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1.  RNA-Binding Protein AUF1 Promotes Myogenesis by Regulating MEF2C Expression Levels 
Molecular and Cellular Biology  2014;34(16):3106-3119.
The mammalian RNA-binding protein AUF1 (AU-binding factor 1, also known as heterogeneous nuclear ribonucleoprotein D [hnRNP D]) binds to numerous mRNAs and influences their posttranscriptional fate. Given that many AUF1 target mRNAs encode muscle-specific factors, we investigated the function of AUF1 in skeletal muscle differentiation. In mouse C2C12 myocytes, where AUF1 levels rise at the onset of myogenesis and remain elevated throughout myocyte differentiation into myotubes, RNP immunoprecipitation (RIP) analysis indicated that AUF1 binds prominently to Mef2c (myocyte enhancer factor 2c) mRNA, which encodes the key myogenic transcription factor MEF2C. By performing mRNA half-life measurements and polysome distribution analysis, we found that AUF1 associated with the 3′ untranslated region (UTR) of Mef2c mRNA and promoted MEF2C translation without affecting Mef2c mRNA stability. In addition, AUF1 promoted Mef2c gene transcription via a lesser-known role of AUF1 in transcriptional regulation. Importantly, lowering AUF1 delayed myogenesis, while ectopically restoring MEF2C expression levels partially rescued the impairment of myogenesis seen after reducing AUF1 levels. We propose that MEF2C is a key effector of the myogenesis program promoted by AUF1.
PMCID: PMC4135590  PMID: 24891619
2.  Cytokines secreted by macrophages isolated from tumor microenvironment of inflammatory breast cancer patients possess chemotactic properties 
Although there is a growing literature describing the role of macrophages in breast cancer, the role of macrophages in inflammatory breast cancer (IBC) is unclear. The aim of present study was to isolate and characterize tumor associated macrophages of IBC and non-IBC patients and define their role in IBC. Tumor infiltrating monocytes/macrophages (CD14+ and CD68+) were measured by immunohistochem-istry using specific monoclonal antibodies. Blood drained from axillary vein tributaries was collected during breast cancer surgery and the percentage of CD14+ in the total isolated leukocytes was assessed by flow cytometric analysis. CD14+ cells were separated from total leukocytes by immuno-magnetic beads technique and were cultured overnight. Media conditioned by CD14+ were collected and subjected to cytokine profiling using cytokine antibody array. Wound healing and invasion assays were used to test whether cytokines highly secreted by tumor drained macrophages induce motility and invasion of breast cancer cells. We found that macrophages highly infiltrate into carcinoma tissues of IBC patients. In addition blood collected from axillary tributaries of IBC patients is highly enriched with CD14+ cells as compared to blood collected from non-IBC patients. Cytokine profiling of CD14+ cells isolated from IBC patients revealed a significant increase in secretion of tumor necrosis factor-α; monocyte chemoat-tractant protein-1/CC-chemokine ligand 2; interleukin-8 and interleukin-10 as compared to CD14+ cells isolated from non-IBC patients. Tumor necrosis factor-a, interleukin-8 and interleukin-10 significantly increased motility and invasion of IBC cells in vitro. In conclusion, macrophages isolated from the tumor microenvironment of IBC patients secrete chemotactic cytokines that may augment dissemination and metastasis of IBC carcinoma cells.
PMCID: PMC3928544  PMID: 24291763
Inflammatory breast cancer; Macrophages; Cytokines; Invasion; Motility
3.  Clustering-Based Method for Developing a Genomic Copy Number Alteration Signature for Predicting the Metastatic Potential of Prostate Cancer 
Journal of probability and statistics  2012;2012(2012):873570-.
The transition of cancer from a localized tumor to a distant metastasis is not well understood for prostate and many other cancers, partly, because of the scarcity of tumor samples, especially metastases, from cancer patients with long-term clinical follow-up. To overcome this limitation, we developed a semi-supervised clustering method using the tumor genomic DNA copy number alterations to classify each patient into inferred clinical outcome groups of metastatic potential. Our data set was comprised of 294 primary tumors and 49 metastases from 5 independent cohorts of prostate cancer patients. The alterations were modeled based on Darwin's evolutionary selection theory and the genes overlapping these altered genomic regions were used to develop a metastatic potential score for a prostate cancer primary tumor. The function of the proteins encoded by some of the predictor genes promote escape from anoikis, a pathway of apoptosis, deregulated in metastases. We evaluated the metastatic potential score with other clinical predictors available at diagnosis using a Cox proportional hazards model and show our proposed score was the only significant predictor of metastasis free survival. The metastasis gene signature and associated score could be applied directly to copy number alteration profiles from patient biopsies positive for prostate cancer.
PMCID: PMC4240515  PMID: 25419216
4.  Methylation of histone H4 at aspartate 24 by Protein L-isoaspartate O-methyltransferase (PCMT1) links histone modifications with protein homeostasis 
Scientific Reports  2014;4:6674.
