Arabidopsis mRNA decay landscape shaped by XRN 5'-3' exoribonucleases.

5'-3' exoribonucleases (XRNs) play crucial roles in the control of RNA processing, quality, and quantity in eukaryotes. Although genome-wide profiling of RNA decay fragments is now feasible, how XRNs shape the plant mRNA degradome remains elusive. Here, we profiled and analyzed the RNA degradomes of Arabidopsis wild-type and mutant plants with defects in XRN activity. Deficiency of nuclear XRN3 or cytoplasmic XRN4 activity but not nuclear XRN2 activity greatly altered Arabidopsis mRNA decay profiles. Short excised linear introns and cleaved pre-mRNA fragments downstream of polyadenylation sites were polyadenylated and stabilized in the xrn3 mutant, demonstrating the unique function of XRN3 in the removal of cleavage remnants from pre-mRNA processing. Further analysis of stabilized XRN3 substrates confirmed that pre-mRNA 3' end cleavage frequently occurs after adenosine. The most abundant decay intermediates in wild-type plants include not only the primary substrates of XRN4 but also the products of XRN4-mediated cytoplasmic decay. An increase in decay intermediates with 5' ends upstream of a consensus motif in the xrn4 mutant suggests that there is an endonucleolytic cleavage mechanism targeting the 3' untranslated regions of many Arabidopsis mRNAs. However, analysis of decay fragments in the xrn4 mutant indicated that, except for microRNA-directed slicing, endonucleolytic cleavage events in the coding sequence rarely result in major decay intermediates. Together, these findings reveal the major substrates and products of nuclear and cytoplasmic XRNs along Arabidopsis transcripts and provide a basis for precise interpretation of RNA degradome data.

Sex Difference in the Associations among Obesity-Related Indices with Incident Hypertension in a Large Taiwanese Population Follow-Up Study.

Hypertension is a major risk factor for stroke, atherosclerosis, and other cardiovascular diseases, and obesity is a major risk factor for hypertension. The aim of this longitudinal study was to investigate sex differences in the correlations among obesity-related indices and incident hypertension in a large Taiwanese cohort. We included 21,466 enrollees in the Taiwan Biobank and followed them for 4 years. Of the 21,466 patients enrolled in this study, 6899 (mean age, 49.6 ± 10.9 years) were male and 14,567 (mean age, 49.7 ± 10.0 years) were female. Data on visceral adiposity index (VAI), waist-to-height ratio (WHtR), waist-to-hip ratio (WHR), lipid accumulation product (LAP), conicity index (CI), body roundness index (BRI), body mass index (BMI), body adiposity index (BAI), and abdominal volume index (AVI) were collected and analyzed. The results showed that all of the studied obesity-related indices were significantly associated with incident hypertension. Among them, WHtR was the strongest predictor of hypertension in both sexes. In addition, interactions between VAI, LAP, CI, BMI, and AVI with sex on incident hypertension were also statistically significant. CI and AVI were more strongly associated with hypertension in the men than in the women, while VAI, LAP, and BMI were more strongly associated with hypertension in the women. In conclusion, the studied obesity-related indices were found to be predictors of incident hypertension, and there were differences in the associations between the male and female participants. Our findings may imply that reducing body weight may be associated with a lower risk of developing hypertension.

Clinical impact of recreational drug use among people living with HIV in southern Taiwan.

