Orientation-independent-DIC imaging reveals that a transient rise in depletion attraction contributes to mitotic chromosome condensation.

Genomic information must be faithfully transmitted into two daughter cells during mitosis. To ensure the transmission process, interphase chromatin is further condensed into mitotic chromosomes. Although protein factors like condensins and topoisomerase IIα are involved in the assembly of mitotic chromosomes, the physical bases of the condensation process remain unclear. Depletion attraction/macromolecular crowding, an effective attractive force that arises between large structures in crowded environments around chromosomes, may contribute to the condensation process. To approach this issue, we investigated the "chromosome milieu" during mitosis of living human cells using an orientation-independent-differential interference contrast module combined with a confocal laser scanning microscope, which is capable of precisely mapping optical path differences and estimating molecular densities. We found that the molecular density surrounding chromosomes increased with the progression from prophase to anaphase, concurring with chromosome condensation. However, the molecular density went down in telophase, when chromosome decondensation began. Changes in the molecular density around chromosomes by hypotonic or hypertonic treatment consistently altered the condensation levels of chromosomes. In vitro, native chromatin was converted into liquid droplets of chromatin in the presence of cations and a macromolecular crowder. Additional crowder made the chromatin droplets stiffer and more solid-like. These results suggest that a transient rise in depletion attraction, likely triggered by the relocation of macromolecules (proteins, RNAs, and others) via nuclear envelope breakdown and by a subsequent decrease in cell volumes, contributes to mitotic chromosome condensation, shedding light on a different aspect of the condensation mechanism in living human cells.

Usefulness of pituitary high-resolution 3D MRI with deep-learning-based reconstruction for perioperative evaluation of pituitary adenomas.

PURPOSE: To evaluate the diagnostic value of T1-weighted 3D fast spin-echo sequence (CUBE) with deep learning-based reconstruction (DLR) for depiction of pituitary adenoma and parasellar regions on contrast-enhanced MRI. METHODS: We evaluated 24 patients with pituitary adenoma or residual tumor using CUBE with and without DLR, 1-mm slice thickness 2D T1WI (1-mm 2D T1WI) with DLR, and 3D spoiled gradient echo sequence (SPGR) as contrast-enhanced MRI. Depiction scores of pituitary adenoma and parasellar regions were assigned by two neuroradiologists, and contrast-to-noise ratio (CNR) was calculated. RESULTS: CUBE with DLR showed significantly higher scores for depicting pituitary adenoma or residual tumor compared to CUBE without DLR, 1-mm 2D T1WI with DLR, and SPGR (p < 0.01). The depiction score for delineation of the boundary between adenoma and the cavernous sinus was higher for CUBE with DLR than for 1-mm 2D T1WI with DLR (p = 0.01), but the difference was not significant when compared to SPGR (p = 0.20). CUBE with DLR had better interobserver agreement for evaluating adenomas than 1-mm 2D T1WI with DLR (Kappa values, 0.75 vs. 0.41). The CNR of the adenoma to the brain parenchyma increased to a ratio of 3.6 (obtained by dividing 13.7, CNR of CUBE with DLR, by 3.8, that without DLR, p < 0.01). CUBE with DLR had a significantly higher CNR than SPGR, but not 1-mm 2D T1WI with DLR. CONCLUSION: On the contrast-enhanced MRI, compared to CUBE without DLR, 1-mm 2D T1WI with DLR and SPGR, CUBE with DLR improves the depiction of pituitary adenoma and parasellar regions.

Chromatin organization and behavior in HRAS-transformed mouse fibroblasts.

