Factor-Dependent Internal Ribosome Entry Site and -1 Programmed Frameshifting Signal in the Bemisia-Associated Dicistrovirus 2.

The dicistrovirus intergenic (IGR) IRES uses the most streamlined translation initiation mechanism: the IRES recruits ribosomes directly without using protein factors and initiates translation from a non-AUG codon. Several subtypes of dicistroviruses IRES have been identified; typically, the IRESs adopt two -to three overlapping pseudoknots with key stem-loop and unpaired regions that interact with specific domains of the ribosomal 40S and 60S subunits to direct translation. We previously predicted an atypical IGR IRES structure and a potential -1 programmed frameshift (-1 FS) signal within the genome of the whitefly Bemisia-associated dicistrovirus 2 (BaDV-2). Here, using bicistronic reporters, we demonstrate that the predicted BaDV-2 -1 FS signal can drive -1 frameshifting in vitro via a slippery sequence and a downstream stem-loop structure that would direct the translation of the viral RNA-dependent RNA polymerase. Moreover, the predicted BaDV-2 IGR can support IRES translation in vitro but does so through a mechanism that is not typical of known factorless dicistrovirus IGR IRES mechanisms. Using deletion and mutational analyses, the BaDV-2 IGR IRES is mapped within a 140-nucleotide element and initiates translation from an AUG codon. Moreover, the IRES does not bind directly to purified ribosomes and is sensitive to eIF2 and eIF4A inhibitors NSC1198983 and hippuristanol, respectively, indicating an IRES-mediated factor-dependent mechanism. Biophysical characterization suggests the BaDV-2 IGR IRES contains several stem-loops; however, mutational analysis suggests a model whereby the IRES is unstructured or adopts distinct conformations for translation initiation. In summary, we have provided evidence of the first -1 FS frameshifting signal and a novel factor-dependent IRES mechanism in this dicistrovirus family, thus highlighting the diversity of viral RNA-structure strategies to direct viral protein synthesis.

Development of a single-domain antibody to target a G-quadruplex located on the hepatitis B virus covalently closed circular DNA genome.

To achieve a virological cure for hepatitis B virus (HBV), innovative strategies are required to target the covalently closed circular DNA (cccDNA) genome. Guanine-quadruplexes (G4s) are a secondary structure that can be adopted by DNA and play a significant role in regulating viral replication, transcription, and translation. Antibody-based probes and small molecules have been developed to study the role of G4s in the context of the human genome, but none have been specifically made to target G4s in viral infection. Herein, we describe the development of a humanized single-domain antibody (S10) that can target a G4 located in the PreCore (PreC) promoter of the HBV cccDNA genome. MicroScale Thermophoresis demonstrated that S10 has a strong nanomolar affinity to the PreC G4 in its quadruplex form and a structural electron density envelope of the complex was determined using Small-Angle X-ray Scattering. Lentiviral transduction of S10 into HepG2-NTCP cells shows nuclear localization, and chromatin immunoprecipitation coupled with next-generation sequencing demonstrated that S10 can bind to the HBV PreC G4 present on the cccDNA. This research validates the existence of a G4 in HBV cccDNA and demonstrates that this DNA secondary structure can be targeted with high structural and sequence specificity using S10.

Penile Osteosarcoma: A Rare Extraosseous Site.

Primary penile extraosseous osteosarcoma (EOS) ranks the most uncommon amongst the differential penile masses, with only nine cases reported so far. In this report, we share the management of a 67-year-old Hispanic male who presented with a painful mass over his distal penile shaft and glans for the last two months. After initial imaging and complete blood investigations, he underwent partial penectomy. Histology revealed high-grade sarcoma, with osteoid production, favoring high-grade extra-skeletal osteosarcoma, with tumor necrosis involving approximately 5% of the tumor volume. The patient had bilateral palpable inguinal lymphadenopathy, which was seen even on a pre-op CT scan. The patient thus underwent bilateral robotic superficial and deep inguinal standard template lymph node dissection three weeks after his partial penectomy. His pathology was negative for malignancy in all examined lymph nodes. At his last follow-up, five months post his primary surgery, he had been doing well without concerns for recurrence.

Chronic pelvic pain and dysmenorrhea disguising a rare submucosal leiomyoma of the urinary bladder.