BACKGROUND: It is unclear about the impact of recreational drug use on the adherence, drug-drug interaction and the occurrence of sexual transmitted diseases (STDs) among people living with HIV. MATERIAL AND METHODS: A retrospective study was conducted between Dec 2016, and July 2018 to assess the clinical impact of recreational drug consumption in people living with HIV with antiretroviral therapy. We collected data of the demographics, recreational drug use, laboratory results and STDs diagnoses. Potential drug-drug interactions were checked with reference databases. The association between recreational drug use and STDs, HIV viral load suppression and drug interactions were evaluated. RESULTS: A total of 462 participants were enrolled, included 384 recreational drug users and 78 non-recreational drug users. Younger age (adjusted odds ratio [aOR], 0.94; 95% CI: 0.91-0.98; p = 0.001), longer HIV infection period (aOR, 1.11; 95% CI: 1.03-1.20; p = 0.009) and poor antiretroviral drug adherence (1-2 pills missing per month: aOR, 6.82; 95% CI: 3.50-13.27; p < 0.001; >2 pills missing per month: aOR, 3.50; 95% CI: 1.28-9.61; p = 0.015) were factors associated with recreational drug use. Methamphetamine and nitrites were two most common recreational drugs. Recreational drug use was significantly associated with STDs in one-year follow-up period (aOR, 2.43; 95% CI: 1.11-5.32; p = 0.027) but was not significantly associated with unsuppressed viral load, though a trend was observed (OR, 2.23; 95% CI: 0.92-5.37; p = 0.074). Potential interactions with recreational drugs included 33.1% antiretroviral drugs and 31.3% medications for comorbidities. CONCLUSION: Recreational drug was associated with STDs. A great proportion of the patients consuming recreational drugs had potential interactions with antiretroviral drugs and medications for comorbidities. The association of recreational drug use and unsuppressed viral load warrants further investigation.

Widespread Exon Junction Complex Footprints in the RNA Degradome Mark mRNA Degradation before Steady State Translation.

Exon junction complexes (EJCs) are deposited on mRNAs during splicing and displaced by ribosomes during the pioneer round of translation. Nonsense-mediated mRNA decay (NMD) degrades EJC-bound mRNA, but the lack of suitable methodology has prevented the identification of other degradation pathways. Here, we show that the RNA degradomes of Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), worm (Caenorhabditis elegans), and human (Homo sapiens) cells exhibit an enrichment of 5' monophosphate (5'P) ends of degradation intermediates that map to the canonical EJC region. Inhibition of 5' to 3' exoribonuclease activity and overexpression of an EJC disassembly factor in Arabidopsis reduced the accumulation of these 5'P ends, supporting the notion that they are in vivo EJC footprints. Hundreds of Arabidopsis NMD targets possess evident EJC footprints, validating their degradation during the pioneer round of translation. In addition to premature termination codons, plant microRNAs can also direct the degradation of EJC-bound mRNAs. However, the production of EJC footprints from NMD but not microRNA targets requires the NMD factor SUPPRESSOR WITH MORPHOLOGICAL EFFECT ON GENITALIA PROTEIN7. Together, our results demonstrating in vivo EJC footprinting in Arabidopsis unravel the composition of the RNA degradome and provide a new avenue for studying NMD and other mechanisms targeting EJC-bound mRNAs for degradation before steady state translation.

Genome Editing and Protoplast Regeneration to Study Plant-Pathogen Interactions in the Model Plant Nicotiana benthamiana.

Biotic diseases cause substantial agricultural losses annually, spurring research into plant pathogens and strategies to mitigate them. Nicotiana benthamiana is a commonly used model plant for studying plant-pathogen interactions because it is host to numerous plant pathogens and because many research tools are available for this species. The clustered regularly interspaced short palindromic repeats (CRISPR) system is one of several powerful tools available for targeted gene editing, a crucial strategy for analyzing gene function. Here, we demonstrate the use of various CRISPR-associated (Cas) proteins for gene editing of N. benthamiana protoplasts, including Staphylococcus aureus Cas9 (SaCas9), Streptococcus pyogenes Cas9 (SpCas9), Francisella novicida Cas12a (FnCas12a), and nCas9-activation-induced cytidine deaminase (nCas9-Target-AID). We successfully mutated Phytoene Desaturase (PDS) and Ethylene Receptor 1 (ETR1) and the disease-associated genes RNA-Dependent RNA Polymerase 6 (RDR6), and Suppressor of Gene Silencing 3 (SGS3), and confirmed that the mutated alleles were transmitted to progeny. sgs3 mutants showed the expected phenotype, including absence of trans-acting siRNA3 (TAS3) siRNA and abundant expression of the GFP reporter. Progeny of both sgs3 and rdr6 null mutants were sterile. Our analysis of the phenotypes of the regenerated progeny indicated that except for the predicted phenotypes, they grew normally, with no unexpected traits. These results confirmed the utility of gene editing followed by protoplast regeneration in N. benthamiana. We also developed a method for in vitro flowering and seed production in N. benthamiana, allowing the regenerants to produce progeny in vitro without environmental constraints.

