Quantitative analysis tool for clinical functional MRI in mild traumatic brain injury.

Functional magnetic resonance imaging (fMRI) has been available commercially for clinical diagnostic use for many years. However, both clinical interpretation of fMRI by a neuroradiologist and quantitative analysis of fMRI data can require significant personnel resources that exceed reimbursement. In this report, a fully automated computer-based quantification methodology (Enumerated Auditory Response, EAR) has been developed to provide an auditory fMRI assessment of patients who have suffered a mild traumatic brain injury. Fifty-five study participants with interpretable auditory fMRI sequence data were assessed by EAR analysis, as well as both clinical radiologist fMRI interpretation and voxelwise general linear model (GLM) analysis. Comparison between the clinical interpretation and the two computer analysis methods resulted in 67% concordance (identical), 32% nearconcordance (one level difference), and 1% discordant. Comparison between the clinical computer-based quantification (EAR) and GLM analysis yielded significant correlations in right and left ear responses (p⟨0.05) for the full subject group. Automated fMRI quantification analysis equivalent to EAR might be appropriate for both future research projects with constrained resources, as well as possible routine clinical use.

Analysis of magnetic resonance spectroscopy relative metabolite ratios in mild traumatic brain injury and normative controls.

INTRODUCTION: We evaluated magnetic resonance spectroscopy (MRS) in United States military personnel with persistent symptoms after mild traumatic brain injury (mTBI), comparing over time two groups randomized to receive hyperbaric oxygen or sham chamber sessions and a third group of normative controls. METHODS: Active-duty or veteran military personnel and normative controls underwent MRS outcome measures at baseline, 13 weeks (mTBI group only), and six months. Participants received 3.0 Tesla brain MRS for analysis of water-suppressed two-dimensional (2D) multivoxel 1H-MRS of the brain using point resolved spectroscopy (PRESS) with volume selection localized above the lateral ventricles and within the brain parenchyma, of which one voxel was chosen in each hemisphere without artifact. Script-based automatic data processing was used to assess N-acetylaspartate (NAA), creatine (Cr), and choline (Cho). Metabolite ratios for white matter were then calculated for NAA/Cr (Area), Cho/Cr (Area), and Cho/NAA (Area). These ratios were compared using standard analysis methodology. RESULTS: There were no observable differences between participants with mTBI and normative controls nor any observable changes over time in the NAA/Cr (area), Cho/Cr (area), and Cho/NAA (area) ratios. Similarly, the control and injured participants were indistinguishable. DISCUSSION: While participants with mild TBI showed no difference in MRS compared to normative controls, our results are limited by the few voxels chosen and potentially by less sensitive MRS markers.

Visible light optical spectroscopy is sensitive to neovascularization in the dysplastic cervix.

Neovascularization in cervical intraepithelial neoplasia (CIN) is studied because it is the precursor to the third most common female cancer worldwide. Diffuse reflectance from 450-600 nm was collected from 46 patients (76 sites) undergoing colposcopy at Duke University Medical Center. Total hemoglobin, derived using an inverse Monte Carlo model, significantly increased in CIN 2+ (N=12) versus CIN 1 (N=16) and normal tissues (N=48) combined with P<0.004. Immunohistochemistry using monoclonal anti-CD34 was used to quantify microvessel density to validate the increased hemoglobin content. Biopsies from 51 sites were stained, and up to three hot spots per slide were selected for microvessel quantification by two observers. Similar to the optical study results, microvessel density was significantly increased in CIN 2+ (N=16) versus CIN 1 (N=21) and normal tissue (N=14) combined with P<0.007. Total vessel density, however, was not significantly associated with dysplastic grade. Hence, our quantitative optical spectroscopy system is primarily sensitive to dysplastic neovascularization immediately beneath the basement membrane, with minimal confounding from vascularity inherent in the normal stromal environment. This tool could have potential for in vivo applications in screening for cervical cancer, prognostics, and monitoring of antiangiogenic effects in chemoprevention therapies.

Sagittal whole-spine magnetic resonance imaging in 750 consecutive outpatients: accurate determination of the number of lumbar vertebral bodies.