Histone modifications play crucial roles in modulating chromatin function and transcriptional activity. Due to their long half-life, histones can, in addition to post-translational modifications, also accumulate spontaneous chemical alterations, which can affect their functionality and require either protein repair or degradation. One of the major sources of such protein damage or ageing is the conversion of aspartate into isoaspartate residues that can then be methylated. Here, we characterize a novel histone modification, the methylation of histone H4 at aspartate 24 (H4D24me). We generated H4D24me specific antibodies and showed that H4D24me is ubiquitously present in different mouse and human cells. Our in vitro and in vivo data identified PCMT1 (Protein L-isoaspartate O-methyltransferase), an enzyme involved in protein repair, as a novel H4D24 specific histone methyltransferase. Furthermore, we demonstrated that VprBP (HIV-1 viral protein R (Vpr)-binding protein), a chromo domain-containing protein, specifically recognizes H4D24me potentially implicating H4D24me in H4 degradation. Thus, this work links for the first time a histone modification with histone protein aging and histone homeostasis, suggesting novel functions for histone modifications beyond transcriptional regulation.
PMCID: PMC4202215  PMID: 25327473
5.  Auf1/Hnrnpd-Deficient Mice Develop Pruritic Inflammatory Skin Disease 
Mice lacking heterogenous nuclear ribonuclear protein D (Hnrnpd), also known as Auf1, a regulator of inflammatory cytokine mRNA stability, develop chronic dermatitis with age that is characterized by pruritis and excoriations. Histological analysis showed marked epidermal acanthosis and spongiosis, neovascularization, and elevated number of inflammatory cells, including T cells, macrophages, neutrophils, mast cells, and eosinophils. Hnrnpd-deficient (Hnrnpdtm1Rjsc) mice with dermatitis display elevated serum IgE levels. Lesions in Hnrnpdtm1Rjsc mice were associated with a shift towards a Th2 immune environment. Evaluation of T-cell-mediated skin inflammation by assaying contact hypersensitivity indicated an increased response in Hnrnpdtm1Rjsc mice. T cells and macrophages from Hnrnpdtm1Rjsc mice demonstrate a number of abnormalities associated with dermatitis, including increased IL2, tumor-necrosis factor-α (TNFα), and IL1β production. Finally, many features of spontaneous dermatitis could be recapitulated in experimentally induced lesions by subcutaneous injection of CCL27 and TNF in unaffected Hnrnpdtm1Rjsc mice. Collectively, these data highlight the importance of HNRNPD and proper regulation of mRNA stability in the intricate processes of leukocyte recruitment and inflammatory activation within the skin.
PMCID: PMC4074411  PMID: 18830269
6.  Clinical, Etiological, and Therapeutic Features of Jaw-opening and Jaw-closing Oromandibular Dystonias: A Decade of Experience at a Single Treatment Center 
Dystonia is a heterogeneous hyperkinetic disorder. The anatomical location of the dystonia helps clinicians guide their evaluation and treatment plan. When dystonia involves masticatory, lingual, and pharyngeal muscles, it is referred to as oromandibular dystonia (OMD).
We identified patients diagnosed with OMD in a Movement Disorders Clinic and Laryngeal Movement Disorders Clinic from a single institution. Demographic, etiological, clinical, and therapeutic information was retrospectively reviewed for patients with jaw-opening (O-OMD) and jaw-closing (C-OMD) OMD.
Twenty-seven patients were included. Their average age of onset was in the sixth decade of life and there was a female predominance. Etiological factors linked in this study to OMD included a family history of dystonia or essential tremor, occupation, cerebellar disease, a dental disorder, and tardive syndrome. Clinically, patients with C-OMD presented with more prominent feeding difficulties, but seemed to respond better to therapy than those with O-OMD. In addition to the known benefits of botulinum toxin therapy, patients who described sensory tricks obtained benefit from the use of customized dental prosthesis.
This works provides useful information on potential etiological factors for OMD and its response to therapy, and highlights the potential benefit of dental prosthesis for the treatment of OMD.
PMCID: PMC4011122  PMID: 24847442
Oromandibular dystonia; botulinum toxin; sensory trick; etiology
7.  Phase 2 trial of everolimus and carboplatin combination in patients with triple negative metastatic breast cancer 
Rapamycin acts synergistically with platinum agents to induce apoptosis and inhibit proliferation in breast cancer cell lines. Combination of everolimus also known as RAD001 (oral mammalian target of rapamycin (mTOR) inhibitor) and carboplatin may have activity in metastatic triple-negative breast cancer (TNBC).
The primary objective of this study was to determine clinical benefit rate (CBR), that is (complete remission (CR) + partial remission (PR) + stable disease (SD) lasting ≥6 months) and the toxicity of everolimus/carboplatin in women with metastatic TNBC. Prior carboplatin was allowed. Treatment consisted of intravenous carboplatin area under the curve (AUC) 6 (later decreased to AUC 5 and subsequently to AUC 4) every 3 weeks with daily 5 mg everolimus.
