Three-dimensional virtual histology enabled through cytoplasm-specific X-ray stain for microscopic and nanoscopic computed tomography.

Many histological methods require staining of the cytoplasm, which provides instrumental details for diagnosis. One major limitation is the production of 2D images obtained by destructive preparation of 3D tissue samples. X-ray absorption micro- and nanocomputed tomography (microCT and nanoCT) allows for a nondestructive investigation of a 3D tissue sample, and thus aids to determine regions of interest for further histological examinations. However, application of microCT and nanoCT to biological samples (e.g., biopsies) is limited by the missing contrast within soft tissue, which is important to visualize morphological details. We describe an eosin-based preparation overcoming the challenges of contrast enhancement and selectivity for certain tissues. The eosin-based staining protocol is suitable for whole-organ staining, which then enables high-resolution microCT imaging of whole organs and nanoCT imaging of smaller tissue pieces retrieved from the original sample. Our results demonstrate suitability of the eosin-based staining method for diagnostic screening of 3D tissue samples without impeding further diagnostics through histological methods.

Characterising thermal water circulation in fractured bedrock using a multidisciplinary approach: a case study of St. Gorman's Well, Ireland.

A hydrogeological conceptual model of the source, circulation pathways and temporal variation of a low-enthalpy thermal spring in a fractured limestone setting is derived from a multidisciplinary approach. St. Gorman's Well is a thermal spring in east-central Ireland with a complex and variable temperature profile (maximum of 21.8 °C). Geophysical data from a three-dimensional(3D)audio-magnetotelluric(AMT) survey are combined with time-lapse hydrogeological data and information from a previously published hydrochemical analysis to investigate the operation of this intriguing hydrothermal system. Hydrochemical analysis and time-lapse measurements suggest that the thermal waters flow within the fractured limestones of the Carboniferous Dublin Basin at all times but display variability in discharge and temperature. The 3D electrical resistivity model of the subsurface revealed two prominent structures: (1) a NW-aligned faulted contact between two limestone lithologies; and (2) a dissolutionally enhanced, N-aligned, fault of probable Cenozoic age. The intersection of these two structures, which has allowed for karstification of the limestone bedrock, has created conduits facilitating the operation of relatively deep hydrothermal circulation (likely estimated depths between 240 and 1,000 m) within the limestone succession of the Dublin Basin. The results of this study support a hypothesis that the maximum temperature and simultaneous increased discharge observed at St. Gorman's Well each winter is the result of rapid infiltration, heating and recirculation of meteoric waters within a structurally controlled hydrothermal circulation system. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10040-021-02393-1.

Un modèle conceptuel hydrogéologique de la source, des voies de circulation et de la variation temporelle d'une source thermale à faible enthalpie dans un contexte de calcaire fracturé est dérivé d'une approche multidisciplinaire. St. Gorman's Well est une source thermale du centre-est de l'Irlande avec un profil de température complexe et variable (maximum de 21.8 °C). Les données géophysiques d'un levé audio-magnétotellurique (AMT) en trois dimensions (3D) sont combinées avec des données hydrogéologiques à intervalles de temps et des informations provenant d'une analyse hydrochimique publiée précédemment pour étudier le fonctionnement de cet intrigant système hydrothermal. L'analyse hydrochimique et les mesures à différentes périodes suggèrent que les eaux thermales s'écoulent à tout moment dans les calcaires fracturés du bassin carbonifère de Dublin, mais présentent une variabilité de débit et de température. Le modèle de résistivité électrique 3D du sous-sol a révélé deux structures importantes: (1) un contact faillé orienté NW entre deux lithologies calcaires; et (2) une faille alignée au Nord, améliorée par dissolution, d'âge cénozoïque probable. L'intersection de ces deux structures, qui a permis la karstification du socle calcaire, a créé des conduits facilitant le fonctionnement d'une circulation hydrothermale relativement profonde (profondeurs estimées vraisemblablement entre 240 et 1,000 m) au sein de la succession calcaire du bassin de Dublin. Les résultats de cette étude appuient l'hypothèse selon laquelle la température maximale et l'augmentation simultanée du débit observés à St. Gorman's Well chaque hiver sont le résultat d'une infiltration, d'un réchauffement et d'une recirculation rapides des eaux météoriques dans un système de circulation hydrothermale structurellement contrôlé.