Representing 0.43% of all urinary bladder neoplasms, leiomyomas are rare mesenchymal tumours with a benign pathophysiology. There have only been approximately 250 cases published on this subject, necessitating further inquiry into this disease and effective management protocols. Treatment options may include a broad spectrum of surgical interventions, from minimally invasive resection to radical cystectomy, depending on the location, size and symptoms associated with the tumour. To date, few cases of leiomyoma have resulted in recurrence after removal, and zero have reported malignant transformation. Described here in detail is a woman in her early 40s who presented with a history of chronic pelvic pain and irregular vaginal bleeding. The urology team completed further evaluation after imaging discovered a concerning bladder lesion. Eventually, she underwent transurethral resection, with the subsequent pathology revealing a rare diagnosis of leiomyoma in the urinary bladder.

The 3' terminal region of Zika virus RNA contains a conserved G-quadruplex and is unfolded by human DDX17.

Zika virus (ZIKV) infection remains a worldwide concern, and currently no effective treatments or vaccines are available. Novel therapeutics are an avenue of interest that could probe viral RNA-human protein communication to stop viral replication. One specific RNA structure, G-quadruplexes (G4s), possess various roles in viruses and all domains of life, including transcription and translation regulation and genome stability, and serves as nucleation points for RNA liquid-liquid phase separation. Previous G4 studies on ZIKV using a quadruplex forming G-rich sequences Mapper located a potential G-quadruplex sequence in the 3' terminal region (TR) and was validated structurally using a 25-mer oligo. It is currently unknown if this structure is conserved and maintained in a large ZIKV RNA transcript and its specific roles in viral replication. Using bioinformatic analysis and biochemical assays, we demonstrate that the ZIKV 3' TR G4 is conserved across all ZIKV isolates and maintains its structure in a 3' TR full-length transcript. We further established the G4 formation using pyridostatin and the BG4 G4-recognizing antibody binding assays. Our study also demonstrates that the human DEAD-box helicases, DDX3X132-607 and DDX17135-555, bind to the 3' TR and that DDX17135-555 unfolds the G4 present in the 3' TR. These findings provide a path forward in potential therapeutic targeting of DDX3X or DDX17's binding to the 3' TR G4 region for novel treatments against ZIKV.

tRNA shape is an identity element for an archaeal pyrrolysyl-tRNA synthetase from the human gut.

Protein translation is orchestrated through tRNA aminoacylation and ribosomal elongation. Among the highly conserved structure of tRNAs, they have distinguishing features which promote interaction with their cognate aminoacyl tRNA synthetase (aaRS). These key features are referred to as identity elements. In our study, we investigated the tRNA:aaRS pair that installs the 22nd amino acid, pyrrolysine (tRNAPyl:PylRS). Pyrrolysyl-tRNA synthetases (PylRSs) are naturally encoded in some archaeal and bacterial genomes to acylate tRNAPyl with pyrrolysine. Their large amino acid binding pocket and poor recognition of the tRNA anticodon have been instrumental in incorporating >200 noncanonical amino acids. PylRS enzymes can be divided into three classes based on their genomic structure. Two classes contain both an N-terminal and C-terminal domain, however the third class (ΔpylSn) lacks the N-terminal domain. In this study we explored the tRNA identity elements for a ΔpylSn tRNAPyl from Candidatus Methanomethylophilus alvus which drives the orthogonality seen with its cognate PylRS (MaPylRS). From aminoacylation and translation assays we identified five key elements in ΔpylSn tRNAPyl necessary for MaPylRS activity. The absence of a base (position 8) and a G-U wobble pair (G28:U42) were found to affect the high-resolution structure of the tRNA, while molecular dynamic simulations led us to acknowledge the rigidity imparted from the G-C base pairs (G3:C70 and G5:C68).

