Efficient engineering of human auricular cartilage through mesenchymal stem cell chaperoning.

A major challenge to the clinical translation of tissue-engineered ear scaffolds for ear reconstruction is the limited auricular chondrocyte (hAuC) yield available from patients. Starting with a relatively small number of chondrocytes in culture results in dedifferentiation and loss of phenotype with subsequent expansion. To significantly decrease the number of chondrocytes required for human elastic cartilage engineering, we co-cultured human mesenchymal stem cells (hMSCs) with HAuCs to promote healthy elastic cartilage formation. HAuCs along with human bone marrow-derived hMSCs were encapsulated into 1% Type I collagen at 25 million/mL total cell density with different ratios (HAuCs/hMSCs: 10/90, 25/75, 50/50) and then injected into customized 3D-printed polylactic acid (PLA) ridged external scaffolds, which simulate the shape of the auricular helical rim, and implanted subcutaneously in nude rats for 1, 3 and 6 months. The explanted constructs demonstrated near complete volume preservation and topography maintenance of the ridged "helical" feature after 6 months with all ratios. Cartilaginous appearing tissue formed within scaffolds by 3 months, verified by histologic analysis demonstrating mature elastic cartilage within the constructs with chondrocytes seen in lacunae within a Type II collagen and proteoglycan-enriched matrix, and surrounded by a neoperichondrial external layer. Compressive mechanical properties comparable to human elastic cartilage were achieved after 6 months. Co-implantation of hAuCs and hMSCs in collagen within an external scaffold efficiently produced shaped human elastic cartilage without volume loss even when hAuC comprised only 10% of the implanted cell population, marking a crucial step toward the clinical translation of auricular tissue engineering.

Mitochondrial Dysfunction Is a Driver of SP-2509 Drug Resistance in Ewing Sarcoma.

Expression of the fusion oncoprotein EWS/FLI causes Ewing sarcoma, an aggressive pediatric tumor characterized by widespread epigenetic deregulation. These epigenetic changes are targeted by novel lysine-specific demethylase-1 (LSD1) inhibitors, which are currently in early-phase clinical trials. Single-agent-targeted therapy often induces resistance, and successful clinical development requires knowledge of resistance mechanisms, enabling the design of effective combination strategies. Here, we used a genome-scale CRISPR-Cas9 loss-of-function screen to identify genes whose knockout (KO) conferred resistance to the LSD1 inhibitor SP-2509 in Ewing sarcoma cell lines. Multiple genes required for mitochondrial electron transport chain (ETC) complexes III and IV function were hits in our screen. We validated this finding using genetic and chemical approaches, including CRISPR KO, ETC inhibitors, and mitochondrial depletion. Further global transcriptional profiling revealed that altered complex III/IV function disrupted the oncogenic program mediated by EWS/FLI and LSD1 and blunted the transcriptomic response to SP-2509. IMPLICATIONS: These findings demonstrate that mitochondrial dysfunction modulates SP-2509 efficacy and suggest that new therapeutic strategies combining LSD1 with agents that prevent mitochondrial dysfunction may benefit patients with this aggressive malignancy.

Rapid profiling of G2 phase to mitosis progression by flow cytometry in asynchronous cells.

The precise control of the cell cycle G2 phase to Mitosis (M phase) transition is central for cell fate determination. The commonly used methods for assessing G2 to M phase progression are based on synchronizing cells and involve perturbation of the natural cell cycle progression. Additionally, these methods are often time-consuming and labor-intensive. Here, we report a flow cytometry-based method that offers a kinetic analysis of G2 to M phase progression in asynchronous cells using nocodazole, 5-Ethynyl-2´-deoxyuridine staining, and histone H3 serine 28 phosphorylation (pH3) staining. Nocodazole is used to collect mitotic cells and prevent their progression into G1, at the same time EdU is added for use as a dump channel during analysis. The remaining cells can then be identified as either G1 or G2/M based on their DNA content. Finally, G2 and M phase cells can be separated based on a mitotic marker, phosphorylation of ser28 on histone H3. While developed to assay G2/M phase progression, this method also resolves G1/S phase progression with no additional steps other than analysis. Compared to double thymidine block, this method does not require extended pre-treatments and is compatible with a greater variety of cell lines, while at the same time offering enhanced consistency and temporal resolution.

Radioisotope-Based Protocol for Determination of Central Carbon Metabolism in T Cells.