In higher eukaryotic cells, a string of nucleosomes, where long genomic DNA is wrapped around core histones, are rather irregularly folded into a number of condensed chromatin domains, which have been revealed by super-resolution imaging and Hi-C technologies. Inside these domains, nucleosomes fluctuate and locally behave like a liquid. The behavior of chromatin may be highly related to DNA transaction activities such as transcription and repair, which are often upregulated in cancer cells. To investigate chromatin behavior in cancer cells and compare those of cancer and non-cancer cells, we focused on oncogenic-HRAS (Gly12Val)-transformed mouse fibroblasts CIRAS-3 cells and their parental 10T1/2 cells. CIRAS-3 cells are tumorigenic and highly metastatic. First, we found that HRAS-induced transformation altered not only chromosome structure, but also nuclear morphology in the cell. Using single-nucleosome imaging/tracking in live cells, we demonstrated that nucleosomes are locally more constrained in CIRAS-3 cells than in 10T1/2 cells. Consistently, heterochromatin marked with H3K27me3 was upregulated in CIRAS-3 cells. Finally, Hi-C analysis showed enriched interactions of the B-B compartment in CIRAS-3 cells, which likely represents transcriptionally inactive chromatin. Increased heterochromatin may play an important role in cell migration, as they have been reported to increase during metastasis. Our study also suggests that single-nucleosome imaging provides new insights into how local chromatin is structured in living cells.

Association of prediabetes with reduced brain volume in a general elderly Japanese population.

OBJECTIVES: Diabetes frequently results in cognitive impairment, but it is less clear if brain health is adversely affected during the prediabetic stage. Our aim is to identify possible changes in brain volume as measured by magnetic resonance imaging (MRI) in a large elderly population stratified according to level of "dysglycemia." METHODS: This is a cross-sectional study of 2144 participants (median age 69 years, 60.9% female) who underwent 3-T brain MRI. Participants were divided into 4 dysglycemia groups based on HbA1c levels (%): normal glucose metabolism (NGM) (< 5.7%), prediabetes (5.7 to < 6.5%), undiagnosed diabetes (6.5% or higher), and known diabetes (defined by self-report). RESULTS: Of the 2144 participants, 982 had NGM, 845 prediabetes, 61 undiagnosed diabetes, and 256 known diabetes. After adjustment for age, sex, education, body weight, cognitive status, smoking, drinking, and disease history, total gray matter volume was significantly lower among participants with prediabetes (0.41% lower, standardized ß = - 0.0021 [95% CI - 0.0039, - 0.00039], p = 0.016), undiagnosed diabetes (1.4% lower, standardized ß = - 0.0069 [95% CI - 0.012, - 0.002], p = 0.005), and known diabetes (1.1% lower, standardized ß = - 0.0055 [95% CI - 0.0081, - 0.0029], p < 0.001) compared to the NGM group. After adjustment, total white matter volume and hippocampal volume did not differ significantly between the NGM group and either the prediabetes group or the diabetes group. CONCLUSION: Sustained hyperglycemia may have deleterious effects on gray matter integrity even prior to the onset of clinical diabetes. KEY POINTS: • Sustained hyperglycemia has deleterious effects on gray matter integrity even prior to the onset of clinical diabetes.

Optic radiation atrophy in Lewy body disease with visual hallucination on phase difference enhanced magnetic resonance images.

Visual hallucinations (VH) occur commonly in Lewy body disease (LBD), including Parkinson's disease (PD), PD with dementia, and dementia with Lewy bodies. We aimed to use phase difference enhanced imaging (PADRE) to assess structural abnormalities of optic radiation (OR) in patients with Lewy body disease (LBD) concomitant with VH. Firstly, two radiologists reviewed the OR appearances in healthy subjects (HS) on PADRE. Next, based on the OR abnormalities, two reviewers assessed the PADRE images from 18 HS and 38 and 110 patients with LBD, with and without VH, respectively, in a blinded manner. Finally, all patients with LBD without VH were eventually followed up for at least 5 years after magnetic resonance imaging to determine the appearance of VH. The radiologists identified three layers, namely external sagittal stratum, internal sagittal stratum, and tapetum, in OR on the PADRE in HS. Moreover, they were able to consensually define the OR as abnormal when the layers were obscured and the disappearance of the cranial side. The sensitivity/specificity of abnormal OR for each case was 68%/81% (LBD with VH vs. LBD without VH). Furthermore, VH appeared in 12 of the 21 (57%) patients with LBD and abnormal OR during the follow-up period. However, no patients without abnormal OR reported VH. Patients with LBD and VH demonstrated the abnormal OR. This, in turn, might be a useful marker to distinguish the patients with VH from those without VH and HS. Moreover, abnormal OR on PADRE may precede the appearance of VH in LBD.