Se deriva un modelo conceptual hidrogeológico de la fuente, las vías de circulación y la variación temporal de un manantial termal de baja entalpía en un entorno de caliza fracturada a partir de un enfoque multidisciplinar. Gorman's Well es un manantial termal en el centro-este de Irlanda con un perfil de temperatura complejo y variable (máximo de 21.8 °C). Los datos geofísicos de un estudio audio-magnetotelúrico (AMT) tridimensional (3D) se combinan con los datos hidrogeológicos de un lapso de tiempo y la información de un análisis hidroquímico previamente publicado para investigar el funcionamiento de este intrigante sistema hidrotermal. El análisis hidroquímico y las mediciones a intervalos de tiempo sugieren que las aguas termales fluyen dentro de las calizas fracturadas de la cuenca carbonífera de Dublín en todo momento, pero muestran variabilidad en la descarga y la temperatura. El modelo de resistividad eléctrica tridimensional del subsuelo reveló dos estructuras prominentes: (1) un contacto de falla alineado al NW entre dos litologías calcáreas; y (2) una falla de disolución incrementada, alineada al N, de probable edad cenozoica. La intersección de estas dos estructuras, que ha permitido la karstificación del lecho rocoso calcáreo, ha creado conductos que facilitan el funcionamiento de una circulación hidrotermal relativamente profunda (probablemente a profundidades estimadas entre 240 y 1,000 m) dentro de la sucesión calcárea de la cuenca de Dublín. Los resultados de este estudio apoyan la hipótesis de que la temperatura máxima y el aumento simultáneo de la descarga observados en St. Gorman's Well cada invierno son el resultado de una rápida infiltración, calentamiento y recirculación de aguas meteóricas dentro de un sistema de circulación hidrotermal estructuralmente controlado.

Um modelo hidrogeológico conceitual da fonte, vias de circulação e variação temporal de uma fonte termal de baixa entalpia em um ambiente de calcário fraturado é derivado de uma abordagem multidisciplinar. O poço de St. Gorman é uma fonte termal no centro-leste da Irlanda com um perfil de temperatura complexo e variável (máximo de 21.8°C). Os dados geofísicos de uma pesquisa áudio-magnetotelúrica (AMT) tridimensional (3D) são combinados com dados hidrogeológicos em intervalos de tempo e informações de uma análise hidroquimica publicada anteriormente, para investigar a operação deste intrigante sistema hidrotérmico. A análise hidroquimica e as medições em intervalos de tempo sugerem que as águas termais fluem de dentro dos calcários fraturados da Bacia Carbonifera de Dublin o tempo todo, mas exibem variabilidade na descarga e na temperatura. O modelo de resistividade elétrica 3D da subsuperfície revelou duas estruturas proeminentes: (1) um contato defeituoso alinhado a NO entre duas litologias de calcário; e (2) uma falha dissolucionalmente aumentada, alinhada a N, de provável idade Cenozóica. A intersecção dessas duas estruturas, que permitiu a carstificação da rocha calcária, criou condutos que facilitam a operação de circulação hidrotérmica relativamente profunda (profundidade estimada entre 240 e 1,000 m) dentro da sucessão de calcário da Bacia Dublin. Os resultados desse estudo suportam a hipótese de que a temperatura máxima e o aumento simultâneo da descarga observada no poço de St. Gorman a cada inverno é o resultado da rápida infiltração, aquecimento e recirculação de águas meteóricas dentro de um sistema de circulação hidrotérmica estruturalmente controlado.

Correction: DNA Damage, Homology-Directed Repair, and DNA Methylation.

[This corrects the article DOI: 10.1371/journal.pgen.0030110.].

Myoanatomy of the velvet worm leg revealed by laboratory-based nanofocus X-ray source tomography.