We enrolled 25 patients in this study. Median age was 58 years. There were one CR, six PRs, seven SDs and eight PDs (progression of disease). CBR was 36% (95% confidence interval (CI) 21.1 to 57.4%). One SD was achieved in a patient progressing on single agent carboplatin. The median progression free survival (PFS) was 3 months (95% CI 1.6 to 4.6 months) and overall survival (OS) was 16.6 months (95% CI 7.3 months to not reached). There were seven patients (28%) with ≥ grade 3 thrombocytopenia; three (12%) with grade 3 neutropenia (no bleeding/febrile neutropenia) and one (4%) with grade 3 anemia. Greater hematological toxicity was seen in the first seven patients treated with carboplatin AUC5/6. After the amendment for starting dose of carboplatin to AUC 4, the regimen was well tolerated with only one out of 18 patients with grade 3 neutropenia and two patients with grade 3 thrombocytopenia. There was only one case of mucositis.
Everolimus-carboplatin was efficacious in metastatic TNBC. Dose limiting hematological toxicity was observed when AUC5/6 of carboplatin was combined with everolimus. However, carboplatin AUC 4 was well tolerated in combination with everolimus with continuing responses.
Trial registrations NCT01127763.
PMCID: PMC4053575  PMID: 24684785
8.  mRNA decay factor AUF1 maintains normal aging, telomere maintenance and suppression of senescence by activation of telomerase transcription 
Molecular cell  2012;47(1):5-15.
Inflammation is associated with DNA damage, cellular senescence and aging. Cessation of the inflammatory cytokine response is mediated in part through cytokine mRNA degradation facilitated by RNA binding proteins, including AUF1. We report a major unrecognized function of AUF1 – it activates telomerase expression, suppresses cellular senescence and maintains normal aging. AUF1 deficient mice undergo striking telomere erosion, markedly increased DNA damage responses at telomere ends, pronounced cellular senescence and rapid premature aging that increases with successive generations, which can be rescued in AUF1 knockout mice and their cultured cells by resupplying AUF1 expression. AUF1 binds and strongly activates the transcription promoter for telomerase catalytic subunit Tert. In addition to directing inflammatory cytokine mRNA decay, AUF1 destabilizes cell cycle checkpoint mRNAs, preventing cellular senescence. Thus, a single gene, AUF1, links maintenance of telomere length and normal aging to attenuation of inflammatory cytokine expression and inhibition of cellular senescence.
PMCID: PMC3966316  PMID: 22633954
9.  Acetylation of histone H3 at lysine 64 regulates nucleosome dynamics and facilitates transcription 
eLife  2014;3:e01632.
Post-translational modifications of proteins have emerged as a major mechanism for regulating gene expression. However, our understanding of how histone modifications directly affect chromatin function remains limited. In this study, we investigate acetylation of histone H3 at lysine 64 (H3K64ac), a previously uncharacterized acetylation on the lateral surface of the histone octamer. We show that H3K64ac regulates nucleosome stability and facilitates nucleosome eviction and hence gene expression in vivo. In line with this, we demonstrate that H3K64ac is enriched in vivo at the transcriptional start sites of active genes and it defines transcriptionally active chromatin. Moreover, we find that the p300 co-activator acetylates H3K64, and consistent with a transcriptional activation function, H3K64ac opposes its repressive counterpart H3K64me3. Our findings reveal an important role for a histone modification within the nucleosome core as a regulator of chromatin function and they demonstrate that lateral surface modifications can define functionally opposing chromatin states.
eLife digest
DNA is a very long molecule, so it needs to be packaged carefully to fit into the nucleus of a cell. To achieve this, the DNA is wrapped around proteins called histones to form a structure termed a nucleosome, which is the building block of a more compacted substance called chromatin. However, to express the genes in the DNA it is necessary to open up parts of the chromatin to give various enzymes access to the DNA.
Cells often chemically modify histones by adding acetyl or methyl groups, and these modifications are known to influence what proteins can bind to the nucleosomes, which ultimately influences what genes are expressed in the cell at a given time. It has been suspected for some time that histone modifications can also influence gene expression more directly, but there has been little evidence for this idea.
Now Di Cerbo et al. have studied what happens when acetyl or methyl groups are added to a specific site within a histone called H3K64, which is close to where the DNA wraps around this histone. These experiments showed that this site tends to be acetylated when a nearby gene is active, and to be unmodified or methylated when this gene is not active. It appears that the addition of the acetyl group makes this region of the chromatin less stable: this, in turn, makes it easier for the chromatin to be unpacked, thus giving access to the enzymes that transcribe the DNA and allowing transcription to take place. The work of Di Cerbo et al. shows that methylation and acetylation at the same site within a histone can define two opposing states of chromatin and DNA: an active state and a repressive state.
