Biochemical characterization of the meiosis-essential yet evolutionarily divergent topoisomerase VIB-like protein MTOPVIB from Arabidopsis thaliana.

Formation of programmed DNA double-strand breaks is essential for initiating meiotic recombination. Genetic studies on Arabidopsis thaliana and Mus musculus have revealed that assembly of a type IIB topoisomerase VI (Topo VI)-like complex, composed of SPO11 and MTOPVIB, is a prerequisite for generating DNA breaks. However, it remains enigmatic if MTOPVIB resembles its Topo VI subunit B (VIB) ortholog in possessing robust ATPase activity, ability to undergo ATP-dependent dimerization, and activation of SPO11-mediated DNA cleavage. Here, we successfully prepared highly pure A. thaliana MTOPVIB and MTOPVIB-SPO11 complex. Contrary to expectations, our findings highlight that MTOPVIB differs from orthologous Topo VIB by lacking ATP-binding activity and independently forming dimers without ATP. Most significantly, our study reveals that while MTOPVIB lacks the capability to stimulate SPO11-mediated DNA cleavage, it functions as a bona fide DNA-binding protein and plays a substantial role in facilitating the dsDNA binding capacity of the MOTOVIB-SPO11 complex. Thus, we illustrate mechanistic divergence between the MTOPVIB-SPO11 complex and classical type IIB topoisomerases.

Electrical impedance tomography for non-invasive identification of fatty liver infiltrate in overweight individuals.

Non-alcoholic fatty liver disease (NAFLD) is one of the most common causes of cardiometabolic diseases in overweight individuals. While liver biopsy is the current gold standard to diagnose NAFLD and magnetic resonance imaging (MRI) is a non-invasive alternative still under clinical trials, the former is invasive and the latter costly. We demonstrate electrical impedance tomography (EIT) as a portable method for detecting fatty infiltrate. We enrolled 19 overweight subjects to undergo liver MRI scans, followed by EIT measurements. The MRI images provided the a priori knowledge of the liver boundary conditions for EIT reconstruction, and the multi-echo MRI data quantified liver proton-density fat fraction (PDFF%) to validate fat infiltrate. Using the EIT electrode belts, we circumferentially injected pairwise current to the upper abdomen, followed by acquiring the resulting surface-voltage to reconstruct the liver conductivity. Pearson's correlation analyses compared EIT conductivity or MRI PDFF with body mass index, age, waist circumference, height, and weight variables. We reveal that the correlation between liver EIT conductivity or MRI PDFF with demographics is statistically insignificant, whereas liver EIT conductivity is inversely correlated with MRI PDFF (R = -0.69, p = 0.003, n = 16). As a pilot study, EIT conductivity provides a portable method for operator-independent and cost-effective detection of hepatic steatosis.

Reproducibility and Radiation Effect of High-Resolution In Vivo Micro Computed Tomography Imaging of the Mouse Lumbar Vertebra and Long Bone.

A moderate radiation dose, in vivo µCT scanning protocol was developed and validated for long-term monitoring of multiple skeletal sites (femur, tibia, vertebra) in mice. A customized, 3D printed mouse holder was designed and utilized to minimize error associated with animal repositioning, resulting in good to excellent reproducibility in most cortical and trabecular bone microarchitecture and density parameters except for connectivity density. Repeated in vivo µCT scans of mice were performed at the right distal femur and the 4th lumbar vertebra every 3 weeks until euthanized at 9 weeks after the baseline scan. Comparing to the non-radiated counterparts, no radiation effect was found on trabecular bone volume fraction, osteoblast and osteoblast number/surface, or bone formation rate at any skeletal site. However, trabecular number, thickness, and separation, and structure model index were sensitive to ionizing radiation associated with the µCT scans, resulting in subtle but significant changes over multiple scans. Although the extent of radiation damage on most trabecular bone microarchitecture measures are comparable or far less than the age-related changes during the monitoring period, additional considerations need to be taken to minimize the confounding radiation factors when designing experiments using in vivo µCT imaging for long-term monitoring of mouse bone.

Displacement analysis of myocardial mechanical deformation (DIAMOND) reveals segmental susceptibility to doxorubicin-induced injury and regeneration.