Therapeutic Targeting of Non-oncogene Dependencies in High-risk Neuroblastoma.

PURPOSE: Neuroblastoma is a pediatric malignancy of the sympathetic nervous system with diverse clinical behaviors. Genomic amplification of MYCN oncogene has been shown to drive neuroblastoma pathogenesis and correlate with aggressive disease, but the survival rates for those high-risk tumors carrying no MYCN amplification remain equally dismal. The paucity of mutations and molecular heterogeneity has hindered the development of targeted therapies for most advanced neuroblastomas. We use an alternative method to identify potential drugs that target nononcogene dependencies in high-risk neuroblastoma. EXPERIMENTAL DESIGN: By using a gene expression-based integrative approach, we identified prognostic signatures and potentially effective single agents and drug combinations for high-risk neuroblastoma. RESULTS: Among these predictions, we validated in vitro efficacies of some investigational and marketed drugs, of which niclosamide, an anthelmintic drug approved by the FDA, was further investigated in vivo. We also quantified the proteomic changes during niclosamide treatment to pinpoint nucleoside diphosphate kinase 3 (NME3) downregulation as a potential mechanism for its antitumor activity. CONCLUSIONS: Our results establish a gene expression-based strategy to interrogate cancer biology and inform drug discovery and repositioning for high-risk neuroblastoma.

DCL2- and RDR6-dependent transitive silencing of SMXL4 and SMXL5 in Arabidopsis dcl4 mutants causes defective phloem transport and carbohydrate over-accumulation.

DICER-LIKE (DCL) enzymes process double-stranded RNA into small RNAs that act as regulators of gene expression. Arabidopsis DCL4 and DCL2 each allow the post-transcriptional gene silencing (PTGS) of viruses and transgenes, but primary PTGS-prone DCL4 outcompetes transitive PTGS-prone DCL2 in wild-type plants. This hierarchy likely prevents DCL2 having any detrimental effects on endogenous genes. Indeed, dcl4 mutants exhibit developmental defects and increased sensitivity to genotoxic stress. In this study, the mechanism underlying dcl4 defects was investigated using genetic, biochemical and high-throughput sequencing approaches. We show that the purple phenotype of dcl4 leaves correlates with carbohydrate over-accumulation and defective phloem transport, and depends on the activity of SUPPRESSOR OF GENE SILENCING 3, RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) and DCL2. This phenotype correlates with the downregulation of two genes expressed in the apex and the vasculature, SMAX1-LIKE 4 (SMXL4) and SMXL5, and the accumulation of DCL2- and RDR6-dependent small interfering RNAs derived from these two genes. Supporting a causal effect, smxl4 smxl5 double mutants exhibit leaf pigmentation, enhanced starch accumulation and defective phloem transport, similar to dcl4 plants. Overall, this study elucidates the detrimental action of DCL2 when DCL4 is absent, and indicates that DCL4 outcompeting DCL2 in wild-type plants is crucial to prevent the degradation of endogenous transcripts by DCL2- and RDR6-dependent transitive PTGS.

Global Analysis of Truncated RNA Ends Reveals New Insights into Ribosome Stalling in Plants.

High-throughput approaches for profiling the 5' ends of RNA degradation intermediates on a genome-wide scale are frequently applied to analyze and validate cleavage sites guided by microRNAs (miRNAs). However, the complexity of the RNA degradome other than miRNA targets is currently largely uncharacterized, and this limits the application of RNA degradome studies. We conducted a global analysis of 5'-truncated mRNA ends that mapped to coding sequences (CDSs) of Arabidopsis thaliana, rice (Oryza sativa), and soybean (Glycine max). Based on this analysis, we provide multiple lines of evidence to show that the plant RNA degradome contains in vivo ribosome-protected mRNA fragments. We observed a 3-nucleotide periodicity in the position of free 5' RNA ends and a bias toward the translational frame. By examining conserved peptide upstream open reading frames (uORFs) of Arabidopsis and rice, we found a predominance of 5' termini of RNA degradation intermediates that were separated by a length equal to a ribosome-protected mRNA fragment. Through the analysis of RNA degradome data, we discovered uORFs and CDS regions potentially associated with stacked ribosomes in Arabidopsis. Furthermore, our analysis of RNA degradome data suggested that the binding of Arabidopsis ARGONAUTE7 to a noncleavable target site of miR390 might directly hinder ribosome movement. This work demonstrates an alternative use of RNA degradome data in the study of ribosome stalling.