PMCID: PMC3965291  PMID: 24668167
histone; chromatin; acetylation; Human; Mouse
10.  On the Design of an Interactive, Patient-Specific Surgical Simulator for Mitral Valve Repair 
Surgical repair of the mitral valve is a difficult procedure that is often avoided in favor of less effective valve replacement because of the associated technical challenges facing non-expert surgeons. In the interest of increasing the rate of valve repair, an accurate, interactive surgical simulator for mitral valve repair was developed. With a haptic interface, users can interact with a mechanical model during simulation to aid in the development of a surgical plan and then virtually implement the procedure to assess its efficacy. Sub-millimeter accuracy was achieved in a validation study, and the system was successfully used by a cardiac surgeon to repair three virtual pathological valves.
PMCID: PMC3915525  PMID: 24511427
11.  Interstitial fluid pressure correlates with intra-voxel incoherent motion imaging metrics in a mouse mammary carcinoma model 
NMR in biomedicine  2011;25(5):10.1002/nbm.1793.
Effective delivery of therapeutic drug to the core of a tumor is often impeded by physiological barriers, such as interstitial fluid pressure (IFP). There are a number of therapies to lower IFP and induce tumor vascular normalization. However, lack of a non-invasive means to measure IFP hinders utilization of such a window of opportunity for maximizing the treatment response. Thus, the purpose of this study was to investigate the feasibility of using intravoxel incoherent motion (IVIM) diffusion parameters as noninvasive imaging biomarkers for IFP. Mice bearing the 4T1 mammary carcinoma model were studied with diffusion weighted magnetic resonance imaging (DWI) immediately followed by wick-in-needle IFP measurement. Voxelwise analysis was conducted with a conventional monoexponential diffusion model as well as a biexponential model taking IVIM into account. There was no significant correlation of IFP with either median apparent diffusion coefficient from the monoexponential model (r = 0.11, p = 0.78) or median tissue diffusivity from the biexponential model (r = 0.30, p = 0.44). On the other hand, IFP was correlated with the median pseudo-diffusivity (Dp) of apparent vascular voxels (r = 0.76, p = 0.02) and with the median product of perfusion-fraction and pseudo-diffusivity (fp·Dp) of apparent vascular voxels (r = 0.77, p = 0.02). Although the effect of IVIM in tumors has been reported previously, to our knowledge, this study represents the first direct comparison of IVIM metrics with IFP, with the results supporting the feasibility of using IVIM-DWI metrics as noninvasive biomarkers for tumor IFP.
PMCID: PMC3883504  PMID: 22072561
interstitial fluid pressure; diffusion; pseudo-diffusion; intravoxel incoherent motion; breast cancer
12.  Inflammatory Breast Cancer: High Incidence of Detection of Mixed Human Cytomegalovirus Genotypes Associated with Disease Pathogenesis 
Frontiers in Oncology  2014;4:246.
Inflammatory breast cancer (IBC) is a highly metastatic, aggressive, and fatal form of breast cancer. Patients presenting with IBC are characterized by a high number of axillary lymph node metastases. Recently, we found that IBC carcinoma tissues contain significantly higher levels of human cytomegalovirus (HCMV) DNA compared to other breast cancer tissues that may regulate cell signaling pathways. In fact, HCMV pathogenesis and clinical outcome can be statistically associated with multiple HCMV genotypes within IBC. Thus, in the present study, we established the incidence and types of HCMV genotypes present in carcinoma tissues of infected non-IBC versus IBC patients. We also assessed the correlation between detection of mixed genotypes of HCMV and disease progression. Genotyping of HCMV in carcinoma tissues revealed that glycoprotein B (gB)-1 and glycoprotein N (gN)-1 were the most prevalent HCMV genotypes in both non-IBC and IBC patients with no significant difference between patients groups. IBC carcinoma tissues, however, showed statistically significant higher incidence of detection of the gN-3b genotype compared to non-IBC patients. The incidence of detection of mixed genotypes of gB showed that gB-1 + gB-3 was statistically significantly higher in IBC than non-IBC patients. Similarly, the incidence of detection of mixed genotypes of gN showed that gN-1 + gN-3b and gN-3 + gN-4b/c were statistically significant higher in the carcinoma tissues of IBC than non-IBC. Mixed presence of different HCMV genotypes was found to be significantly correlated with the number of metastatic lymph nodes in non-IBC but not in IBC patients. In IBC, detection of mixed HCMV different genotypes significantly correlates with lymphovascular invasion and formation of dermal lymphatic emboli, which was not found in non-IBC patients.
PMCID: PMC4160966  PMID: 25309872
human cytomegalovirus; glycoproteins; inflammatory breast neoplasms; UL55; UL73; lymphovascular invasion; metastasis
13.  Chromatin dynamics during epigenetic reprogramming in the mouse germ line 
Nature  2008;452(7189):10.1038/nature06714.