Enzymes known as PylRS offer the remarkable ability to expand the natural genetic code of a living cell with unnatural amino acids. Currently, over 200 unnatural amino acids can be genetically encoded with the help of PylRS and its partner tRNAPyl, enabling us to endow proteins with novel properties, or regulate protein activity using light or inducible cross-linking. One intriguing feature of PylRS enzymes is their ability to avoid cross-reactivity when two PylRS homologs from different organisms-such as those from the archaea Methanosarcina mazei and Methanomethylophilus alvus-are co-expressed in a single cell. This makes it possible to simultaneously encode two unnatural amino acids in a single protein. This study illuminates the elusive mechanism of PylRS specificity by using cryo-electron microscopy, biochemistry and molecular simulations. The interaction of PylRS from M. alvus with its tRNAPyl is best described as two pieces of a jigsaw puzzle; in which PylRS recognizes the unique shape of its cognate tRNA instead of specific nucleotides in the tRNA sequence like other tRNA-binding enzymes. This finding may streamline the rational design of tools for simultaneous genetic incorporation of multiple unnatural amino acids, thereby facilitating the development of valuable proteins for research, medicine, and biotechnology.

Primary adenocarcinoma of the renal pelvis in spina bifida patient with history of simple cystectomy.

Primary renal adenocarcinomas comprise of less than 1% of renal and ureteral epithelial tumors. We present a case of a 67-year-old male with a history of simple cystectomy who underwent left nephroureterectomy for primary enteric-type renal adenocarcinoma with cystitis glandularis features. Pathological examination confirmed grade 2 pT1N0MX primary enteric-type renal adenocarcinoma. The patient underwent left open radical nephroureterectomy, with an uneventful postoperative course. Surgical excision is the mainstay treatment, while chemotherapy and radiation are potential adjuncts. Prognosis remains poor, with a 50% overall survival rate within two years of surgery. Further research is needed to enhance treatment recommendations.

Proceedings of the 25th Analytical Ultracentrifugation Workshops and Symposium.

The 25th International Analytical Ultracentrifugation (AUC) Workshops and Symposium (AUC2022) took place at the University of Lethbridge in Lethbridge, Canada, in July 2022. In total, 104 attendees (Attendance Profile: 104 attendees, 69 in-person, 35 remote. Brazil 1, Canada 24, China 1, Czech Republic 2, Finland 1, France 3, Germany 22, India 3, Italy 1, Japan 4, Spain 1, Switzerland 3, Taiwan 1, United Kingdom 5, United States 32) participated in the event and presented the latest advances in the field. While the primary focus of the conference was to showcase the applications of AUC in chemical, life sciences, and nanoparticle disciplines, several presentations also integrated complementary methods, such as isothermal titration calorimetry, microscale thermophoresis, light scattering (static and dynamic), small-angle X-ray scattering, X-ray crystallography, and cryo-electron microscopy. Additionally, the delegates gained valuable hands-on experience from 20 workshops covering a broad range of applications, experimental designs and systems, and the latest software innovations in solution biophysics. The AUC2022 special volume highlights the sustained innovation, utility and relevance of AUC and related solution biophysical methods across various disciplines, including biochemistry, structural biology, synthetic polymer chemistry, carbohydrate chemistry, protein and nucleic acid characterization, nano-science, and macromolecular interactions.

Retroperitoneal drop metastases following robotic partial nephrectomy for renal cell carcinoma.

Rare retroperitoneal recurrence of clear cell renal cell carcinoma highlights the risk of tumor violation during surgery. A 61-year-old female with recurrent RCC in the retroperitoneum is presented six years after partial nephrectomy. Initial surveillance CT revealed a renal cyst and subsequent imaging confirmed clear cell RCC. Multiple small lesions indicated retroperitoneal recurrence. Surgical excision confirmed metastatic clear cell RCC. The proximity of the recurrence to the lower pole of the primary tumor suggests tumor violation as the cause. Respecting tumor boundaries during surgery is crucial to prevent metastasis and improve patient survival.

A Case Report of a Patient With Prostate Adenocarcinoma Metastatic to the Posterior Peritoneum Despite the Negative Preoperative Prostate-Specific Membrane Antigen (PSMA) Positron Emission Tomography/Computed Tomography (PET/CT) Scan.