Zebrafish are increasingly utilized to model cardiomyopathies and regeneration. Current methods evaluating cardiac function have known limitations, fail to reliably detect focal mechanics, and are not readily feasible in zebrafish. We developed a semiautomated, open-source method - displacement analysis of myocardial mechanical deformation (DIAMOND) - for quantitative assessment of 4D segmental cardiac function. We imaged transgenic embryonic zebrafish in vivo using a light-sheet fluorescence microscopy system with 4D cardiac motion synchronization. Our method permits the derivation of a transformation matrix to quantify the time-dependent 3D displacement of segmental myocardial mass centroids. Through treatment with doxorubicin, and by chemically and genetically manipulating the myocardial injury-activated Notch signaling pathway, we used DIAMOND to demonstrate that basal ventricular segments adjacent to the atrioventricular canal display the highest 3D displacement and are also the most susceptible to doxorubicin-induced injury. Thus, DIAMOND provides biomechanical insights into in vivo segmental cardiac function scalable to high-throughput research applications.

Spatial and temporal variations in hemodynamic forces initiate cardiac trabeculation.

Hemodynamic shear force has been implicated as modulating Notch signaling-mediated cardiac trabeculation. Whether the spatiotemporal variations in wall shear stress (WSS) coordinate the initiation of trabeculation to influence ventricular contractile function remains unknown. Using light-sheet fluorescent microscopy, we reconstructed the 4D moving domain and applied computational fluid dynamics to quantify 4D WSS along the trabecular ridges and in the groves. In WT zebrafish, pulsatile shear stress developed along the trabecular ridges, with prominent endocardial Notch activity at 3 days after fertilization (dpf), and oscillatory shear stress developed in the trabecular grooves, with epicardial Notch activity at 4 dpf. Genetic manipulations were performed to reduce hematopoiesis and inhibit atrial contraction to lower WSS in synchrony with attenuation of oscillatory shear index (OSI) during ventricular development. γ-Secretase inhibitor of Notch intracellular domain (NICD) abrogated endocardial and epicardial Notch activity. Rescue with NICD mRNA restored Notch activity sequentially from the endocardium to trabecular grooves, which was corroborated by observed Notch-mediated cardiomyocyte proliferations on WT zebrafish trabeculae. We also demonstrated in vitro that a high OSI value correlated with upregulated endothelial Notch-related mRNA expression. In silico computation of energy dissipation further supports the role of trabeculation to preserve ventricular structure and contractile function. Thus, spatiotemporal variations in WSS coordinate trabecular organization for ventricular contractile function.

Increased expression of enolase alpha in human breast cancer confers tamoxifen resistance in human breast cancer cells.

Enolase-alpha (ENO-1) is a key glycolytic enzyme that has been used as a diagnostic marker to identify human lung cancers. To investigate the role of ENO-1 in breast cancer diagnosis and therapy, the mRNA levels of ENO-1 in 244 tumor and normal paired tissue samples and 20 laser capture-microdissected cell clusters were examined by quantitative real-time PCR analysis. Increased ENO-1 mRNA expression was preferentially detected in estrogen receptor-positive (ER+) tumors (tumor/normal ratio >90-fold) when compared to ER-negative (tumor/normal ratio >20-fold) tumor tissues. The data presented here demonstrate that those patients whose tumors highly expressed ENO-1 had a poor prognosis with greater tumor size (>2 cm, *P = .017), poor nodal status (N > 3, *P = .018), and a shorter disease-free interval (<==1 year, *P < .009). We also found that higher-expressing ENO-1 tumors confer longer distance relapse (tumor/normal ratio = 82.8-92.4-fold) when compared to locoregional relapse (tumor/normal ratio = 43.4-fold) in postsurgical 4-hydroxy-tamoxifen (4-OHT)-treated ER+ patients (*P = .014). These data imply that changes in tumor ENO-1 levels are related to clinical 4-OHT therapeutic outcome. In vitro studies demonstrated that decreasing ENO-1 expression using small interfering RNA (siRNA) significantly augmented 4-OHT (100 nM)-induced cytotoxicity in tamoxifen-resistant (Tam-R) breast cancer cells. These results suggest that downregulation of ENO-1 could be utilized as a novel pharmacological approach for overcoming 4-OHT resistance in breast cancer therapy.