The germ cell lineage exhibits unique characteristics, which are essential towards generating totipotency. Among the distinctive events in this lineage is DNA demethylation and the erasure of parental imprints, which occur on embryonic day 11.5 (E11.5) after the primordial germ cells (PGCs) have entered into the developing gonads 12. Little is yet known about the mechanism involved, except that this appears to be an active process. Here we have examined the associated changes in the chromatin to gain further insights into this reprogramming event. We show that chromatin changes during this process occur in two-steps. The first changes observed in nascent PGCs at E8.5 establish a distinctive chromatin signature with some characteristics associated with pluripotency. Subsequently, at E11.5 when these PGCs are residing in the gonads, major changes occur in nuclear architecture with an extensive erasure of several histone modification marks along with exchange of histone variants. Furthermore, at this time, the histone chaperones, HIRA and NAP1, which are implicated in histone exchange, show accumulation in PGC nuclei undergoing reprogramming. We thus suggest that the mechanism of histone replacement is critical for these chromatin rearrangements to occur. The striking chromatin changes we show here are intimately linked with the process of genome-wide DNA demethylation. Based on the timing of the observed events, we propose, that if DNA demethylation entails DNA repair based mechanism, the evident histone replacement would rather than being a prerequisite, represent a repair-induced response event.
PMCID: PMC3847605  PMID: 18354397
14.  Atomic Resolution Description of the Interaction between the Nucleoprotein and Phosphoprotein of Hendra Virus 
PLoS Pathogens  2013;9(9):e1003631.
Hendra virus (HeV) is a recently emerged severe human pathogen that belongs to the Henipavirus genus within the Paramyxoviridae family. The HeV genome is encapsidated by the nucleoprotein (N) within a helical nucleocapsid. Recruitment of the viral polymerase onto the nucleocapsid template relies on the interaction between the C-terminal domain, NTAIL, of N and the C-terminal X domain, XD, of the polymerase co-factor phosphoprotein (P). Here, we provide an atomic resolution description of the intrinsically disordered NTAIL domain in its isolated state and in intact nucleocapsids using nuclear magnetic resonance (NMR) spectroscopy. Using electron microscopy, we show that HeV nucleocapsids form herringbone-like structures typical of paramyxoviruses. We also report the crystal structure of XD of P that consists of a three-helix bundle. We study the interaction between NTAIL and XD using NMR titration experiments and provide a detailed mapping of the reciprocal binding sites. We show that the interaction is accompanied by α-helical folding of the molecular recognition element of NTAIL upon binding to a hydrophobic patch on the surface of XD. Finally, using solution NMR, we investigate the interaction between intact nucleocapsids and XD. Our results indicate that monomeric XD binds to NTAIL without triggering an additional unwinding of the nucleocapsid template. The present results provide a structural description at the atomic level of the protein-protein interactions required for transcription and replication of HeV, and the first direct observation of the interaction between the X domain of P and intact nucleocapsids in Paramyxoviridae.
Author Summary
The polymerase of negative strand RNA viruses reads the viral RNA that is associated with the nucleoprotein N forming a helical nucleocapsid. The interaction between N and the cofactor of the polymerase, the phosphoprotein P, is essential for transcription and replication of the viral genome. The mechanism by which the polymerase dislodges the RNA from the nucleoprotein for its polymerising activity remains unknown, although it has been proposed that binding to P causes a conformational change in the nucleocapsid. Here, we use nuclear magnetic resonance (NMR) spectroscopy to develop an atomic resolution description of the intrinsically disordered C-terminal domain, NTAIL, of N of Hendra virus, an emerging paramyxovirus, and X-ray crystallography to determine the structure of the X domain (XD) of P. Characterization of the interaction between XD and NTAIL provides evidence for folding of NTAIL upon binding to P. Crucially, we were also able to study, for the first time, the interaction between XD and recombinant paramyxoviral nucleocapsids. NMR spectra of NTAIL in its isolated form and in the context of nucleocapsids demonstrate that binding of XD does not change the dynamics of NTAIL and that the nucleocapsid does not undergo any major rearrangements or unwinding upon interaction with P.
PMCID: PMC3784471  PMID: 24086133
15.  Management Strategy for Symptomatic Bisphosphonate-Associated Incomplete Atypical Femoral Fractures 
HSS Journal  2012;8(2):103-110.
Long-term bisphosphonate use has often been associated with atypical femoral fractures. These fractures evolve from incomplete femoral fractures. A previous study demonstrated that the presence of a radiolucent line in an incomplete fracture can indicate a high risk of progression to complete fracture.
The aim of this study is to present a management strategy for symptomatic bisphosphonate-associated incomplete atypical femoral fractures. Specific study questions include the following: (1) Is there a difference in the prognosis of these fractures based on the presence or absence of a radiolucent fracture line? (2) Can treatment with teriparatide assist in clinical/radiographic healing of these incomplete fractures? (3) Is there a characteristic biochemical profile in these patients?
