Microstructure of the radial head: Insights into anatomical variations and implications for advanced interventions.

Appropriate management of radial head fractures is integral to prevent long-term consequences like chronic pain and loss of motion. Advanced imaging systems, like micro-computed tomography (µCT), are valuable for understanding radial head fracture patterns as they utilize micrometer scale resolution to define important parameters of bone health like cortical density and trabecular thickness. The purpose of this study was to identify and describe the structural morphology of the radial head utilizing µCT. Nine fresh-frozen cadaveric human radii were divided into four equal quadrants, based, and labeled as posteromedial, posterolateral, anteromedial, and anterolateral. Quadrants were scanned with a SCANCO MicroCT40 with both cortical and cancellous bone density measurements at a resolution of 36.0 µm. Bone density, direct trabecular number, and trabecular thickness were recorded as milligrams of hydroxyapatite/cm3. A one-way repeated measures ANOVA was performed to compare the bone densities, trabecular number, and trabecular thickness of each of the four quadrants (p < 0.05). The posteromedial quadrant contained substantially more bone than other quadrants. Significantly greater bone densities were found in the posteromedial quadrant (148.1 mg of HA/cm3) compared to the anteromedial quadrant (54.6 mg of HA/cm3), posterolateral quadrant (137.5 mg of HA/cm3) compared to the anteromedial quadrant (54.6 mg of HA/cm3), and posterolateral quadrant (137.5 mg of HA/cm3) compared to the anterolateral quadrant (58.1 mg of HA/cm3). The trabecular number was not significantly different between quadrants. Trabecular thickness was significantly lower in the anterolateral (0.1417 mg of HA/cm3) and anteromedial (0.1416 mg of HA/cm3) quadrants compared to the posteromedial (0.1809 mg of HA/cm3) quadrant. The posterior half of the radial head was found to have a higher density of columns and arches compared to the anterior half. The microstructure of trabecular bone in the distal radius forms columns, struts, and arches, which allow for efficient transmission of stress through the bone. The microstructure of the radial head has similar microarchitecture to the distal radius with the present study identifying the presence of columns and arches in the radial head. These structures, along with trabecular density, in the posterior radial head may explain the lower incidence of fractures involving the posterior half of the radial head. Furthermore, our study supports the idea that the high incidence of fractures involving the anterolateral quadrant is due to microarchitecture characteristics and the relative lack of supportive structures compared to other areas. The novel insight gained from this study will aid in the development of advanced interventions for preventative measures and better treatment of radial head fractures like more satisfactory purchase when screws are directed towards the denser posteromedial quadrant.

Primary acute lymphoblastic leukemia cells are susceptible to microtubule depolymerization in G1 and M phases through distinct cell death pathways.

Microtubule targeting agents (MTAs) are widely used cancer chemotherapeutics which conventionally exert their effects during mitosis, leading to mitotic or postmitotic death. However, accumulating evidence suggests that MTAs can also generate death signals during interphase, which may represent a key mechanism in the clinical setting. We reported previously that vincristine and other microtubule destabilizers induce death not only in M phase but also in G1 phase in primary acute lymphoblastic leukemia cells. Here, we sought to investigate and compare the pathways responsible for phase-specific cell death. Primary acute lymphoblastic leukemia cells were subjected to centrifugal elutriation, and cell populations enriched in G1 phase (97%) or G2/M phases (80%) were obtained and treated with vincristine. We found death of M phase cells was associated with established features of mitochondrial-mediated apoptosis, including Bax activation, loss of mitochondrial transmembrane potential, caspase-3 activation, and nucleosomal DNA fragmentation. In contrast, death of G1 phase cells was not associated with pronounced Bax or caspase-3 activation but was associated with loss of mitochondrial transmembrane potential, parylation, nuclear translocation of apoptosis-inducing factor and endonuclease G, and supra-nucleosomal DNA fragmentation, which was enhanced by inhibition of autophagy. The results indicate that microtubule depolymerization induces distinct cell death pathways depending on during which phase of the cell cycle microtubule perturbation occurs. The observation that a specific type of drug can enter a single cell type and induce two different modes of death is novel and intriguing. These findings provide a basis for advancing knowledge of clinical mechanisms of MTAs.

Chemical inhibition of DNA-PKcs impairs the activation and cytotoxicity of CD4+ helper and CD8+ effector T cells.

