discs were removed six weeks after implantation for radiologic and histological analysis. Implantation of the cartilage gel promoted degenerative disc repair compared to hFCPCs or hFCPC-derived ECM by increasing the cellularity and matrix integrity, promoting reconstruction of nucleus pulposus, restoring disc hydration, and downregulating inflammatory cytokines and pain. Our results demonstrate that cartilage gel has higher therapeutic potential than its cellular or ECM component alone, and support further translation to large animal models and human subjects

An Injectable Engineered Cartilage Gel Improves Intervertebral Disc Repair in a Rat Nucleotomy Model

Lower back pain is a major problem caused by intervertebral disc degeneration. A common surgical procedure is lumbar partial discectomy (excision of the herniated disc causing nerve root compression), which results in further disc degeneration, severe lower back pain, and disability after discectomy. Thus, the development of disc regenerative therapies for patients who require lumbar partial discectomy is crucial. Here, we investigated the effectiveness of an engineered cartilage gel utilizing human fetal cartilage-derived progenitor cells (hFCPCs) on intervertebral disc repair in a rat tail nucleotomy model. Eight-week-old female Sprague-Dawley rats were randomized into three groups to undergo intradiscal injection of (1) cartilage gel, (2) hFCPCs, or (3) decellularized extracellular matrix (ECM) (n = 10/each group). The treatment materials were injected immediately after nucleotomy of the coccygeal discs. The coccygeal discs were removed six weeks after implantation for radiologic and histological analysis. Implantation of the cartilage gel promoted degenerative disc repair compared to hFCPCs or hFCPC-derived ECM by increasing the cellularity and matrix integrity, promoting reconstruction of nucleus pulposus, restoring disc hydration, and downregulating inflammatory cytokines and pain. Our results demonstrate that cartilage gel has higher therapeutic potential than its cellular or ECM component alone, and support further translation to large animal models and human subjects

Structural and Morphological Features of Concentric Iron Oxide/Carbon Nanotubes Obtained from Phospholipids

Biologically active 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DC8,9PC) nanotube-forming phospholipids (PLs) have been utilized as templates to prepare ferromagnetic nanotubes (FMNTs). Combining X-ray diffraction (XRD), selected area electron diffraction (SAD), high-resolution transmission electron microscopy (HRTEM), Raman, and Mössbauer spectroscopy measurements, FMNTs morphological features and chemical composition were determined. These studies showed that FMNTs consist of iron oxide/carbon/iron oxide concentric nanotubes with the amorphous carbon phase sandwiched between two iron oxide layers. The iron oxide phase consists of nanocrystalline magnetite (Fe3O4) which coexist as tetrahedral Fe3+ and octahedral Fe2.5+ sites containing minute quantities of hematite (α-Fe2O3) phase. The carbon phase consists of amorphous carbon forming an amorphous carbon nanotube (ACNT). Magnetic measurements showed that saturation magnetization (Ms) of FMNTs is 79 emu/g, but upon removal of the iron oxide outer and inner layers, ACNTs become paramagnetic. The electrical resistivity (ρ) of single FMNT is 3.3 × 10−2 Ω·m, which decreases to 5.06 × 10−4 Ω·m for ACNT. These magneto-electric properties can be easily tailored, depending upon desired applications and needs