Patients and Methods
We retrospectively examined all femur radiographs ordered by the metabolic bone disease service at our hospital between July 1, 2006 and July 1, 2011 and identified 10 patients with a total of 14 incomplete fractures. Nine patients received bisphosphonates for a mean duration of 10 ± 5 years (range, 4–17). The mean follow-up since the time of diagnosis was 20 ± 11 months (range, 6–36 months).
Five fractures did not have a radiolucent fracture line and were treated conservatively with partial weight-bearing restrictions and pharmacologic therapy. All five of these fractures healed with conservative management. Nine fractures had a radiolucent fracture line, and only two of these were treated successfully with conservative management including teriparatide. Six of the eight patients with a radiolucent line elected for surgical prophylaxis after 3 months of conservative management, whereas one patient underwent surgical prophylaxis without a trial of conservative management. Regarding the biochemical profiles, bone turnover markers for our patient cohort were in the lower quartile.
Fractures without a radiolucent line appear to respond to conservative management and not require surgical prophylaxis. Teriparatide treatment may hold promise in promoting healing of these fractures.
PMCID: PMC3715620  PMID: 23874247
bisphosphonates; atypical femoral fractures; incomplete fractures; teriparatide; radiolucent fracture line
16.  Dyskerin depletion increases VEGF mRNA internal ribosome entry site-mediated translation 
Nucleic Acids Research  2013;41(17):8308-8318.
Dyskerin is a nucleolar protein encoded by the DKC1 gene that (i) stabilizes the RNA component of the telomerase complex, and (ii) drives the site-specific pseudouridilation of rRNA. It is known that the partial lack of dyskerin function causes a defect in the translation of a subgroup of mRNAs containing internal ribosome entry site (IRES) elements such as those encoding for the tumor suppressors p27 and p53. In this study, we aimed to analyze what is the effect of the lack of dyskerin on the IRES-mediated translation of mRNAs encoding for vascular endothelial growth factor (VEGF). We transiently reduced dyskerin expression and measured the levels of the IRES-mediated translation of the mRNA encoding for VEGF in vitro in transformed and primary cells. We demonstrated a significant increase in the VEGF IRES-mediated translation after dyskerin knock-down. This translational modulation induces an increase in VEGF production in the absence of a significant upregulation in VEGF mRNA levels. The analysis of a list of viral and cellular IRESs indicated that dyskerin depletion can differentially affect IRES-mediated translation. These results indicate for the first time that dyskerin inhibition can upregulate the IRES translation initiation of specific mRNAs.
PMCID: PMC3783170  PMID: 23821664
17.  Preoperative concurrent paclitaxel-radiation in locally advanced breast cancer: pathologic response correlates with five-year overall survival 
We have previously demonstrated high pathologic response rates after neoadjuvant concurrent chemoradiation in patients with locally advanced breast cancer (LABC). We now report disease-free survival (DFS) and overall survival (OS) in the context of pathologic response. 105 LABC patients (White 46%, Non-White 54%) were treated with paclitaxel (30 mg/m2 intravenously twice a week) for 10–12 weeks. Daily radiotherapy was delivered to breast, axillary, and supraclavicular lymph nodes during weeks 2–7 of paclitaxel treatment, at 1.8 Gy per fraction to a total dose of 45 Gy with a tumor boost of 14 Gy at 2 Gy/fraction. Pathological complete response (pCR) was defined as the absence of invasive cancer in breast and lymph nodes and pathological partial response (pPR) as the persistence of <10 microscopic foci of invasive carcinoma in breast or lymph nodes. Pathologic response (pCR and pPR) after neoadjuvant chemoradiation was achieved in 36/105 patients (34%) and was associated with significantly better DFS and OS. Pathological responders had a lower risk of recurrence or death (HR = 0.35, P = 0.01) and a longer OS (HR = 4.27, P = 0.01) compared with non-responders. Median DFS and OS were 57 and 84 months for non-responders, respectively, and have not yet been reached for responders. Importantly, pathologic response was achieved in 54% of patients with HR negative tumors (26/48). In conclusion, pathologic response to concurrent paclitaxel-radiation translated into superior DFS and OS. Half of the patients with HR negative tumors achieved a pathologic response.
PMCID: PMC3655407  PMID: 20878462
Concurrent chemoradiation; Neoadjuvant; Locally advanced breast cancer; Survival; Pathologic response
19.  Anti-Tumor Effects of Ganoderma lucidum (Reishi) in Inflammatory Breast Cancer in In Vivo and In Vitro Models 
PLoS ONE  2013;8(2):e57431.
