Assessing osteoporosis in postmenopausal women: preliminary results using a novel lumbar spine phantom-based MRI scoring method.

OBJECTIVE: To develop a novel magnetic resonance imaging (MRI) phantom for producing F-score (for fat) and W-score (for water) and to evaluate the performance of these scores in assessing osteoporosis and related vertebral fractures. MATERIALS AND METHODS: First, a real-time phantom consisting of oil and water tubes was manufactured. Then, 30 female volunteers (age: 62.3 ± 6.3 years) underwent lumbar spine examination with MRI (using a novel phantom) and dual-energy X-ray absorptiometry (DXA), following ethical approval. MRI phantom-based F-score and W-score were defined by normalizing the vertebral signal intensities (SIs) by the oil and water SIs of the phantom on T1- and T2-weighted images, respectively. The diagnostic performances of the new scores for assessing osteoporosis and vertebral fractures were examined using receiver operating characteristic analysis and compared with DXA-measured areal bone mineral density (DXA-aBMD). RESULTS: The F-score and W-score were greater in the osteoporotic patients (3.93 and 2.29) than the non-osteoporotic subjects (3.05 and 1.79) and achieved AUC values of 0.85 and 0.74 (p < 0.05), respectively, when detecting osteoporosis. Similarly, F-score and W-score had greater values for the fracture patients (3.94 and 2.53) than the non-fracture subjects (3.14 and 1.69) and produced better AUC values (0.90 for W-score and 0.79 for F-score) compared to DXA-aBMD (AUC: 0.27, p < 0.05). In addition, the F-score and W-score had a strong correlation (r = 0.77; p < 0.001). CONCLUSION: A novel real-time lumber spine MRI phantom was developed, based upon which newly defined F-score and W-score were able to detect osteoporosis and demonstrated an improved ability over DXA-aBMD in differentiating patients with vertebral fractures.

Bortezomib Is Effective in the Treatment of T Lymphoblastic Leukaemia by Inducing DNA Damage, WEE1 Downregulation, and Mitotic Catastrophe.

T lymphoblastic leukemia (T-ALL) is an aggressive haematolymphoid malignancy comprising 15% of acute lymphoblastic leukemia (ALL). Although its prognosis has improved with intensive chemotherapy, the relapse/refractory disease still carries a dismal prognosis. Thus, there is an urgent need to develop novel therapy for T-ALL. Bortezomib, a 26S proteasome inhibitor, is licensed to treat plasma cell myeloma and mantle cell lymphoma. Due to its favorable side effect profile, it is a novel agent of research interest in the treatment of ALL. Despite an increasing number of clinical trials of bortezomib in T-ALL, its detailed mechanistic study in terms of DNA damage, cell cycle, and mitotic catastrophe remains elusive. Moreover, WEE1, a protein kinase overexpressed in ALL and involved in cell-cycle regulation, has been known to be a novel therapeutic target in many cancers. But the role of bortezomib in modulating WEE1 expression in ALL still remains elusive. In this study, we demonstrate the therapeutic efficacy of bortezomib on T-ALL primary samples and cell lines. Our findings reveal that bortezomib treatment induces DNA damage and downregulates WEE1, leading to G2-M cell-cycle progression with damaged DNA. This abnormal mitotic entry induced by bortezomib leads to mitotic catastrophe in T-ALL. In conclusion, our findings dissect the mechanism of action of bortezomib and provide further insights into the use of bortezomib to treat T-ALL. Our findings suggest the possibility of novel combination therapy using proteasome inhibitors together with DNA-damaging agents in the future, which may fill the research gaps and unmet clinical needs in treating ALL.

Diagnostic Role of Magnetic Resonance Imaging in Low Back Pain Caused by Vertebral Endplate Degeneration.