X-ray computed tomography (CT) is a powerful noninvasive technique for investigating the inner structure of objects and organisms. However, the resolution of laboratory CT systems is typically limited to the micrometer range. In this paper, we present a table-top nanoCT system in conjunction with standard processing tools that is able to routinely reach resolutions down to 100 nm without using X-ray optics. We demonstrate its potential for biological investigations by imaging a walking appendage of Euperipatoides rowelli, a representative of Onychophora-an invertebrate group pivotal for understanding animal evolution. Comparative analyses proved that the nanoCT can depict the external morphology of the limb with an image quality similar to scanning electron microscopy, while simultaneously visualizing internal muscular structures at higher resolutions than confocal laser scanning microscopy. The obtained nanoCT data revealed hitherto unknown aspects of the onychophoran limb musculature, enabling the 3D reconstruction of individual muscle fibers, which was previously impossible using any laboratory-based imaging technique.

Targeted DNA methylation by homology-directed repair in mammalian cells. Transcription reshapes methylation on the repaired gene.

We report that homology-directed repair of a DNA double-strand break within a single copy Green Fluorescent Protein (GFP) gene in HeLa cells alters the methylation pattern at the site of recombination. DNA methyl transferase (DNMT)1, DNMT3a and two proteins that regulate methylation, Np95 and GADD45A, are recruited to the site of repair and are responsible for selective methylation of the promoter-distal segment of the repaired DNA. The initial methylation pattern of the locus is modified in a transcription-dependent fashion during the 15-20 days following repair, at which time no further changes in the methylation pattern occur. The variation in DNA modification generates stable clones with wide ranges of GFP expression. Collectively, our data indicate that somatic DNA methylation follows homologous repair and is subjected to remodeling by local transcription in a discrete time window during and after the damage. We propose that DNA methylation of repaired genes represents a DNA damage code and is source of variation of gene expression.

New therapeutic perspectives in CCDC6 deficient lung cancer cells.

Non-small cell lung cancer (NSCLC) is the main cause of cancer-related death worldwide and new therapeutic strategies are urgently needed. In this study, we have characterized a panel of NSC lung cancer cell lines for the expression of coiled-coil-domain containing 6 (CCDC6), a tumor suppressor gene involved in apoptosis and DNA damage response. We show that low CCDC6 protein levels are associated with a weak response to DNA damage and a low number of Rad51 positive foci. Moreover, CCDC6 deficient lung cancer cells show defects in DNA repair via homologous recombination. In accordance with its role in the DNA damage response, CCDC6 attenuation confers resistance to cisplatinum, the current treatment of choice for NSCLC, but sensitizes the cells to olaparib, a small molecule inhibitor of the repair enzymes PARP1/2. Remarkably, the combination of the two drugs is more effective than each agent individually, as demonstrated by a combination index <1. Finally, CCDC6 is expressed at low levels in about 30% of the NSCL tumors we analyzed by TMA immunostaining. The weak CCDC6 protein staining is significatively correlated with the presence of lymph node metastasis (p ≤ 0.02) and negatively correlated to the disease free survival (p ≤ 0.01) and the overall survival (p ≤ 0.05). Collectively, the data indicate that CCDC6 levels provide valuable insight for OS. CCDC6 could represent a predictive biomarker of resistance to conventional single mode therapy and yield insight on tumor sensitivity to PARP inhibitors in NSCLC.

Gold(III) macrocycles: nucleotide-specific unconventional catalytic inhibitors of human topoisomerase I.

Topoisomerase IB (Top1) is a key eukaryotic nuclear enzyme that regulates the topology of DNA during replication and gene transcription. Anticancer drugs that block Top1 are either well-characterized interfacial poisons or lesser-known catalytic inhibitor compounds. Here we describe a new class of cytotoxic redox-stable cationic Au(3+) macrocycles which, through hierarchical cluster analysis of cytotoxicity data for the lead compound, 3, were identified as either poisons or inhibitors of Top1. Two pivotal enzyme inhibition assays prove that the compounds are true catalytic inhibitors of Top1. Inhibition of human topoisomerase IIα (Top2α) by 3 was 2 orders of magnitude weaker than its inhibition of Top1, confirming that 3 is a type I-specific catalytic inhibitor. Importantly, Au(3+) is essential for both DNA intercalation and enzyme inhibition. Macromolecular simulations show that 3 intercalates directly at the 5'-TA-3' dinucleotide sequence targeted by Top1 via crucial electrostatic interactions, which include π-π stacking and an Au···O contact involving a thymine carbonyl group, resolving the ambiguity of conventional (drug binds protein) vs unconventional (drug binds substrate) catalytic inhibition of the enzyme. Surface plasmon resonance studies confirm the molecular mechanism of action elucidated by the simulations.