SETDB1-dependent heterochromatin stimulates alternative lengthening of telomeres.

Alternative lengthening of telomeres, or ALT, is a recombination-based process that maintains telomeres to render some cancer cells immortal. The prevailing view is that ALT is inhibited by heterochromatin because heterochromatin prevents recombination. To test this model, we used telomere-specific quantitative proteomics on cells with heterochromatin deficiencies. In contrast to expectations, we found that ALT does not result from a lack of heterochromatin; rather, ALT is a consequence of heterochromatin formation at telomeres, which is seeded by the histone methyltransferase SETDB1. Heterochromatin stimulates transcriptional elongation at telomeres together with the recruitment of recombination factors, while disrupting heterochromatin had the opposite effect. Consistently, loss of SETDB1, disrupts telomeric heterochromatin and abrogates ALT. Thus, inhibiting telomeric heterochromatin formation in ALT cells might offer a new therapeutic approach to cancer treatment.

Evaluation of Peritumoral Brain Parenchyma Using Contrast-Enhanced 3D Fast Imaging Employing Steady-State Acquisition at 3T for Differentiating Metastatic Brain Tumors and Glioblastomas.

BACKGROUND: Metastatic brain tumors and glioblastomas are the 2 of the most common brain neoplasms in adults. However, distinguishing solitary metastatic brain tumors from glioblastomas on conventional magnetic resonance imaging remains particularly challenging. Thus, we aimed to retrospectively assess the role of contrast-enhanced fast imaging employing steady-state acquisition (CE-FIESTA) imaging in distinguishing between metastatic brain tumors and glioblastomas. MATERIALS AND METHODS: Forty-three patients with metastatic brain tumors and 14 patients with glioblastomas underwent conventional magnetic resonance imaging and CE-FIESTA before surgery. First, 1 neuroradiologist and 1 neurosurgeon classified the CE-FIESTA findings for the peritumoral brain parenchyma by consensus. Next, the 2 neuroradiologists performed an observer performance study comparing tumor shape classification (smooth or irregular margins), a classic imaging finding, with the CE-FIESTA classification of the peritumoral brain parenchyma. RESULTS: The CE-FIESTA findings for the peritumoral brain parenchyma were classified as follows: type A, no hyperintense rim; type B, partial hyperintense rim; and type C, extended hyperintense rim. With regard to the diagnosis of metastatic brain tumors, the observer performance study demonstrated that the mean sensitivity, specificity, and accuracy of an extended hyperintense rim classification (type C) on CE-FIESTA images were 95.3%, 85.7%, and 93.0%, respectively. The accuracy of the CE-FIESTA classification was significantly higher than that of the tumor shape classification. CONCLUSIONS: CE-FIESTA images may provide useful information for distinguishing metastatic brain tumors from glioblastomas, especially when focusing on differences in the peritumoral brain parenchyma.

Targeting 24 bp within Telomere Repeat Sequences with Tandem Tetramer Pyrrole-Imidazole Polyamide Probes.