OBJECT: When the number of lumbar and sacral vertebrae is being assessed, variations from typical lumbosacral anatomy may confuse the practitioner, potentially leading to significant clinical errors. In this study, the authors describe the statistical variation in lumbar spine anatomy in an outpatient imaging setting, evaluate the potential implications for clinical practice based on the variation in the number of lumbar-type vertebrae identified, and recommend a method for rapidly determining the number of lumbar spine vertebral bodies (VBs) in outpatients referred for lumbar spine MR imaging. METHODS: A total of 762 patients (male and female) who presented with low back-related medical conditions underwent whole-spine MR imaging in an outpatient setting. RESULTS: The high-speed whole-spine evaluation was successful for determining the number of lumbar-type VBs in 750 (98%) of 762 consecutive patients. The sagittal whole-spine 3-T MR imaging system images obtained between the beginning of January 2005 and the end of February 2007 were reviewed. The VBs were counted successively from the level of C-2 inferiorly to the intervertebral disc below the most inferior lumbar-type VB. Numbers of disc herniations were also evaluated in the context of the number of VBs. CONCLUSIONS: One in 5 of these outpatients did not have 5 lumbar-type vertebrae: 14.5% had 6; 5.3% had 4; and 1 (0.13%) had the rare finding of 3 lumbar-type vertebrae. Two-thirds of the individuals with 6 lumbar-type vertebrae were male and two-thirds of the individuals with 4 lumbar-type vertebrae were female. Sagittal whole-spine MR imaging can be performed rapidly and efficiently in the majority of patients (98%), and provides improved accuracy for the determination of the number of lumbar-type VBs. A supplementary coronal MR, Ferguson view radiograph or intraoperative fluoroscopic determination for the presence of lumbosacral transitional vertebrae may add additional information when indicated for clinical treatment or surgical planning.

Quantitative physiology of the precancerous cervix in vivo through optical spectroscopy.

Cervical cancer is the second most common female cancer worldwide. The ability to quantify physiological and morphological changes in the cervix is not only useful in the diagnosis of cervical precancers but also important in aiding the design of cost-effective detection systems for use in developing countries that lack well-established screening and diagnostic programs. We assessed the capability of a diffuse reflectance spectroscopy technique to identify contrasts in optical biomarkers that vary with different grades of cervical intraepithelial neoplasia (CIN) from normal cervical tissues. The technology consists of an optical probe and an instrument (with broadband light source, dispersive element, and detector), and a Monte Carlo algorithm to extract optical biomarker contributions including total hemoglobin (Hb) concentration, Hb saturation, and reduced scattering coefficient from the measured spectra. Among 38 patients and 89 sites examined, 46 squamous normal sites, 18 CIN 1, and 15 CIN 2(+) sites were included in the analysis. Total Hb was statistically higher in CIN 2(+) (18.3 +/- 3.6 microM, mean +/- SE) compared with normal (9.58 +/- 1.91 microM) and CIN 1 (12.8 +/- 2.6 microM), whereas scattering was significantly reduced in CIN 1 (8.3 +/- 0.8 cm(-1)) and CIN 2(+) (8.6 +/- 1.0 cm(-1)) compared with normal (10.2 +/- 1.1 cm(-1)). Hemoglobin saturation was not significantly altered in CIN 2(+) compared with normal and CIN 1. The difference in total Hb is likely because of stromal angiogenesis, whereas decreased scattering can be attributed to breakdown of collagen network in the cervical stroma.

Developmental activation of the Rb-E2F pathway and establishment of cell cycle-regulated cyclin-dependent kinase activity during embryonic stem cell differentiation.