Measurement of apparent diffusion coefficient with simultaneous MR/positron emission tomography in patients with peritoneal carcinomatosis: comparison with 18F-FDG-PET.

PURPOSE: To characterize peritoneal carcinomatosis (PC) of different histologically proven primary tumors based on diffusion-weighted imaging (DWI) and (18) F-FDG positron emission tomography (PET). MATERIALS AND METHODS: Forty-one patients underwent simultaneous MR/PET after clinically indicated (18) F-FDG-PET/CT. For all patients, histology of the primary tumor was obtained. MR protocol comprised anatomical imaging and axial DWI. Apparent diffusion coefficient (ADC) maps and FDG-PET were co-registered for evaluation of ADC and standard uptake value (SUV) of peritoneal lesions. Both lesion- and patient-based analysis was performed. Up to four peritoneal lesions were evaluated per patient. Mean and maximum standard uptake value (SUVmean , SUVmax ), mean and minimum ADC (ADCmean , ADCmin ) of each lesion were assessed. Spearman rank correlation (rs ) of ADC and SUV were calculated. SUV and ADC of ovarian and colorectal cancer lesions were compared using Wilcoxon test. RESULTS: Measurable lesions (n = 52) were found in 20 of 41 PC patients. Moderate, but significant correlation existed between ADC and SUV in the lesion-based as well as the patient-based analysis (lesion-based: SUVmean versus ADCmean rs = -0.58; SUVmax versus ADCmin rs = -0.56, all P < 0.0001; patient-based: SUVmean versus ADCmean rs = -0.64, P = 0.002; SUVmax versus ADCmin rs = -0.60, P = 0.005). ADC and SUV differed significantly between ovarian and colorectal cancer lesions (ADCmin : P < 0.0001; ADCmean : P < 0.0001; SUVmax : P = 0.002; SUVmean : P = 0.005). Overall, mucinous tumor entities showed a tendency to higher ADC and lower SUV. CONCLUSION: PC lesions showed significant differences in glucose uptake and diffusion characteristics depending on primary tumor histology. These differences should be considered when interpreting FDG-PET and DWI in PC patients.

GADD45α inhibition of DNMT1 dependent DNA methylation during homology directed DNA repair.

In this work, we examine regulation of DNA methyltransferase 1 (DNMT1) by the DNA damage inducible protein, GADD45α. We used a system to induce homologous recombination (HR) at a unique double-strand DNA break in a GFP reporter in mammalian cells. After HR, the repaired DNA is hypermethylated in recombinant clones showing low GFP expression (HR-L expressor class), while in high expressor recombinants (HR-H clones) previous methylation patterns are erased. GADD45α, which is transiently induced by double-strand breaks, binds to chromatin undergoing HR repair. Ectopic overexpression of GADD45α during repair increases the HR-H fraction of cells (hypomethylated repaired DNA), without altering the recombination frequency. Conversely, silencing of GADD45α increases methylation of the recombined segment and amplifies the HR-L expressor (hypermethylated) population. GADD45α specifically interacts with the catalytic site of DNMT1 and inhibits methylation activity in vitro. We propose that double-strand DNA damage and the resulting HR process involves precise, strand selected DNA methylation by DNMT1 that is regulated by GADD45α. Since GADD45α binds with high avidity to hemimethylated DNA intermediates, it may also provide a barrier to spreading of methylation during or after HR repair.

Gold(III) complexes of pyridyl- and isoquinolylamido ligands: structural, spectroscopic, and biological studies of a new class of dual topoisomerase I and II inhibitors.