Synthetic molecules that bind sequence-specifically to DNA have been developed for varied biological applications, including anticancer activity, regulation of gene expression, and visualization of specific genomic regions. Increasing the number of base pairs targeted by synthetic molecules strengthens their sequence specificity. Our group has been working on the development of pyrrole-imidazole polyamides that bind to the minor groove of DNA in a sequence-specific manner without causing denaturation. Recently, we reported a simple synthetic method of fluorescent tandem dimer polyamide probes composed of two hairpin moieties with a linking hinge, which bound to 12 bp in human telomeric repeats (5'-(TTAGGG)n-3') and could be used to specifically visualize telomeres in chemically fixed cells under mild conditions. We also performed structural optimization and extension of the target base pairs to allow more specific staining of telomeres. In the present study, we synthesized tandem tetramer polyamides composed of four hairpin moieties, targeting 24 bp in telomeric repeats, the longest reported binding site for synthetic, non-nucleic-acid-based, sequence-specific DNA-binding molecules. The novel tandem tetramers bound with a nanomolar dissociation constant to 24 bp sequences made up of four telomeric repeats. Fluorescently labeled tandem tetramer polyamide probes could visualize human telomeres in chemically fixed cells with lower background signals than polyamide probes reported previously, suggesting that they had higher specificity for telomeres. Furthermore, high-throughput sequencing of human genomic DNA pulled down by the biotin-labeled tandem tetramer polyamide probe confirmed its effective binding to telomeric repeats in the complex chromatinized genome.

Telomere Visualization in Tissue Sections using Pyrrole-Imidazole Polyamide Probes.

Pyrrole-Imidazole (PI) polyamides bind to specific DNA sequences in the minor groove with high affinity. Specific DNA labeling by PI polyamides does not require DNA denaturation with harsh treatments of heat and formamide and has the advantages of rapid and less disruptive processing. Previously, we developed tandem hairpin PI polyamide probes (TH59 series), which label telomeres in cultured cell lines more efficiently than conventional methods, such as fluorescence in situ hybridization (FISH). Here, we demonstrate that a TH59 derivative, HPTH59-b, along with immunostaining for specifying cell types in the tissues, visualizes telomeres in mouse and human tissue sections. Quantitative measurements of telomere length with single-cell resolution suggested shorter telomeres in the proliferating cell fractions of tumor than in non-tumor tissues. Thus, PI polyamides are a promising alternative for telomere labeling in clinical research, as well as in cell biology.

Prediction of hard meningiomas: quantitative evaluation based on the magnetic resonance signal intensity.

BACKGROUND: From a surgical perspective, presurgical prediction of meningioma consistency is beneficial. PURPOSE: To quantitatively analyze the correlation between the magnetic resonance (MR) signal intensity (SI) or apparent diffusion coefficient (ADC) and meningioma consistency and to determine which MR sequence could help predicting hard meningiomas. MATERIAL AND METHODS: This study included 43 patients with meningiomas who underwent preoperative MR imaging (MRI), including T1-weighted (T1W) imaging, T2-weighted (T2W) imaging, fluid-attenuated inversion recovery (FLAIR), diffusion-weighted imaging (DWI), contrast-enhanced (CE)-T1W imaging, and CE-fast imaging employing steady-state acquisition (FIESTA). A neurosurgeon evaluated the tumor consistency using a visual analog scale (VAS) with the anchors "soft" (score = 0) and "hard" (score = 10). The SI ratio (tumor to cerebral cortex SI) and ADC value were compared with the tumor consistency. The sensitivity, specificity, and accuracy for predicting hard meningiomas (VAS score ≥8; 9 of 43 patients) were calculated using cutoff values for the SI ratio that were obtained in a receiver operating characteristic curve analysis. RESULTS: A significant negative correlation was observed between the tumor consistency and the SI ratio on T2W imaging, FLAIR, and CE-FIESTA (P < 0.05) but not on T1W imaging, CE-T1W imaging, and the ADC value. The sensitivity, specificity, and accuracy for predicting hard meningiomas were 89%, 79%, and 81% with T2W imaging; 89%, 76%, and 79% with FLAIR; and 100%, 74%, and 79% with CE-FIESTA, respectively. CONCLUSION: Our results suggest that a quantitative assessment using conventional T2W imaging or FLAIR may be a simple and useful method for predicting hard meningiomas.

Relationship between the catechol-O-methyl transferase Val108/158Met genotype and brain volume in treatment-naive major depressive disorder: Voxel-based morphometry analysis.