The medicinal mushroom Ganoderma lucidum (Reishi) was tested as a potential therapeutic for Inflammatory Breast Cancer (IBC) using in vivo and in vitro IBC models. IBC is a lethal and aggressive form of breast cancer that manifests itself without a typical tumor mass. Studies show that IBC tissue biopsies overexpress E-cadherin and the eukaryotic initiation factor 4GI (eIF4GI), two proteins that are partially responsible for the unique pathological properties of this disease. IBC is treated with a multimodal approach that includes non-targeted systemic chemotherapy, surgery, and radiation. Because of its non-toxic and selective anti-cancer activity, medicinal mushroom extracts have received attention for their use in cancer therapy. Our previous studies demonstrate these selective anti-cancer effects of Reishi, where IBC cell viability and invasion, as well as the expression of key IBC molecules, including eIF4G is compromised. Thus, herein we define the mechanistic effects of Reishi focusing on the phosphoinositide-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway, a regulator of cell survival and growth. The present study demonstrates that Reishi treated IBC SUM-149 cells have reduced expression of mTOR downstream effectors at early treatment times, as we observe reduced eIF4G levels coupled with increased levels of eIF4E bound to 4E-BP, with consequential protein synthesis reduction. Severe combined immunodeficient mice injected with IBC cells treated with Reishi for 13 weeks show reduced tumor growth and weight by ∼50%, and Reishi treated tumors showed reduced expression of E-cadherin, mTOR, eIF4G, and p70S6K, and activity of extracellular regulated kinase (ERK1/2). Our results provide evidence that Reishi suppresses protein synthesis and tumor growth by affecting survival and proliferative signaling pathways that act on translation, suggesting that Reishi is a potential natural therapeutic for breast and other cancers.
PMCID: PMC3585368  PMID: 23468988
20.  Human Cytomegalovirus Infection Enhances NF-κB/p65 Signaling in Inflammatory Breast Cancer Patients 
PLoS ONE  2013;8(2):e55755.
Human Cytomegalovirus (HCMV) is an endemic herpes virus that re-emerges in cancer patients enhancing oncogenic potential. Recent studies have shown that HCMV infection is associated with certain types of cancer morbidity such as glioblastoma. Although HCMV has been detected in breast cancer tissues, its role, if any, in the etiology of specific forms of breast cancer has not been investigated. In the present study we investigated the presence of HCMV infection in inflammatory breast cancer (IBC), a rapidly progressing form of breast cancer characterized by specific molecular signature. We screened for anti-CMV IgG antibodies in peripheral blood of 49 non-IBC invasive ductal carcinoma (IDC) and 28 IBC patients. In addition, we screened for HCMV-DNA in postsurgical cancer and non-cancer breast tissues of non-IBC and IBC patients. We also tested whether HCMV infection can modulate the expression and activation of transcriptional factor NF-κB/p65, a hallmark of IBC. Our results reveal that IBC patients are characterized by a statistically significant increase in HCMV IgG antibody titers compared to non-IBC patients. HCMV-DNA was significantly detected in cancer tissues than in the adjacent non-carcinoma tissues of IBC and IDC, and IBC cancer tissues were significantly more infected with HCMV-DNA compared to IDC. Further, HCMV sequence analysis detected different HCMV strains in IBC patients tissues, but not in the IDC specimens. Moreover, HCMV-infected IBC cancer tissues were found to be enhanced in NF-κB/p65 signaling compared to non-IBC patients. The present results demonstrated a correlation between HCMV infection and IBC. Etiology and causality of HCMV infection with IBC now needs to be rigorously examined.
PMCID: PMC3572094  PMID: 23418456
21.  Real-time image-based rigid registration of three-dimensional ultrasound 
Medical Image Analysis  2011;16(2):402-414.
Registration of three-dimensional ultrasound (3DUS) volumes is necessary in several applications, such as when stitching volumes to expand the field of view or when stabilizing a temporal sequence of volumes to cancel out motion of the probe or anatomy. Current systems that register 3DUS volumes either use external tracking systems (electromagnetic or optical), which add expense and impose limitations on acquisitions, or are image-based methods that operate offline and are incapable of providing immediate feedback to clinicians. This paper presents a real-time image-based algorithm for rigid registration of 3DUS volumes designed for acquisitions in which small probe displacements occur between frames. Described is a method for feature detection and descriptor formation that takes into account the characteristics of 3DUS imaging. Volumes are registered by determining a correspondence between these features. A global set of features is maintained and integrated into the registration, which limits the accumulation of registration error. The system operates in real-time (i.e. volumes are registered as fast or faster than they are acquired) by using an accelerated framework on a graphics processing unit. The algorithm’s parameter selection and performance is analyzed and validated in studies which use both water tank and clinical images. The resulting registration accuracy is comparable to similar feature-based registration methods, but in contrast to these methods, can register 3DUS volumes in real-time.
PMCID: PMC3267836  PMID: 22154960
ultrasound; registration; real-time; image-based
22.  Mitral annulus segmentation from four-dimensional ultrasound using a valve state predictor and constrained optical flow 
Medical Image Analysis  2011;16(2):497-504.