The structures, spectroscopy, and cytotoxicity of four novel nominally square-planar gold(III) chelates 1-4 with the general formula cis-AuCl2(X), where the ligand X is an anionic bidentate pyridyl- or isoquinolylamido chelating agent, are described. The Au-N(amido), Au-N(pyridyl), and Au-N(isoquinolyl) distances are 2.002(9)-2.016(3), 2.01(1)-2.037(3), and 2.037(3) Å, respectively. Density functional theory simulations afforded accurate gold(III) coordination geometries for 1-4 (bond distances and angles to within 5% of the X-ray values), while accurate transition energies were limited to those calculated in the UV spectral region. The complexes had variable stability in dimethyl sulfoxide: compound 3 (relatively rigid) was indefinitely stable, compounds 1 and 2 (conformationally flexible) slowly demetalated over 30 days, and 4 (extensively aromatic) formed an insoluble precipitate after 10 days (72 h in an aqueous buffer). The isoquinolylamido derivative 4 was sufficiently cytotoxic in the NCI-60 screen to undergo full five-dose testing. Notably low GI50 (1.8, 2.3, and 3.2 µM) and IC50 (4.0, 9.8, and 15 µM) values were recorded for the OVCAR-3, IGROV1, and SW-620 cell lines, respectively. Hierarchical cluster analysis employing the National Cancer Institute (NCI) data for known anticancer drugs and 4 revealed that compound 4 is mechanistically identical with the topoisomerase IIα (Top2) poison zorubicin and statistically similar to the topoisomerase IB (Top1) poisons camptothecin and 9-methoxycamptothecin. The Top2-catalyzed decatenation reaction of kinetoplast DNA was studied as a function of the concentration of 4: the compound acts as an interfacial poison of Top2 at low concentrations (<1 µM) and a catalytic inhibitor of the enzyme above 5 µM. Gel mobility shift assays (plasmid DNA substrate) showed that the catalytic inhibition of Top2 likely correlates with DNA binding by 4 at concentrations >5 µM. Compound 4 is also a catalytic inhibitor of Top1 at higher concentrations, consistent with DNA binding by the complex.

Discrimination of phase altered targets by an echolocating Atlantic bottlenose dolphin.

Sensitivity of echolocating dolphins to phase changes within echoes may be a vital piece of information when constructing echolocation models. Previous experiments have yielded ambiguous results leaving it unclear what cues might have been used by passively listening dolphins to discriminate between different phase altered signals. This study used a phantom echo generator to produce computer controlled echoes. The dolphin interacted with the system in a real echolocation task to discriminate between simulated targets that were unaltered and those that had a 180° phase shift. The frequency amplitude spectral content between the two targets was the same. There were no temporal differences between the two targets. The only cue that the dolphin could use to discriminate between them was the 180° phase shift. The dolphin preformed at a success level of 40% in discriminating the two echoes. This indicates that the 180° phase shift was not perceived.

Pulmonary lesion assessment: comparison of whole-body hybrid MR/PET and PET/CT imaging--pilot study.

PURPOSE: To compare the performance of magnetic resonance (MR)/positron emission tomography (PET) imaging in the staging of lung cancer with that of PET/computed tomography (CT) as the reference standard and to compare the quantification accuracy of a new whole-body MR/PET system with corresponding PET/CT data sets. MATERIALS AND METHODS: Institutional review board approval and informed consent were obtained. Ten patients in whom bronchial carcinoma was proven or clinically suspected underwent clinically indicated fluorine 18 fluorodeoxyglucose (FDG) PET/CT and, immediately thereafter, whole-body MR/PET imaging with a new hybrid whole-body system (3.0-T MR imager with integrated PET system). Attenuation correction of MR/PET images was segmentation based with fat-water separation. Tumor-to-liver ratios were calculated and compared between PET/CT and MR/PET imaging. Tumor staging on the basis of the PET/CT and MR/PET studies was performed by two readers. Spearman rank correlation was used for comparison of data. RESULTS: MR/PET imaging provided diagnostic image quality in all patients, with good tumor delineation. Most lesions (nine of 10) showed pronounced FDG uptake. One lesion was morphologically suspicious for malignancy at CT and MR imaging but showed no FDG uptake. MR/PET imaging had higher mean tumor-to-liver ratios than did PET/CT (4.4 ± 2.0 [standard deviation] for PET/CT vs 8.0 ± 3.9 for MR/PET imaging). Significant correlation regarding the tumor-to-liver ratio was found between both imaging units (ρ = 0.93; P < .001). Identical TNM scores based on MR/PET and PET/CT data were found in seven of 10 patients. Differences in T and/or N staging occurred mainly owing to modality-inherent differences in lesion size measurement. CONCLUSION: MR/PET imaging of the lung is feasible and provides diagnostic image quality in the assessment of pulmonary masses. Similar lesion characterization and tumor stage were found in comparing PET/CT and MR/PET images in most patients.