Asymmetric bulges and mismatches determine 20-nt microRNA formation in plants.

Plant microRNAs (miRNAs) are predominantly 21 nucleotides (nt) long but non-canonical lengths of 22 and 20 nt are commonly observed in diverse plant species. While miRNAs longer than 21 nt can be attributed to the neglect of unpaired bases within asymmetric bulges by the ruler function of dicer-like 1 (DCL1), how 20-nt miRNA is generated remains obscure. Analysis of small RNA data revealed that 20-nt miRNA can be divided into 3 main groups featured by atypical 3' overhangs or shorter duplex regions. Asymmetric bulges or mismatches at specific positions are commonly observed within each group and were shown to be crucial for 20-nt miRNA formation. Analysis of DCL1 cleavage sites on 20-nt miRNA precursors suggests that these determinants might alter precursor structure or trigger 3'-end decay of mature miRNA. The results herein advance our understanding of miRNA biogenesis and demonstrate that the effect of asymmetric bulges on miRNA length could be position-dependent.

Retinal proteins associated with redox regulation and protein folding play central roles in response to high glucose conditions.

Diabetic retinopathy typically causes poor vision and blindness. A previous study revealed that a high blood glucose concentration induces glycoxidation and weakens the retinal capillaries. Nevertheless, the molecular mechanisms underlying the effects of high blood glucose induced diabetic retinopathy remain to be elucidated. In the present study, we cultured the retinal pigmented epithelial cell line ARPE-19 in mannitol-balanced 5.5, 25, and 100 mM glucose media and investigated protein level alterations. Proteomic analysis revealed significant changes in 137 protein features, of which 124 demonstrated changes in a glucose concentration dependent manner. Several proteins functionally associated with redox regulation, protein folding, or the cytoskeleton are affected by increased glucose concentrations. Additional analyses also revealed that cellular oxidative stress, including endoplasmic reticulum stress, was significantly increased after treatment with high glucose concentrations. However, the mitochondrial membrane potential and cell survival remained unchanged during treatment with high glucose concentrations. To summarize, in this study, we used a comprehensive retinal pigmented epithelial cell based proteomic approach for identifying changes in protein expression associated retinal markers induced by high glucose concentrations. Our results revealed that a high glucose condition can induce cellular oxidative stress and modulate the levels of proteins with functions in redox regulation, protein folding, and cytoskeleton regulation; however, cell viability and mitochondrial integrity are not significantly disturbed under these high glucose conditions.

Identification of up- and down-regulated proteins in doxorubicin-resistant uterine cancer cells: reticulocalbin-1 plays a key role in the development of doxorubicin-associated resistance.

Drug resistance is a frequent cause of failure in cancer chemotherapy treatments. In this study, a pair of uterine sarcoma cancer lines, MES-SA, and doxorubicin-resistant partners, MES-SA/DxR-2µM cells and MES-SA/DxR-8µM cells, as a model system to investigate resistance-dependent proteome alterations and to identify potential therapeutic targets. We used two-dimensional differential gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) to perform this research and the results revealed that doxorubicin-resistance altered the expression of 208 proteins in which 129 identified proteins showed dose-dependent manners in response to the levels of resistance. Further studies have used RNA interference, H2A.X phosphorylation assay, cell viability analysis, and analysis of apoptosis against reticulocalbin-1 (RCN1) proteins, to prove its potency on the formation of doxorubicin resistance as well as the attenuation of doxorubicin-associated DNA double strand breakage. To sum up, our results provide useful diagnostic markers and therapeutic candidates such as RCN1 for the treatment of doxorubicin-resistant uterine cancer.