Despite the role of prostate-specific antigen (PSA) screening and the multitude of therapies available, prostate cancer (PCa) remains a leading cause of cancer-related morbidity and mortality. For many patients diagnosed with PCa, clinical and radiographic staging are critical components for management decisions. PCa staging with the use of imaging modalities such as MRI and bone scintigraphy is recommended in patients with newly diagnosed intermediate or high-risk PCa and in patients with biochemical recurrence; it is also recommended for monitoring the patient's response to treatment for diagnosed PCa. Prostate-specific membrane antigen (PSMA) positron emission tomography/computed tomography (PET/CT), recently approved in 2021, is an imaging modality that has been shown to have a greater sensitivity, specificity, and negative likelihood ratio than conventional imaging modalities such as CT, bone scintigraphy, and MRI in prostate cancer staging. Despite the improvement in staging that PSMA-PET/CT can provide, our current report details a false-negative result in detecting a rare PCa metastasis to the peritoneum, which was found at the time of an attempted radical prostatectomy. Although the patient had a negative preoperative PSMA-PET/CT and was presumed to be non-metastatic, the prostatectomy was aborted because the patient was unexpectedly found to have peritoneal metastasis.

Mapping and characterization of G-quadruplexes in monkeypox genomes.

Monkeypox virus (MPXV) is a double-stranded DNA virus from the family Poxviridae, which is endemic in West and Central Africa. Various human outbreaks occurred in the 1980s, resulting from a cessation of smallpox vaccination. Recently, MPXV cases have reemerged in non-endemic nations, and the 2022 outbreak has been declared a public health emergency. Treatment optionsare limited, and many countries lack the infrastructure to provide symptomatic treatments. The development of cost-effective antivirals could ease severe health outcomes. G-quadruplexes have been a target of interest in treating viral infections with different chemicals. In the present work, a genomic-scale mapping of different MPXV isolates highlighted two conserved putative quadruplex-forming sequences MPXV-exclusive in 590 isolates. Subsequently, we assessed the G-quadruplex formation using circular dichroism spectroscopy and solution small-angle X-ray scattering. Furthermore, biochemical assays indicated the ability of MPXV quadruplexes to be recognized by two specific G4-binding partners-Thioflavin T and DHX36. Additionally, our work also suggests that a quadruplex binding small-molecule with previously reported antiviral activity, TMPyP4, interacts with MPXV G-quadruplexes with nanomolar affinity in the presence and absence of DHX36. Finally, cell biology experiments suggests that TMPyP4 treatment substantially reduced gene expression of MPXV proteins. In summary, our work provides insights into the G-quadruplexes from the MPXV genome that can be further exploited to develop therapeutics.

Investigating RNA-RNA interactions through computational and biophysical analysis.

Numerous viruses utilize essential long-range RNA-RNA genome interactions, specifically flaviviruses. Using Japanese encephalitis virus (JEV) as a model system, we computationally predicted and then biophysically validated and characterized its long-range RNA-RNA genomic interaction. Using multiple RNA computation assessment programs, we determine the primary RNA-RNA interacting site among JEV isolates and numerous related viruses. Following in vitro transcription of RNA, we provide, for the first time, characterization of an RNA-RNA interaction using size-exclusion chromatography coupled with multi-angle light scattering and analytical ultracentrifugation. Next, we demonstrate that the 5' and 3' terminal regions of JEV interact with nM affinity using microscale thermophoresis, and this affinity is significantly reduced when the conserved cyclization sequence is not present. Furthermore, we perform computational kinetic analyses validating the cyclization sequence as the primary driver of this RNA-RNA interaction. Finally, we examined the 3D structure of the interaction using small-angle X-ray scattering, revealing a flexible yet stable interaction. This pathway can be adapted and utilized to study various viral and human long-non-coding RNA-RNA interactions and determine their binding affinities, a critical pharmacological property of designing potential therapeutics.

A comprehensive review of methods to study lncRNA-protein interactions in solution.

The long non-coding RNAs (lncRNAs) other than rRNA and tRNA were earlier assumed to be 'junk genomic material'. However, recent advancements in genomics methods have highlighted their roles not only in housekeeping but also in the progression of diseases like cancer as well as viral infections. lncRNAs owing to their length, have both short-range and long-range interactions resulting in complex folded structures that recruit various biomolecules enabling lncRNAs to undertake their various biological functions. Using cell lysate pull-down assays increasing number of lnRNAs-interacting proteins are being identified. These interactions can be further exploited to develop targeted novel therapeutic strategies to inhibit lncRNA-protein interactions. This review attempts to succinctly techniques that can identify and characterize the lnRNAs-protein interactions (i.e. affinity, stoichiometry, and thermodynamics). Furthermore, using other sophisticated biophysical techniques, one can also perform size estimations, and determine low-resolution structures. Since these methods study the biomolecules in solution, large-scale structural observations can be performed in real-time. This review attempts to briefly introduce the readers to biochemical and biophysical techniques, such that they can utilize these methods to obtain a holistic characterization of the biomolecules of interest. Additionally, it should be noted that the use of these methods is not limited to the characterization of the interacting molecules but can also be used to determine the efficacy of the therapeutic molecules to disrupt these interactions.