T lymphocytes are the major components of the adaptive immune system. It's been known that T cells are able to engage a diverse range of metabolic programs to meet the metabolic demands during their life cycle from early development, activation to functional differentiation. Central carbon metabolic pathways provide energy, reducing power, and biosynthetic precursors to support T cell homeostasis, proliferation, and immune functions. As such, quantitative or semiquantitative analysis of central carbon metabolic flux activities offers mechanistic details, as well as insights into the regulation of metabolic pathways and the impact of changing metabolic programs on T cell life cycle. Global profiling of cellular metabolites by mass spectrometry-based metabolomics and metabolic flux analysis (MFA) using radioactive and nonradioactive/stable isotope approaches are powerful tools for determination of central carbon metabolic pathway activity. Here, we describe in detail the procedure for the radioisotope-based approach of analyzing central carbon metabolic fluxes in T cells.

Inferior Oblique Entrapment After Orbital Fracture With Transection and Repair.

Extraocular muscle (EOM) entrapment with resulting reduction in motility and diplopia is a known complication of orbital fractures. Less commonly, transection of the EOMs due to trauma, iatrogenic injury, or intentional myotomy may lead to persistent diplopia. The inferior oblique (IO) is often encountered during orbital surgery along the medial wall and floor, and may be disinserted to aid in visualization. The authors present a case of IO entrapment which occurred during zygomaticomaxillary fracture reduction. Intraoperatively, an IO transection was performed and the muscle was reattached within the orbit. Postoperatively, the patient did not develop diplopia or motility disruption. This technique may provide a useful solution to an unusual problem during orbital fracture repair.

A Metabolism Toolbox for CAR T Therapy.

The adoptive transfer of T cells expressing chimeric antigen receptors (CARs) through genetic engineering is one of the most promising new therapies for treating cancer patients. A robust CAR T cell-mediated anti-tumor response requires the coordination of nutrient and energy supplies with CAR T cell expansion and function. However, the high metabolic demands of tumor cells compromise the function of CAR T cells by competing for nutrients within the tumor microenvironment (TME). To substantially improve clinical outcomes of CAR T immunotherapy while treating solid tumors, it is essential to metabolically prepare CAR T cells to overcome the metabolic barriers imposed by the TME. In this review, we discuss a potential metabolism toolbox to improve the metabolic fitness of CAR T cells and maximize the efficacy of CAR T therapy.

Phosphorylation of CDC25C by AMP-activated protein kinase mediates a metabolic checkpoint during cell-cycle G2/M-phase transition.

From unicellular to multicellular organisms, cell-cycle progression is tightly coupled to biosynthetic and bioenergetic demands. Accumulating evidence has demonstrated the G1/S-phase transition as a key checkpoint where cells respond to their metabolic status and commit to replicating the genome. However, the mechanism underlying the coordination of metabolism and the G2/M-phase transition in mammalian cells remains unclear. Here, we show that the activation of AMP-activated protein kinase (AMPK), a highly conserved cellular energy sensor, significantly delays mitosis entry. The cell-cycle G2/M-phase transition is controlled by mitotic cyclin-dependent kinase complex (CDC2-cyclin B), which is inactivated by WEE1 family protein kinases and activated by the opposing phosphatase CDC25C. AMPK directly phosphorylates CDC25C on serine 216, a well-conserved inhibitory phosphorylation event, which has been shown to mediate DNA damage-induced G2-phase arrest. The acute induction of CDC25C or suppression of WEE1 partially restores mitosis entry in the context of AMPK activation. These findings suggest that AMPK-dependent phosphorylation of CDC25C orchestrates a metabolic checkpoint for the cell-cycle G2/M-phase transition.

MYC and HIF in shaping immune response and immune metabolism.

Upon antigen stimulation, quiescent naive T cells undergo a phase of cell mass accumulation followed by cell cycle entry, clonal expansion, differentiation into functional subsets and back again to a quiescent state as they develop into memory cells. The transitions between these distinct cellular states place unique metabolic demands on energy, redox and biosynthesis. To fulfill these demands, T cells switch back and forth between their primary catabolic pathways. While quiescent naive and memory T cells largely rely on the oxidation of fatty acids and glucose, active T cells rely on glycolysis and glutaminolysis to sustain cell growth, proliferation and differentiation. Beyond several key signaling kinase cascades, the hypoxia inducible factor 1 (HIF-1) and the proto-oncogene MYC, act alone or in concert, to coordinate T cell metabolic reprogramming, cell proliferation, functional differentiation and apoptosis, enabling a robust T cell-mediated adaptive immune response.

Synthesis of a single G-quartet platform in water.