Increased concentration of sialidases by HeLa cells might influence the cytotoxic ability of NK cells.

OBJECTIVE: Cancer cells reportedly have the ability to escape from the immune system, mainly from natural killer (NK) cells. Although the real mechanisms are complicated, some inhibitors that are secreted from the cancer cells might play an important role. This study's aim was to investigate the potential mediator released by cancer cells (HeLa) that contributes to the decreased cytotoxicity of NK cells. METHODS AND MATERIALS: An NK-HeLa coculture system was used to test the hypothesis that the presence of the potential mediator from cancer cells contributes to the decreased cytotoxicity of NK cells. RESULTS: After coculturing with HeLa cancer cells, the cytotoxicity of NK cells was decreased. When the coculture medium and culture medium containing commercialized sialidase were used to culture NK cells, the cytotoxicity of the NK cells was also inhibited. However, cytotoxicity was partially restored by a sialidase inhibitor (DANA). Western blot analysis of the HeLa cells after coculturing with NK cells demonstrated increased Neu2 and Neu3 expression in HeLa cells. CONCLUSIONS: The finding that Neu2 and Neu3 expression in cancer cells might be involved in the impaired function of NK cells, which could be restored by a sialidase inhibitor, provides a new concept that could be applied to the management of cancer.

Altered ganglioside GD3 in HeLa cells might influence the cytotoxic abilities of NK cells.

OBJECTIVE: Previously, we found that altered sialidases in HeLa cells in a natural killer-HeLa (NK-HeLa) coculture system contributed to the decreased cytotoxic ability of NK cells. However, changes that occur in the glycosylation of the HeLa cells in the NK-HeLa coculture system remain unknown. MATERIALS AND METHODS: An NK-HeLa coculture system was used to examine the changes that occur in the gangliosides of HeLa cells. RESULTS: GD3 expression in HeLa cells was significantly increased in the NK-HeLa coculture system. Exogenous ganglioside GD3 decreased the cytotoxic ability of the NK cells, which could be restored by the addition of the anti-GD3 antibody. Coadministration of GD3 and sialidase further decreased the cytotoxic ability of the NK cells, which could be partially restored by the addition of a sialidase inhibitor (DANA). GD3 expression in HeLa cells also decreased following DANA treatment. CONCLUSIONS: This study suggests that interactions between ganglioside GD3 and sialidases in HeLa cells influence the cytotoxic ability of NK cells.

Cardiomyogenic differentiation potential of human adipose precursor cells.

DNA demethylation agent 5-azacytidine has been widely described in literature as an effective chemical stimulus used to promote cardiomyogenic differentiation in various cell types, ranging from embryonic stem cells, P19 cells, bone marrow-derived mesenchymal stem cells, and recently to adipose-derived stem cells. The purpose of this study was to examine the effects of 5-azacytidine on human adipose precursor cell differentiation along the cardiomyogenic lineage.

Effects of uniaxial cyclic strain on adipose-derived stem cell morphology, proliferation, and differentiation.

Cells and tissues in vivo are subjected to various forms of mechanical forces that are essential to their normal development and functions. The arterial blood vessel wall is continuously exposed to mechanical stresses such as pressure, strain, and shear due to the pulsatile nature of blood flow. Vascular smooth muscle cells (SMCs) populate the media of blood vessels and play important roles in the control of vasoactivity and the remodeling of the vessel wall. It is well documented that the phenotype and functions of vascular SMCs are not only regulated by chemical factors such as transforming growth factor-beta(1) (TGF-beta(1)), but also by mechanical factors such as uniaxial strain. The purpose of our study was to explore the effects of TGF-beta(1) alone or in combination with uniaxial cyclic strain on adipose-derived stem cell (ASC) morphology, proliferation, and differentiation. Low passage ASCs were stimulated with 10% strain at 1 Hz for 7 days, with or without TGF-beta(1). Cyclic strain inhibited proliferation, and caused alignment of the cells and of the F-actin cytoskeleton perpendicular to the direction of strain. Strain alone resulted in a decrease in the expression of early SMC markers alpha-SMA and h (1)-calponin. While the response of SMCs and other progenitor cells such as bone marrow stromal cells to mechanical forces has been extensively studied, the roles of these forces on ASCs remain unexplored. This work advances our understanding of the mechanical regulation of ASCs.