Purification and characterization of inorganic pyrophosphatase for in vitro RNA transcription.

Inorganic pyrophosphatase (iPPase) is an enzyme that cleaves pyrophosphate into two phosphate molecules. This enzyme is an essential component of in vitro transcription (IVT) reactions for RNA preparation as it prevents pyrophosphate from precipitating with magnesium, ultimately increasing the rate of the IVT reaction. Large-scale RNA production is often required for biochemical and biophysical characterization studies of RNA, therefore requiring large amounts of IVT reagents. Commercially purchased iPPase is often the most expensive component of any IVT reaction. In this paper, we demonstrate that iPPase can be produced in large quantities and high quality using a reasonably generic laboratory facility and that laboratory-purified iPPase is as effective as commercially available iPPase. Furthermore, using size exclusion chromatography coupled with multi-angle light scattering and dynamic light scattering, analytical ultracentrifugation, and small-angle X-ray scattering, we demonstrate that yeast iPPase can form tetramers and hexamers in solution as well as the enzymatically active dimer. Our work provides a robust protocol for laboratories involved with RNA in vitro transcription to efficiently produce active iPPase, significantly reducing the financial strain of large-scale RNA production.

Editors' Roundup: June 2022.

This Commentary describes the June 2022 installment of the "Editors' Roundup" feature. The Editors' Roundup is a multi-author collective description of recently published biophysical research contributed by editorial board members of different journals with a bonafide interest in publishing biophysical content. This Commentary contains a series of personal recommendations for articles appearing in the following journals, Biophysics and Physicobiology, European Biophysics Journal, Cell Biochemistry and Biophysics, and Biophysical Reviews.

18F-DCFPyL PET/CT for Initially Diagnosed and Biochemically Recurrent Prostate Cancer: Prospective Trial with Pathologic Confirmation.

Background Prostate-specific membrane antigen (PSMA) PET is standard for newly diagnosed high-risk and biochemically recurrent (BCR) prostate cancer. Although studies suggest high specificity of 2-(3-{1-carboxy-5-[(6-[(18)F]fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-pentanedioic acid (DCFPyL) for targeting PSMA, false-positive findings have been identified and most studies lack histologic confirmation of malignancy. Purpose To estimate the positive predictive value (PPV) of DCFPyL PET/CT by providing histopathologic proof for DCFPyL-avid lesions suspected of being distant metastases at initial diagnosis and recurrence in BCR prostate cancer. Materials and Methods In this prospective trial, men with newly diagnosed high-risk prostate cancer (sample 1) or BCR prostate cancer and negative findings at conventional CT and/or bone scanning (sample 2) were enrolled between January and December 2021. All men underwent DCFPyL PET/CT. Suspected distant metastases and/or recurrences were biopsied. PPV was calculated. Results A total of 92 men with newly diagnosed prostate cancer (median age, 70 years; IQR, 64-75 years) (sample 1) and 92 men with BCR prostate cancer (median age, 71 years; IQR, 66-75 years) (sample 2) were enrolled. In sample 1, 25 of the 92 men (27%) demonstrated DCFPyL-avid lesions suspicious for distant metastases. Biopsy was performed in 23 of the 25 men (92%), with 17 of the 23 (74%) biopsies positive for malignancy and six (26%) benign. Of the six benign biopsies, three were solitary rib foci and three were solitary pelvic bone foci. In sample 2, 57 of the 92 men (62%) demonstrated DCFPyL-avid lesions suspicious for recurrence. Biopsy was performed in 37 of the 57 men (65%), with 33 of the 37 (89%) biopsies positive for malignancy and four (11%) benign. Of the four benign biopsies, two were subcentimeter pelvic nodes and/or nodules, one was a rib, and one was a pelvic bone focus. Conclusion PET/CT with 2-(3-{1-carboxy-5-[(6-[(18)F]fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-pentanedioic acid (DCFPyL) had a high biopsy-proven positive predictive value for distant metastases in newly diagnosed prostate cancer (74%) and for recurrence sites in men with biochemical recurrence (89%). However, there were DCFPyL-avid false-positive findings (particularly in ribs and pelvic bones). Solitary DCFPyL avidity in these locations should not be presumed as malignant. Biopsy may still be needed prior to therapy decisions. ClinicalTrials.gov registration no. NCT04700332 © RSNA, 2022 See also the editorial by Zukotynski and Kuo in this issue.