For over 50 years the G-quartet has been a defining self-assembled structure in biology and non-covalent synthesis. It is shown here for the first time that the G-quartet is isolatable in water in the absence of stabilizing G-quartet stacking or cations through the construction of a phosphate-linked template-assembled synthetic G-quartet. Synthetic design has facilitated preservation of the guanine base, ribose sugar, and phosphate components with correct linkage chemistry relative to G-quadruplex DNA. Thus, a minimal synthetic model of G-quadruplex DNA, as in that associated with human gene promoter or telomere regions, is represented by this system. An application as a probe for interactions between G-quadruplex DNA and potential anticancer therapeutical binding ligands is demonstrated. Binding constants of 10(5)-10(7) M(-1) magnitude and 1:1 stoichiometries for TMPyP4, piper, and azatrux ligands were determined, whereas perturbations in BSU1051 and BRACO19 ligand signal were not observed. These data suggest a unique test for critical end-stacking interactions at the exclusion of intercalative or looping interactions for G-quadruplex binding ligands.

A thymine tetrad assembly templated from thymidylic acid.

A template tetra-coupled with thymidylic acid through a phosphate linkage was characterized in methanol for emergent properties of nucleobase tetrad formation. Intramolecular hydrogen bonded base pairing in the absence of a cation was indicated for the thymidylic acid species supporting a monomeric template-assembled structure. Thus, an initial report of a stabilized individual thymine tetrad assembly is presented here. Consistent with previous investigations, a deoxyguanylic acid variant templated an analogous methanolic monomeric G-tetrad in comparison to the thymine species.

Ester hydrolysis by a histidine-containing cavitein.

We have used a template-assembled synthetic protein (TASP) to investigate catalytic function in ester hydrolysis. A histidine-containing cavitein has been found to catalyze ester hydrolysis with a rate increase of 18 times that of background.

A template-assembled synthetic U-quadruplex.

Fo(u)r U: A lipophilic cavitand with four dinucleoside (uridine) residues has been synthesized and characterized. NMR spectroscopy evidence suggests the self-assembly of a U-quadruplex by the uracil nucleobases in organic solution under ambient conditions.

Monomer-dimer control and crystal engineering in TASPs.

We have reported a template assembled synthetic protein (cavitein Q4) as an unexpected dimer in the solid state and as a monomer-dimer equilibrium in solution. We have since reported an ability to bias a cavitein's monomer-dimer equilibrium in solution by sequence design involving histidine metal chelation or disulfide incorporation. However, little remains known about the forces contributing to dimeric cavitein crystal nucleation and lattice stabilization. We, therefore, designed glutamine variants to probe factors involved in dimeric cavitein crystallization. It was found that a key glutamate hydrogen-bonding interaction between dimers is integral to crystal formation and stabilization. Additionally, we obtained a crystal structure of a cavitein (Q4-E3H) designed to bias the dimeric structure via histidine metal coordination. The resolved structure indicates a histidine cluster interaction that likely accounts for the biased dimeric form observed in solution.

Synthesis and characterization of a template-assembled synthetic U-quartet.

A lipophilic cavitand containing four triazole-linked uridine residues has been synthesized and characterized. Spectral evidence suggests a quartet arrangement of the uracil residues at both ambient and low temperatures. Treatment of the compound with Sr(2+) yields a homodimeric complex as evidenced by NMR spectroscopy.

Conformationally constrained sequence designs to bias monomer-dimer equilibriums in TASP systems.

We have designed template-assembled synthetic proteins (TASPs) with the intent of controlling their oligomeric state by stabilizing specific helical tertiary structures via histidine metal ion chelation or disulfide incorporation. In solution, cavitein Q4 was previously determined to interconvert between a four-helix bundle monomer and an eight-helix bundle dimer. In this paper, we show that judicious mutation of cavitein Q4 can stabilize either the monomeric parallel four-helix bundle or the dimeric antiparallel eight-helix bundle structure. Cavitein Q4-E3H, designed to be dimeric, is indeed biased toward dimerization as a result of incorporation of histidines. Moreover, the addition of nickel was found to further increase the association constant of dimerization. Similarly, a cavitein designed to stabilize the monomeric structure via histidine metal ion chelation (Q4-H) was found to favor a monomer in solution upon addition of nickel. Lastly, a cavitein intended to stabilize a monomeric structure via disulfide incorporation (Q4-C2) is reported. Surprisingly, this disulfide cavitein yielded two products upon oxidation suggesting disulfide formation both above the cavitand template and below may be possible. Nevertheless, the two disulfide caviteins were shown to exist as monomers as per their design.

The restoration of lumbar intervertebral disc load distribution: a comparison of three nucleus replacement technologies.