A solid phase assay for topoisomerase I interfacial poisons and catalytic inhibitors.

We report a mechanism-based screening technique to rapidly identify eukaryotic topoisomerase I targeting agents. The method is based on genetic tagging of topoisomerase I to immobilize the enzyme on a solid surface in a microtiter well format. DNA is added to the wells, and retained DNA is detected by Pico Green fluorescence. Compounds that result in an increase in Pico Green staining represent potential topoisomerase interfacial poisons, whereas those that reduce fluorescence report catalytic inhibitors; therefore, the solid phase assay represents a "bimodal" readout that reveals mechanisms of action. The method has been demonstrated to work with known interfacial poisons and catalytic inhibitors. This method is rapid, robust, economical, and scalable for large library screens.

Facebook management as a tool for client communication and research recruitment in a companion animal university veterinary teaching and research hospital setting.

BACKGROUND: There is little peer-reviewed information about the strategic use of Facebook in companion animal veterinary settings, including for research recruitment. This study evaluates the implementation and execution of a Facebook strategy in a University Teaching and Research Hospital setting and the use of Facebook as a veterinary communication and recruitment tool. METHODS: All posts published on the hospital's Facebook page, messages sent via Messenger, and Facebook insight data from April 2017 to November 2019 (31 months) were reviewed and categorized. Facebook as a recruitment tool was evaluated through a survey among the faculty. RESULTS: A total of 113 posts were published, the Facebook page had 3485 followers and altogether 590,877 users were reached. The use of a Facebook strategy supported consistent management of the Facebook page. The content was well aligned with the strategy. The survey showed that the faculty experienced a facilitation effect by their recruitment posts, although the actual recruitment varied and ranged from none to the vast majority of all recruited subjects. CONCLUSIONS: This case-based, descriptive study gives insight and generates awareness about the possibilities and limitations of communication and research recruitment via Facebook. Further research is needed to evaluate if the findings can be generalized.

Neurotrophin-3 attenuates human peripheral blood T cell and monocyte activation status and cytokine production post stroke.

BACKGROUND AND PURPOSE: Stroke therapy still lacks successful measures to improve post stroke recovery. Neurotrophin-3 (NT-3) is one promising candidate which has proven therapeutic benefit in motor recovery in acute experimental stroke. Post stroke, the immune system has opposing pathophysiological roles: pro-inflammatory cascades and immune cell infiltration into the brain exacerbate cell death while the peripheral immune response has only limited capabilities to fight infections during the acute and subacute phase. With time, anti-inflammatory mechanisms are supposed to support recovery of the ischemic damage within the brain parenchyma. However, interestingly, NT-3 can improve recovery in chronic neurological injury when combined with the pro-inflammatory stimulus lipopolysaccharide (LPS). AIM: We elucidated the impact of NT-3 on human monocyte and T cell activation as well as cytokine production ex vivo after stroke. In addition, we investigated the age-dependent availability of the high affinity NT-3 receptor TrkC upon LPS stimulation. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from acute stroke patients and controls and incubated with different dosages of NT-3 (10 and 100 ng/mL) and with or without LPS or anti-CD3/CD28 for 48 h. Total TrkC expression and cell activation (CD25, CD69 and HLA-DR) were assessed by FACS staining. IFN-γ, TNF-α, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17A, IL-17F, IL-21 and IL-22 were quantified by cytometric bead array. RESULTS: Most monocytes and only a small proportion of T cells expressed TrkC in blood from humans without stroke. Activation of cells from young humans (without strokes) using anti-CD3/CD28 or LPS partially reduced the proportion of monocytes expressing TrkC whilst they increased the proportion of T cells expressing TrkC. In contrast, activation of cells from elderly humans (without strokes) did not affect the proportion of monocytes expressing TrkC and only anti-CD3/CD28 led to an increase in the proportion of CD4+ T cells expressing TrkC. In blood from stroke patients or controls, NT-3 treatment reduced the percentage of monocytes and CD4+ and CD8+ T cells that were activated and reduced all cytokines investigated besides IL-21. CONCLUSIONS: NT-3 attenuated immune responses in cells from stroke patients and controls. The mechanism whereby human immune cells respond to NT-3 may be via TrkC receptors whose levels are regulated by stimulation. Further work is required to determine whether the induction of sensorimotor recovery in rodents by NT-3 after CNS injury is caused by this attenuation of the immune response.