Measurement of the shape and motion of the mitral valve annulus has proven useful in a number of applications, including pathology diagnosis and mitral valve modeling. Current methods to delineate the annulus from four-dimensional (4D) ultrasound, however, either require extensive overhead or user-interaction, become inaccurate as they accumulate tracking error, or they do not account for annular shape or motion. This paper presents a new 4D annulus segmentation method to account for these deficiencies. The method builds on a previously published three-dimensional (3D) annulus segmentation algorithm that accurately and robustly segments the mitral annulus in a frame with a closed valve. In the 4D method, a valve state predictor determines when the valve is closed. Subsequently, the 3D annulus segmentation algorithm finds the annulus in those frames. For frames with an open valve, a constrained optical flow algorithm is used to the track the annulus. The only inputs to the algorithm are the selection of one frame with a closed valve and one user-specified point near the valve, neither of which needs to be precise. The accuracy of the tracking method is shown by comparing the tracking results to manual segmentations made by a group of experts, where an average RMS difference of 1.67 ± 0.63 mm was found across 30 tracked frames.
PMCID: PMC3397406  PMID: 22200622
Mitral valve; Annulus; Tracking; Segmentation; Ultrasound
23.  A unified phylogeny-based nomenclature for histone variants 
Histone variants are non-allelic protein isoforms that play key roles in diversifying chromatin structure. The known number of such variants has greatly increased in recent years, but the lack of naming conventions for them has led to a variety of naming styles, multiple synonyms and misleading homographs that obscure variant relationships and complicate database searches. We propose here a unified nomenclature for variants of all five classes of histones that uses consistent but flexible naming conventions to produce names that are informative and readily searchable. The nomenclature builds on historical usage and incorporates phylogenetic relationships, which are strong predictors of structure and function. A key feature is the consistent use of punctuation to represent phylogenetic divergence, making explicit the relationships among variant subtypes that have previously been implicit or unclear. We recommend that by default new histone variants be named with organism-specific paralog-number suffixes that lack phylogenetic implication, while letter suffixes be reserved for structurally distinct clades of variants. For clarity and searchability, we encourage the use of descriptors that are separate from the phylogeny-based variant name to indicate developmental and other properties of variants that may be independent of structure.
PMCID: PMC3380720  PMID: 22650316
24.  Quantitative proteomics reveals direct and indirect alterations in the histone code following methyltransferase knockdown†‡ 
Molecular Biosystems  2010;6(9):1719-1729.
Histones are highly conserved proteins that organize cellular DNA. These proteins, especially their N-terminal domains, are adorned with many post-translational modifications (PTMs) such as lysine methylation, which are associated with active or repressed transcriptional states. The lysine methyltransferase G9a and its interaction partner Glp1 can mono- or dimethylate histone H3 on lysine (H3K9me1 or me2); possible cross-talk between these modifications and other PTMs on the same or other histone molecules is currently uncharacterized. In this study, we comprehensively analyze the effects of G9a/Glp1 knockdown on the most abundant histone modifications through both Bottom Up and Middle Down mass spectrometry-based proteomics. In addition to the expected decrease in H3K9me1/me2 we find that other degrees of methylation on K9 are affected by the reduction of G9a/Glp1 activity, particularly when K9 methylation occurs in combination with K14 acetylation. In line with this, an increase in K14 acetylation upon G9a knockdown was observed across all H3 variants (H3.1, H3.2 and H3.3), hinting at the potential existence of a binary switch between K9 methylation and K14 acetylation. Interestingly, we also detect changes in the abundance of other modifications (such as H3K79me2) in response to lowered levels of G9a/Glp1 suggesting histone PTM cross-talk amongst the H3 variants. In contrast, we find that G9a/Glp1 knockdown produces little effect on the levels of histone H4 PTMs, indicating low to no trans-histone PTM crosstalk. Lastly, we determined gene expression profiles of control and G9a/Glp1 knockdown cells, and we find that the G9a/Glp1 knockdown influences several genes, including DNA binding proteins and key factors in chromatin. Our results provide new insights into the intra- and inter- histone cross-regulation of histone K9 methylation and its potential downstream gene targets.
PMCID: PMC3321735  PMID: 20577673
25.  Chatting histone modifications in mammals 
Briefings in Functional Genomics  2011;9(5-6):429-443.
Eukaryotic chromatin can be highly dynamic and can continuously exchange between an open transcriptionally active conformation and a compacted silenced one. Post-translational modifications of histones have a pivotal role in regulating chromatin states, thus influencing all chromatin dependent processes. Methylation is currently one of the best characterized histone modification and occurs on arginine and lysine residues. Histone methylation can regulate other modifications (e.g. acetylation, phosphorylation and ubiquitination) in order to define a precise functional chromatin environment. In this review we focus on histone methylation and demethylation, as well as on the enzymes responsible for setting these marks. In particular we are describing novel concepts on the interdependence of histone modifications marks and discussing the molecular mechanisms governing this cross-talks.
PMCID: PMC3080777  PMID: 21266346
histone modifications; histone methylation; cross-talk; epigenetic; chromatin

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