Catechol-O-methyltransferase (COMT) is a methylation enzyme engaged in the degradation of dopamine and noradrenaline by catalyzing the transfer of a methyl group from S-adenosylmethionine. An association was found between the Valine (Val) 108/158Methionine (Met) COMT polymorphism (rs4680) and major depressive disorder (MDD). The authors prospectively investigated the relationship between the Val108/158Met COMT genotype and voxel-based morphometry (VBM) findings for patients with first-episode and treatment-naïve MDD and healthy subjects (HS). Participants comprised 30 MDD patients and 48 age- and sex-matched HS who were divided according to the COMT genotype. Effects of diagnosis, COMT genotype, and the genotype-diagnosis interaction in relation to brain morphology in the Val/Met and Val/Val individuals were evaluated using a VBM analysis of high-resolution magnetic resonance imaging findings. Among the Val/Met individuals, the volume of the bilateral caudate was significantly smaller for MDD patients than for HS. In the Val/Val individuals, the caudate volume was comparable between MDD patients and HS. Significant genotype-diagnosis interaction effects on brain morphology were noted in the right caudate.

End-targeting proteomics of isolated chromatin segments of a mammalian ribosomal RNA gene promoter.

The unbiased identification of proteins associated with specific loci is crucial for understanding chromatin-based processes. The proteomics of isolated chromatin fragment (PICh) method has previously been developed to purify telomeres and identify associated proteins. This approach is based on the affinity capture of endogenous chromatin segments by hybridization with oligonucleotide containing locked nucleic acids. However, PICh is only efficient with highly abundant genomic targets, limiting its applicability. Here we develop an approach for identifying factors bound to the promoter region of the ribosomal RNA genes that we call end-targeting PICh (ePICh). Using ePICh, we could specifically enrich the RNA polymerase I pre-initiation complex, including the selectivity factor 1. The high purity of the ePICh material allowed the identification of ZFP106, a novel factor regulating transcription initiation by targeting RNA polymerase I to the promoter. Our results demonstrate that ePICh can uncover novel proteins controlling endogenous regulatory elements in mammals.

Abnormal white matter integrity in the corpus callosum among smokers: tract-based spatial statistics.

In the present study, we aimed to investigate the difference in white matter between smokers and nonsmokers. In addition, we examined relationships between white matter integrity and nicotine dependence parameters in smoking subjects. Nineteen male smokers were enrolled in this study. Eighteen age-matched non-smokers with no current or past psychiatric history were included as controls. Diffusion tensor imaging scans were performed, and the analysis was conducted using a tract-based special statistics approach. Compared with nonsmokers, smokers exhibited a significant decrease in fractional anisotropy (FA) throughout the whole corpus callosum. There were no significant differences in radial diffusivity or axial diffusivity between the two groups. There was a significant negative correlation between FA in the whole corpus callosum and the amount of tobacco use (cigarettes/day; R = - 0.580, p = 0.023). These results suggest that the corpus callosum may be one of the key areas influenced by chronic smoking.

Stereotactic body radiotherapy for lung tumors in patients with subclinical interstitial lung disease: the potential risk of extensive radiation pneumonitis.