Modulation of T cell activity is an effective strategy for the treatment of autoimmune diseases, immune-related disorders and cancer. This highlights a critical need for the identification of proteins that regulate T cell function. The kinase DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is emerging as a potent regulator of the immune system, spurring interest in its use as a therapeutic target. In murine models of immune-related diseases including asthma and rheumatoid arthritis, treatment with small-molecule DNA-PKcs inhibitors decreased the disease severity. Additionally, DNA-PKcs inhibitors reduced T cell-mediated graft rejection in a murine allogenic skin graft model. These in vivo studies suggest the use of DNA-PKcs inhibitors as immunotherapy for autoimmune and T cell-mediated disorders. In this study, we sought to characterize further the effects of DNA-PKcs inhibitors on T cells to better understand their clinical potential. We determined that inhibition of DNA-PKcs using inhibitor NU7441 and the inhibitors currently in clinical trials for cancer therapy, M3184 and AZD7648, abrogated the activation of murine and human CD4+ and CD8+ T cells as evidenced by the reduced expression of the activation markers CD69 and CD25. Furthermore, inhibition of DNA-PKcs impeded metabolic pathways and the proliferation of activated T cells. This reduced the ability of OTI-CD8+ T cells to kill cancer cells and the expression of IFNγ and cytotoxic genes. These results highlight a critical role for DNA-PKcs in T cells and validate future studies using DNA-PKcs inhibitors as immune modulation therapy for the treatment of immune-related diseases.

Probing Membrane Protein Association Using Concentration-Dependent Number and Brightness.

We introduce concentration-dependent number and brightness (cdN&B), a fluorescence fluctuation technique that can be implemented on a standard confocal microscope and can report on the thermodynamics of membrane protein association in the native plasma membrane. It uses transient transfection to enable measurements of oligomer size as a function of receptor concentration over a broad range, yielding the association constant. We discuss artifacts in cdN&B that are concentration-dependent and can distort the oligomerization curves, and we outline procedures that can correct for them. Using cdN&B, we characterize the association of neuropilin 1 (NRP1), a protein that plays a critical role in the development of the embryonic cardiovascular and nervous systems. We show that NRP1 associates into a tetramer in a concentration-dependent manner, and we quantify the strength of the association. This work demonstrates the utility of cdN&B as a powerful tool in biophysical chemistry.

Discovery of the DNA-PKcs inhibitor DA-143 which exhibits enhanced solubility relative to NU7441.

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) plays a vital role in DNA damage repair and lymphocyte function, presenting a significant target in cancer and immune diseases. Current DNA-PKcs inhibitors are undergoing Phase I/II trials as adjuncts to radiotherapy and chemotherapy in cancer. Nevertheless, clinical utility is limited by suboptimal bioavailability. This study introduces DNA-PKcs inhibitors designed to enhance bioavailability. We demonstrate that a novel DNA-PKcs inhibitor, DA-143, surpasses NU7441 in aqueous solubility as well as other available inhibitors. In addition, DA-143 displayed an improvement in DNA-PKcs inhibition relative to NU7441 achieving an IC50 of 2.5 nM. Consistent with current inhibitors, inhibition of DNA-PKcs by DA-143 resulted in increased tumor cell sensitivity to DNA-damage from chemotherapy and inhibition of human T cell function. The improved solubility of DA-143 is critical for enhanced efficacy at reduced doses and facilitates more effective evaluation of DNA-PKcs inhibition in both preclinical and clinical development.

The legume-rhizobia symbiosis can be supported on Mars soil simulants.

Legumes (soybeans, peas, lentils, etc.) play important roles in agriculture on Earth because of their food value and their ability to form a mutualistic beneficial association with rhizobia bacteria. In this association, the host plant benefits from atmospheric nitrogen fixation by rhizobia. The presence of nitrogen in the Mars atmosphere offers the possibility to take advantage of this important plant-microbe association. While some studies have shown that Mars soil simulants can support plant growth, none have investigated if these soils can support the legume-rhizobia symbiosis. In this study, we investigated the establishment of the legume-rhizobia symbiosis on different Mars soil simulants (different grades of the Mojave Mars Simulant (MMS)-1: Coarse, Fine, Unsorted, Superfine, and the MMS-2 simulant). We used the model legume, Medicago truncatula, and its symbiotic partners, Sinorhizobium meliloti and Sinorhizobium medicae, in these experiments. Our results show that root nodules could develop on M. truncatula roots when grown on these Mars soil simulants and were comparable to those formed on plants that were grown on sand. We also detected nifH (a reporter gene for nitrogen fixation) expression inside these nodules. Our results indicate that the different Mars soil simulants used in this study can support legume-rhizobia symbiosis. While the average number of lateral roots and nodule numbers were comparable on plants grown on the different soil simulants, total plant mass was higher in plants grown on MMS-2 soil than on MMS-1 soil and its variants. Our results imply that the chemical composition of the simulants is more critical than their grain size for plant mass. Based on these results, we recommend that the MMS-2 Superfine soil simulant is a better fit than the MMS-1 soil and it's variants for future studies. Our findings can serve as an excellent resource for future studies investigating beneficial plant-microbe associations for sustainable agriculture on Mars.