Biophysical characterisation of human LincRNA-p21 sense and antisense Alu inverted repeats.

Human Long Intergenic Noncoding RNA-p21 (LincRNA-p21) is a regulatory noncoding RNA that plays an important role in promoting apoptosis. LincRNA-p21 is also critical in down-regulating many p53 target genes through its interaction with a p53 repressive complex. The interaction between LincRNA-p21 and the repressive complex is likely dependent on the RNA tertiary structure. Previous studies have determined the two-dimensional secondary structures of the sense and antisense human LincRNA-p21 AluSx1 IRs using SHAPE. However, there were no insights into its three-dimensional structure. Therefore, we in vitro transcribed the sense and antisense regions of LincRNA-p21 AluSx1 Inverted Repeats (IRs) and performed analytical ultracentrifugation, size exclusion chromatography, light scattering, and small angle X-ray scattering (SAXS) studies. Based on these studies, we determined low-resolution, three-dimensional structures of sense and antisense LincRNA-p21. By adapting previously known two-dimensional information, we calculated their sense and antisense high-resolution models and determined that they agree with the low-resolution structures determined using SAXS. Thus, our integrated approach provides insights into the structure of LincRNA-p21 Alu IRs. Our study also offers a viable pipeline for combining the secondary structure information with biophysical and computational studies to obtain high-resolution atomistic models for long noncoding RNAs.

Targeting Xist with compounds that disrupt RNA structure and X inactivation.

Although more than 98% of the human genome is non-coding1, nearly all of the drugs on the market target one of about 700 disease-related proteins. The historical reluctance to invest in non-coding RNA stems partly from requirements for drug targets to adopt a single stable conformation2. Most RNAs can adopt several conformations of similar stabilities. RNA structures also remain challenging to determine3. Nonetheless, an increasing number of diseases are now being attributed to non-coding RNA4 and the ability to target them would vastly expand the chemical space for drug development. Here we devise a screening strategy and identify small molecules that bind the non-coding RNA prototype Xist5. The X1 compound has drug-like properties and binds specifically the RepA motif6 of Xist in vitro and in vivo. Small-angle X-ray scattering analysis reveals that RepA can adopt multiple conformations but favours one structure in solution. X1 binding reduces the conformational space of RepA, displaces cognate interacting protein factors (PRC2 and SPEN), suppresses histone H3K27 trimethylation, and blocks initiation of X-chromosome inactivation. X1 inhibits cell differentiation and growth in a female-specific manner. Thus, RNA can be systematically targeted by drug-like compounds that disrupt RNA structure and epigenetic function.

Upper tract urothelial carcinoma with Oligometastasis to the right ventricle, surgical considerations, and management.

Right ventricular (RV) metastasis from an upper tract urothelial carcinoma without inferior vena cava or right atrial involvement is an extremely rare event which highlights the heterogeneity of this disease process. We report a case of a 43-year-old man presenting for long-standing hematuria and left flank pain. Computed tomography revealed a left renal mass with para-aortic lymphadenopathy, in addition to a potential mass in the RV. The mass involving the RV was confirmed on subsequent cardiac evaluation with magnetic resonance imaging (MRI) and echocardiography. After discussion in a multidisciplinary tumor board, the patient underwent a left nephrectomy, regional lymphadenectomy, and excision of metastatic RV tumor with bovine patch reconstruction. Final pathology reported invasive urothelial carcinoma in the left kidney with involvement of regional para-aortic lymph nodes and metastatic tumor in the RV (T4N3M1, AJCC 8th edition). The patient did well postoperatively and completed adjuvant Cisplatin-Gemcitabine systemic chemotherapy. This is an important addition to the literature as it highlights the aggressive and heterogeneous nature of urothelial carcinoma and the utility of cardiac MRI in surgical planning.