Low back pain (LBP) is a common health issue worldwide with a huge economic burden on healthcare systems. In the United States alone, the cost is estimated to be $100 billion each year. Intervertebral disc degeneration is considered one of the primary causes of LBP. Moreover, the critical role of the vertebral endplates in disc degeneration and LBP is becoming apparent. Endplate abnormalities are closely correlated with disc degeneration and pain in the lumbar spine. Imaging modalities such as plain film radiography, computed tomography, and fluoroscopy are helpful but not very effective in detecting the causes behind LBP. Magnetic resonance imaging (MRI) can be used to acquire high-quality three-dimensional images of the lumbar spine without using ionizing radiation. Therefore, it is increasingly being used to diagnose spinal disorders. However, according to the American College of Radiology, current referral and justification guidelines for MRI are not sufficiently clear to guide clinical practice. This review aimed to evaluate the role of MRI in diagnosing LBP by considering the correlative contributions of vertebral endplates. The findings of the review indicate that MRI allows for fine evaluations of endplate morphology, endplate defects, diffusion and perfusion properties of the endplate, and Modic changes. Changes in these characteristics of the endplate were found to be closely correlated with disc degeneration and LBP. The collective evidence from the literature suggests that MRI may be the imaging modality of choice for patients suffering from LBP. LEVEL OF EVIDENCE: 1 TECHNICAL EFFICACY STAGE: 3.

Structural basis of DNA replication origin recognition by human Orc6 protein binding with DNA.

The six-subunit origin recognition complex (ORC), a DNA replication initiator, defines the localization of the origins of replication in eukaryotes. The Orc6 subunit is the smallest and the least conserved among ORC subunits. It is required for DNA replication and essential for viability in all species. Orc6 in metazoans carries a structural homology with transcription factor TFIIB and can bind DNA on its own. Here, we report a solution structure of the full-length human Orc6 (HsOrc6) alone and in a complex with DNA. We further showed that human Orc6 is composed of three independent domains: N-terminal, middle and C-terminal (HsOrc6-N, HsOrc6-M and HsOrc6-C). We also identified a distinct DNA-binding domain of human Orc6, named as HsOrc6-DBD. The detailed analysis of the structure revealed novel amino acid clusters important for the interaction with DNA. Alterations of these amino acids abolish DNA-binding ability of Orc6 and result in reduced levels of DNA replication. We propose that Orc6 is a DNA-binding subunit of human/metazoan ORC and may play roles in targeting, positioning and assembling the functional ORC at the origins.

The crystal structure of an antiparallel chair-type G-quadruplex formed by Bromo-substituted human telomeric DNA.

Human telomeric guanine-rich DNA, which could adopt different G-quadruplex structures, plays important roles in protecting the cell from recombination and degradation. Although many of these structures were determined, the chair-type G-quadruplex structure remains elusive. Here, we present a crystal structure of the G-quadruplex composed of the human telomeric sequence d[GGGTTAGG8GTTAGGGTTAGG20G] with two dG to 8Br-dG substitutions at positions 8 and 20 with syn conformation in the K+ solution. It forms a novel three-layer chair-type G-quadruplex with two linking trinucleotide loops. Particularly, T5 and T17 are coplanar with two water molecules stacking on the G-tetrad layer in a sandwich-like mode through a coordinating K+ ion and an A6•A18 base pair. While a twisted Hoogsteen A12•T10 base pair caps on the top of G-tetrad core. The three linking TTA loops are edgewise and each DNA strand has two antiparallel adjacent strands. Our findings contribute to a deeper understanding and highlight the unique roles of loop and water molecule in the folding of the G-quadruplex.

A novel approach for high-level expression and purification of GST-fused highly thermostable Taq DNA polymerase in Escherichia coli.

Polymerases are enzymes that synthesize long chains or polymers of nucleic acids including DNA or RNA from nucleotides. They assemble nucleic acids by copying a DNA or RNA template strand using base-pairing interactions. One of the polymerase enzymes, Taq DNA polymerase, originally isolated from Thermus aquaticus (Taq) is a widely used enzyme in molecular biology so far. The thermostable properties of this enzyme have contributed majorly to the specificity, automation, and efficacy of the polymerase chain reaction (PCR), making it a powerful tool for today's molecular biology researches across the globe. The purification of Taq DNA polymerase from the native host results in low yield, more labor and time consumption. Therefore, many studies have been previously conducted to obtain this enzyme using alternative hosts. So far, all the existing methodologies are more laborious, time-consuming and require heavy expense. We used a novel approach to purify the enzyme with relatively high efficiency, yield and minimum time consumption using Escherichia coli (E. coli) as an alternative host. We cloned a 2500 base pair Taq DNA polymerase gene into pGEX-4T-1 vector, containing a GST-tag, downstream of tac promoter and overexpressed it using isopropyl ß-d-1-thiogalactopyranoside (IPTG) as an inducer. The enzyme was efficiently purified using novel chromatography approaches and was used in routine PCR assays in our laboratory. Our findings suggest a novel approach to facilitate the availability of polymerases for molecular and diagnostic studies. In the future, it may be used for the purification of other recombinant peptides or proteins used in structural biology and proteomics-based researches.