DNA methyltransferase 1-associated protein (DMAP1) is a co-repressor that stimulates DNA methylation globally and locally at sites of double strand break repair.

Correction of double strand DNA breaks proceeds in an error-free pathway of homologous recombination (HR), which can result in gene silencing of half of the DNA molecules caused by action by DNA methyltransferase 1 (DNMT1) (Cuozzo, C., Porcellini, A., Angrisano, T., Morano, A., Lee, B., Di Pardo, A., Messina, S., Iuliano, R., Fusco, A., Santillo, M. R., Muller, M. T., Chiariotti, L., Gottesman, M. E., and Avvedimento, E. V. (2007) PLoS Genet. 3, e110). To explore the mechanism that leads to HR-induced silencing, a genetic screen was carried out based on the silencing of a GFP reporter to identify potential partners. DMAP1, a DNMT1 interacting protein, was identified as a mediator of this process. DMAP1 is a potent activator of DNMT1 methylation in vitro, suggesting that DMAP1 is a co-repressor that supports the maintenance and de novo action of DNMT1. To examine critical roles for DMAP1 in vivo, lentiviral shRNA was used to conditionally reduce cellular DMAP1 levels. The shRNA transduced cells grew poorly and eventually ceased their growth. Analysis of the tumor suppressor gene p16 methylation status revealed a clear reduction in methylated CpGs in the shRNA cells, suggesting that reactivation of a tumor suppressor gene pathway caused the slow growth phenotype. Analysis of HR, using a fluorescence-based reporter, revealed that knocking down DMAP1 also caused hypomethylation of the DNA repair products following gene conversion. DMAP1 was selectively enriched in recombinant GFP chromatin based on chromatin immunoprecipitation analysis. The picture that emerges is that DMAP1 activates DNMT1 preferentially at sites of HR repair. Because DMAP1 depleted cells display enhanced HR, we conclude that it has additional roles in genomic stability.

Differentiation linked regulation of telomerase activity by Makorin-1.

To understand telomere homeostasis, a significant aspect of cancer and growth control, it is important to examine telomerase induction as well as mechanisms of regulated elimination. Makorin-1 (MKRN1) was previously shown to be an E3 ubiquitin ligase that targets the telomerase catalytic subunit (hTERT) for proteasome processing (Kim et al., Genes Dev 19:776-781, 2005). In this study we examined expression and regulation of endogenous MKRN1 during the cell cycle and terminal differentiation. When WI-38 cells transition from active growth into a resting G1 state, basal levels of MKRN1 were found to increase by sixfold. In contrast, cancer cells typically contained low or in some cases undetectable levels of MKRN1 protein. HL-60 cells growing exponentially in culture contain no detectable MKRN1; however, following terminal differentiation, MKRN1 mRNA and protein levels are strongly up-regulated while hTERT mRNA, hTERC, and telomerase are shut down. The initial decrease in telomerase activity is due to a gradual reduction in transcription of the hTERT gene that occurs during the first 12 h of terminal differentiation. MKRN1 protein appears between 12 and 24 h and is attended by a more rapid loss of telomerase activity. As more MKRN1 protein accumulates, significantly less telomerase activity is seen. Addition of the proteasome inhibitor, MG132, reverses the loss of telomerase activity; therefore, reductions in telomerase activity are dynamic, ongoing, and correlated with robust up-regulation of MKRN1 as the cells terminally differentiate. The data are consistent with the idea that MKRN1 represents a telomerase elimination pathway to rapidly draw down the activity during differentiation or cell cycle arrest when telomerase action at chromosome ends is no longer necessary.