To understand cell cycle control mechanisms in early development and how they change during differentiation, we used embryonic stem cells to model embryonic events. Our results demonstrate that as pluripotent cells differentiate, the length of G(1) phase increases substantially. At the molecular level, this is associated with a significant change in the size of active cyclin-dependent kinase (Cdk) complexes, the establishment of cell cycle-regulated Cdk2 activity and the activation of a functional Rb-E2F pathway. The switch from constitutive to cell cycle-dependent Cdk2 activity coincides with temporal changes in cyclin A2 and E1 protein levels during the cell cycle. Transcriptional mechanisms underpin the down-regulation of cyclin levels and the establishment of their periodicity during differentiation. As pluripotent cells differentiate and pRb/p107 kinase activities become cell cycle dependent, the E2F-pRb pathway is activated and imposes cell cycle-regulated transcriptional control on E2F target genes, such as cyclin E1. These results suggest the existence of a feedback loop where Cdk2 controls its own activity through regulation of cyclin E1 transcription. Changes in rates of cell division, cell cycle structure and the establishment of cell cycle-regulated Cdk2 activity can therefore be explained by activation of the E2F-pRb pathway.

LIF/STAT3 controls ES cell self-renewal and pluripotency by a Myc-dependent mechanism.

Murine ES cells can be maintained as a pluripotent, self-renewing population by LIF/STAT3-dependent signaling. The downstream effectors of this pathway have not been previously defined. In this report, we identify a key target of the LIF self-renewal pathway by showing that STAT3 directly regulates the expression of the Myc transcription factor. Murine ES cells express elevated levels of Myc and following LIF withdrawal, Myc mRNA levels collapse and Myc protein becomes phosphorylated on threonine 58 (T58), triggering its GSK3beta dependent degradation. Maintained expression of stable Myc (T58A) renders self-renewal and maintenance of pluripotency independent of LIF. By contrast, expression of a dominant negative form of Myc antagonizes self-renewal and promotes differentiation. Transcriptional control by STAT3 and suppression of T58 phosphorylation are crucial for regulation of Myc activity in ES cells and therefore in promoting self-renewal. Together, our results establish a mechanism for how LIF and STAT3 regulate ES cell self-renewal and pluripotency.

A novel repressive E2F6 complex containing the polycomb group protein, EPC1, that interacts with EZH2 in a proliferation-specific manner.

The transcriptional repressor E2F6 has been identified as a component of two distinct polycomb group protein (PcG)-containing complexes, suggesting a mechanism for the recruitment of repressive complexes to target sequences in DNA. Whereas one complex is involved in the repression of classic E2F target genes in G0, a role for E2F6 within the cell cycle has yet to be defined. We searched for novel E2F6-binding proteins using a yeast two-hybrid screen and identified the PcG protein, EPC1. We showed that, both in vitro and in vivo, E2F6, DP1, and EPC1 form a stable core complex with repressive activity. Furthermore, we identified the proliferation-specific PcG, EZH2, as an EPC1-interacting protein. Using affinity purification, we showed that E2F6, DP1, EPC1, EZH2, and Sin3B co-elute, suggesting the identification of a novel E2F6 complex that exists in vivo in both normal and transformed human cell lines. EZH2 is required for cellular proliferation and consistent with this, EZH2 elutes with the E2F6-EPC1 complex only in proliferating cells. Thus we have identified a novel E2F6-PcG complex (E2F6-EPC1) that interacts with EZH2 and may regulate genes required for cell cycle progression.

Cdk6-cyclin D3 activity in murine ES cells is resistant to inhibition by p16(INK4a).

Through a screen aimed at identifying genes that are specifically upregulated in embryomic stem (ES) cells but not primitive ectoderm, we identified cyclin D3. This was surprising since cyclin D activity is generally believed to be inactive in ES cells even though retinoblastoma tumor suppressor protein (pRb) accumulates in a predominantly hyperphosphorylated state. Cdk6 is the major catalytic partner for cyclin D3 in ES cells and exhibits robust pRb kinase activity that is downregulated during the early stages of ES embryoid body differentiation. To investigate the basis underlying the insensitivity of ES cells to ectopic p16 expression, we show that Cdk6-cyclin D3 complexes are not subject to inhibition by p16, similar to Cdk-viral cyclin complexes. These observations show that specificity exists between Cdk4/6-cyclin D complexes and their ability to be targeted by p16. Our data suggest that Cdk6-cyclin D3 activity in other cell types, including tumors, may also be refractory to p16-mediated growth inhibition and raises the possibility of additional specificity within the INK4 family.