STUDY DESIGN: A validated L3-L4 nonlinear finite element model was used to evaluate strain and pressure in the surrounding structures for 4 nucleus replacement technologies. OBJECTIVE: The objective of the current study was to compare subsidence and anular damage potential between 4 current nucleus replacement technologies. It was hypothesized that a fully conforming nucleus replacement would minimize the risk of both subsidence and anular damage. SUMMARY OF BACKGROUND DATA: Nucleus pulposus replacements are emerging as a less invasive alternative to total disc replacement and fusion as a solution to degenerative intervertebral discs. Multiple technologies have been developed and are currently undergoing clinical investigation. METHODS: The testing conditions were applied by excavating the nucleus of the intact model and virtually implanting models representing the various nucleus replacement technologies. The implants consisted of a conforming injectable polyurethane (E = 4 MPa), soft hydrogel (E = 4 MPa), stiff hydrogel (E = 20 MPa), and polyether-etherketone (PEEK) on PEEK articulating designs. The model was exercised in flexion, extension, lateral bending, axial rotation (7.5 Nm with 450 N preload), and compression (1000 N). Vertebral body strain, anular maximum shear strain, endplate contact pressure, anulus-implant contact pressure, and bone remodeling stimulus were reported. RESULTS: The PEEK implant induced strain maxima in the vertebral bodies with associated endplate contact pressure concentrations. For the PEEK and hydrogel implants, areas of nonconformity with the endplate indicated adjacent bone resorption. Lack of conformity between the implant and inner anulus for the PEEK and hydrogel implants resulted in inward anular bulging with associated increased maximum shear strain. The conforming polyurethane implant maintained outward bulging of the inner anular wall and indicated no bone resorption or stress shielding adjacent to the implant. CONCLUSION: A fully conforming nucleus replacement resulted in a decreased propensity for subsidence, anular bulging, and further degeneration of the anulus when compared with nonconforming implants.

Economic impact of improving outcomes of lumbar discectomy.

BACKGROUND: Lumbar discectomy is usually a successful operation with a relatively low cost. Potential adjunctive procedures, such as repairing the anulus fibrosus or nucleus replacements, necessitate a cost-benefit analysis. PURPOSE: This economic analysis was performed to understand the potential value of advanced implantable technologies designed to improve outcomes after discectomy. STUDY DESIGN/SETTING: Using an insurance claims-based database, the economics of less-than-favorable outcomes after lumbar discectomy were studied. Estimates of improved clinical outcomes because of adjunctive surgical procedural items were modeled. PATIENT SAMPLE: Using Current Procedural Terminology (CPT-4) codes and International Classification of Diseases, Clinical Modification procedure codes (ICD-9 CM), all lumbar discectomy patients were identified in a 6-month period from a large, 2002, commercially available claims-based data set representing 3.1 million insured lives. OUTCOME MEASURES: Not applicable. METHODS: Longitudinal data analysis from 3 years (2002-2004) of the database was performed for evidence of claims after the insured's discectomy (up to 18 months post) as a utilization estimate of surgical and medical treatment resultant of less-than-favorable outcomes. Incidence and cost of secondary operations, medical management, and complications were determined. Using these inputs, an economic model was generated to estimate the effect of improvement in discectomy outcomes. RESULTS: Of the 494 patients who had a discectomy within a 6-month period, 137 (28%) had subsequent claims that suggested the outcome was less than favorable within 18 months. Patients whose insurance claims included codes for a second operation (n=52 patients with 56 operations; 11%) and patients being medically/nonsurgically managed (n=85, 17%) were studied. Average reimbursed charges incurred (2006 dollars) of repeated discectomy (80% of cases) was $6,907 and for arthrodesis (20% of cases) was $24,375. Average additional medical treatment cost to diagnose or manage poor outcome requiring another surgery was $3,365. Procedure-related complications within 40 days of surgery were evident in 15% of the group; with additional average cost to manage of $3,939. CONCLUSIONS: Substantial cost associated with poor discectomy outcomes is often overlooked or underappreciated. Surgical technologies that can improve outcomes of discectomy by 50% to 70% thus improving patient quality of life can be overall cost-neutral between $971 and $1,655 additionally per patient.

Cation-complexation behavior of template-assembled synthetic G-quartets.

We report the preparation and solution study of a set of template-assembled synthetic G-quartets (TASQs) bound to different cations. These G-quartet baskets effectively extract cations of different sizes and valencies. They form isolated G-quartets with small cations such as Na+ and Sr(2+), and dimeric assemblies with larger cations such as Cs+. Their structures were determined by using (1)H NMR spectroscopy, and their sizes were evaluated by using a series of pulsed-field gradient NMR experiments. The effect of anion has been studied, and the cation selectivities have been investigated by a series of competition experiments.