SUMOylation enhances DNA methyltransferase 1 activity.

DNA methylation regulates gene expression through a complex network of protein-protein and protein-DNA interactions in chromatin. The maintenance methylase, DNMT1 (DNA methyltransferase 1), is a prominent enzyme in the process that is linked to DNA replication and drives the heritable nature of epigenetic modifications. The mechanistic details that explain how DNMT1 catalytic action is directed and regulated in chromatin are important in our overall understanding of gene control. In this work, we show that DNMT1 is modified by SUMOylation and we have mapped these SUMOylation sites by defined mutations. SUMOylated DNMT1 is catalytically active on genomic DNA in vivo and we find that SUMOylation significantly enhances the methylase activity of DNMT1 both in vitro and in chromatin. These data suggest that SUMOylation modulates the endogenous activity of a prominent epigenetic maintenance pathway in somatic cells.

DNA damage, homology-directed repair, and DNA methylation.

To explore the link between DNA damage and gene silencing, we induced a DNA double-strand break in the genome of Hela or mouse embryonic stem (ES) cells using I-SceI restriction endonuclease. The I-SceI site lies within one copy of two inactivated tandem repeated green fluorescent protein (GFP) genes (DR-GFP). A total of 2%-4% of the cells generated a functional GFP by homology-directed repair (HR) and gene conversion. However, approximately 50% of these recombinants expressed GFP poorly. Silencing was rapid and associated with HR and DNA methylation of the recombinant gene, since it was prevented in Hela cells by 5-aza-2'-deoxycytidine. ES cells deficient in DNA methyl transferase 1 yielded as many recombinants as wild-type cells, but most of these recombinants expressed GFP robustly. Half of the HR DNA molecules were de novo methylated, principally downstream to the double-strand break, and half were undermethylated relative to the uncut DNA. Methylation of the repaired gene was independent of the methylation status of the converting template. The methylation pattern of recombinant molecules derived from pools of cells carrying DR-GFP at different loci, or from an individual clone carrying DR-GFP at a single locus, was comparable. ClustalW analysis of the sequenced GFP molecules in Hela and ES cells distinguished recombinant and nonrecombinant DNA solely on the basis of their methylation profile and indicated that HR superimposed novel methylation profiles on top of the old patterns. Chromatin immunoprecipitation and RNA analysis revealed that DNA methyl transferase 1 was bound specifically to HR GFP DNA and that methylation of the repaired segment contributed to the silencing of GFP expression. Taken together, our data support a mechanistic link between HR and DNA methylation and suggest that DNA methylation in eukaryotes marks homologous recombined segments.

Comparison of posterolateral corner reconstructions using computer-assisted navigation.

PURPOSE: This study evaluated different fibular-based reconstruction techniques for grade III posterolateral corner (PLC) injuries. METHODS: Seven fresh-frozen cadaveric knees were used in this study. A surgical navigation system was used to determine varus opening and external rotation at 0 degrees , 30 degrees , and 60 degrees with a 9.8-Nm varus stress and 5-Nm external rotation stress applied to the tibia. Intact and disrupted PLC knees were used as controls. Four different fibular-based reconstruction techniques were evaluated. The femoral attachments consisted of a single- or double-tunnel technique, and the fibula attachment consisted of an anteroposterior or oblique tunnel technique. RESULTS: Sectioning of the PLC resulted in an increase in varus and external rotation at all flexion angles. All reconstruction techniques restored varus and external rotation stability compared with the PLC-deficient state, but the single-femoral tunnel reconstruction with an anteroposterior fibular tunnel did not restore varus or external rotation stability at 30 degrees and 60 degrees . No reconstruction technique overconstrained the knee at any flexion angle. CONCLUSIONS: A double femoral tunnel with an oblique fibular tunnel best restored native knee kinematics to the lateral side of the knee. CLINICAL RELEVANCE: Although there are many different techniques to reconstruct the PLC-deficient knee, this study suggests that a single-graft, fibular-based reconstruction that replicates the femoral insertions of the lateral collateral ligament and popliteus will be able to restore varus and external rotation stability to the knee.