Biomechanical MRI detects reduced bone strength in subjects with vertebral fractures.

Vertebral fracture is one of the most serious consequences of osteoporosis. Estimation of vertebral strength from magnetic resonance imaging (MRI) scans may provide a new approach for the prediction of vertebral fractures. To that end, we sought to establish a biomechanical MRI (BMRI) method to compute vertebral strength and test its ability to distinguish fracture from non-fracture subjects. This case-control study included 30 subjects without vertebral fractures and 15 subjects with vertebral fractures. All subjects underwent MRI with a mDIXON-Quant sequence and quantitative computed tomography (QCT), from which proton fat fraction-based bone marrow adipose tissue (BMAT) content and volumetric bone mineral density (vBMD) were measured, respectively. Nonlinear finite element analysis was applied to MRI and QCT scans of L2 vertebrae to compute vertebral strength (BMRI- and BCT-strength). The differences in BMAT content, vBMD, BMRI-strength and BCT-strength between the two groups were examined by t-tests. Receiver operating characteristic (ROC) analysis was performed to assess the ability of each measured parameter to distinguish fracture from non-fracture subjects. Results showed that the fracture group had 23 % lower BMRI-strength (P < .001) and 19 % higher BMAT content (P < .001) than the non-fracture group, whereas no significant difference in vBMD was detected between the two groups. A poor correlation was found between vBMD and BMRI-strength (R2 = 0.33). Compared to vBMD and BMAT content, BMRI- and BCT-strength had the larger area under the curve (0.82 and 0.84, respectively) and provided better sensitivity and specificity in separating fracture from non-fracture subjects. In conclusion, BMRI is capable of detecting reduced bone strength in patients with vertebral fracture, and may serve as a new approach for risk assessment of vertebral fracture.

Mechanical testing and biomechanical CT analysis to assess vertebral flexion strength of Chinese cadavers.

Biomechanical CT (BCT), i.e., quantitative computed tomography-based finite element analysis (QCT-FEA), promises an improved technique over bone mineral density (BMD) in predicting bone strength and the risk of osteoporotic vertebral fractures. However, most of the BCT models only consider a uniform compressive loading condition and they have not been validated for Chinese subjects. This study examined the ability of BCT to predict wedge fracture-related vertebral flexion strength in a cohort of Chinese cadaveric vertebrae. Twelve human vertebrae were scanned with dual energy X-ray absorptiometry (DXA) and QCT to measure areal and volumetric BMD, respectively. To produce wedge fractures, the cadaveric vertebrae were experimentally loaded until failure under a 15° flexion. Vertebral flexion stiffness and strength were measured from the force-displacement curve. Voxel-based heterogeneous FE models of the vertebrae were created and virtually tested in uniform compression and 15° flexion to compute compressive and flexion strength (and stiffness), respectively. The predictions of vertebral flexion strength with BMD or BCT measures were evaluated with linear regression analyses. Results showed weak correlations between experimentally-measured flexion strength vs. DXA-aBMD (R2 = 0.26) or QCT-vBMD (R2 = 0.39). However, there were strong correlations between experimentally-measured flexion strength vs. BCT-computed vertebral strength under either flexion (R2 = 0.71) or compression (R2 = 0.70) loading conditions, although flexion reduced the BCT-computed vertebral strength by 9.2%. These results suggest that, regardless of whether a uniform compression or a flexion loading is simulated, BCT can predict in vitro vertebral flexion strength better than BMD.