PURPOSE: To evaluate the toxicity and efficacy of thoracic stereotactic body radiotherapy (SBRT) in patients with subclinical interstitial lung disease (ILD). METHODS AND MATERIALS: One hundred patients with 124 lung tumors were treated with SBRT at our institution according to our own protocols; patients with subclinical (untreated and oxygen-free) ILD were treated with SBRT, while those with clinical ILD (post- or under treatment) were not. The administration of 48 Gy in four fractions was used in 103 (83%) of the 124 tumors. The presence of subclinical ILD in the pre-SBRT CT findings was reviewed by two chest radiologists. The relationships between radiation pneumonitis (RP) and clinical factors were investigated. RESULTS: Subclinical ILD was recognized in 16 (16%) of 100 patients. Grade 2-5 RP was recognized in 13 (13%) of 100 patients. Grade 2-5 RP was observed in three (19%) of 16 patients with subclinical ILD. Subclinical ILD was not found to be a significant factor influencing Grade 2-5 RP; however, extensive RP beyond the irradiated field, including the contralateral lung, was recognized in only three patients with subclinical ILD, and the rate of extensive RP was significantly high in the patients with subclinical ILD. Grade 4 or 5 extensive RP was recognized in only two patients with subclinical ILD. Dosimetric factors of the lungs (V5, V10, V15, V20, V25, MLD) were significantly associated with Grade 2-5 RP. The three-year overall survival and local control rates of all patients were 53% and 86%, respectively. No significant differences were seen in either overall survival or local control rates between the patients with ILD and those without ILD. CONCLUSIONS: Subclinical ILD was not found to be a significant factor for Grade 2-5 RP or clinical outcomes in the current study; however, uncommon extensive RP can occur in patients with subclinical ILD.

Delineation of optic radiation and stria of Gennari on high-resolution phase difference enhanced imaging.

RATIONALE AND OBJECTIVES: Phase difference enhanced (PADRE) imaging technique can selectively enhanced the phase difference between the target and surrounding tissue. Our purpose is to assess the delineations of the optic radiation and primary visual cortex (stria of Gennari) using PADRE. MATERIALS AND METHODS: The subjects were 6 healthy volunteers. Axial and coronal high-spatial resolution PADRE images were acquired covering the entire optic radiation using a 3T magnetic resonance system. Two radiologists evaluated the PADRE and susceptibility-weighted imaging (SWI)-like images for the delineation of four layers at the optic radiation (tapetum, internal sagittal stratum, external sagittal stratum, and adjacent white matter) on the basis of the anatomic appearances of the cadaveric specimens stained with Bodian's method and Kluver-Barrera method. The radiologists also assessed the delineations of the stria of Gennari on PADRE and SWI-like images. RESULTS: In all 6 healthy subjects, the PADRE images clearly identified the four layers at the optic radiation, as well as the stria of Gennari, which were difficult to appreciate in SWI-like images. The anatomic appearances of the optic radiation on PADRE images were more similar to those seen in the specimens stained with Kluver-Barrera method than with Bodian's method. CONCLUSION: The PADRE technique can delineate the four layers at the optic radiation and the stria of Gennari; the differences in myelin densities can also be enhanced. The PADRE technique may have the potential to reinforce the clinical utility of MRI in the diagnosis of diseases that affect the optic radiation and primary visual cortex.

Abundance of ribosomal RNA gene copies maintains genome integrity.

The ribosomal RNA (rDNA) gene repeats are essential housekeeping genes found in all organisms. A gene amplification system maintains large cluster(s) of tandemly repeated copies in the chromosome, with each species having a specific number of copies. Yeast has many untranscribed rDNA copies (extra copies), and we found that when they are lost, the cells become sensitive to DNA damage induced by mutagens. We show that this sensitivity is dependent on rDNA transcriptional activity, which interferes with cohesion between rDNA loci of sister chromatids. The extra rDNA copies facilitate condensin association and sister-chromatid cohesion, thereby facilitating recombinational repair. These results suggest that high concentrations of heavily transcribed genes are toxic to the cells, and therefore amplified genes, such as rDNA, have evolved.

Extraskeletal osteosarcoma of the pleura: a case report.

We herein present an exceedingly rare case of pleural osteosarcoma that was surgically resected in an elderly patient. A 74-year-old man, complaining of a dry cough and breathlessness on effort, was found to have massive pleural effusion on a chest X-ray. The chest CT and MRI scans indicated a massive effusion and a pleural tumor encasing the left lower lung. The resected tumor was histologically an osteosarcoma, measuring 11.3 x 9.0 x 6.0 cm. Because there was no evidence of any other primary tumor, the diagnosis of extraskeletal osteosarcoma was appropriate.