Regulation of alpha-smooth muscle actin protein expression in adipose-derived stem cells.

The objective of this work was to study the response of adipose-derived stem cells (ASCs) to exogenous biochemical stimulation, and the potential of ASCs to differentiate toward the smooth muscle cell (SMC) lineage. Immunofluorescence staining and Western blot analysis detected protein expression of the early SMC marker alpha-smooth muscle actin (alpha-SMA) in both control and experiment groups. Expression of alpha-SMA in ASCs significantly increased when treated with transforming growth factor-beta1, while alpha-SMA expression only slightly increased in the presence of retinoic acid (RA), beta-mercaptoethanol and ascorbic acid. Treatment with platelet-derived growth factor-BB, RA and dibutyryl-cyclic adenosine monophosphate decreased the expression of alpha-SMA significantly. While beta-mercaptoethanol and ascorbic acid, as well as RA have resulted in increased alpha-SMA expression in marrow-derived mesenchymal stem cells and other progenitor cells, our results demonstrate that these treatments do not significantly increase alpha-SMA expression, indicating that the differentiation potential of ASCs and mesenchymal stem cells may be fundamentally different.

Controlled release of bioactive doxorubicin from microspheres embedded within gelatin scaffolds.

We have encapsulated the chemotherapeutic agent doxorubicin into biodegradable polymer microspheres, and incorporated these microspheres into gelatin scaffolds, resulting in a controlled delivery system. Doxorubicin was encapsulated in poly(D,L-lactide-co-glycolide) (PLGA) using a double emulsion/solvent extraction method. Characterization of the microspheres including diameter, surface morphology, and in vitro drug release was determined. The release of doxorubicin up to 30 days in phosphate buffered solution was assessed by measuring the absorbance of the releasate solution. Gelatin scaffolds were crosslinked using glutaraldehyde and microspheres were added to gelatin during gelation. The murine mammary mouse tumor cell line, 4T1, was treated with various doses of doxorubicin. A propidium iodide assay was utilized to visualize dead cells. Using a Transwell basket assay, PLGA microspheres and gelatin constructs were suspended above 4T1 cells for 48 h. Viable cells were determined using the CyQUANT cell proliferation assay. Results indicate that the release was controlled by the incorporation of PLGA microspheres into gelatin constructs. A significant difference was seen in the cumulative release over days 5-16 (p < 0.05). The bioactivity of doxorubicin released from the microspheres and scaffolds was maintained as proven by significant reduction in viable cells after treatment with PLGA microspheres as well as with the gelatin constructs (p < 0.001). The drug-polymer conjugate can be used as a controlled drug delivery system in a biocompatible scaffold that could potentially promote preservation of soft tissue contour.

A copper chaperone for superoxide dismutase that confers three types of copper/zinc superoxide dismutase activity in Arabidopsis.

The copper chaperone for superoxide dismutase (CCS) has been identified as a key factor integrating copper into copper/zinc superoxide dismutase (CuZnSOD) in yeast (Saccharomyces cerevisiae) and mammals. In Arabidopsis (Arabidopsis thaliana), only one putative CCS gene (AtCCS, At1g12520) has been identified. The predicted AtCCS polypeptide contains three distinct domains: a central domain, flanked by an ATX1-like domain, and a C-terminal domain. The ATX1-like and C-terminal domains contain putative copper-binding motifs. We have investigated the function of this putative AtCCS gene and shown that a cDNA encoding the open reading frame predicted by The Arabidopsis Information Resource complemented only the cytosolic and peroxisomal CuZnSOD activities in the Atccs knockout mutant, which has lost all CuZnSOD activities. However, a longer AtCCS cDNA, as predicted by the Munich Information Centre for Protein Sequences and encoding an extra 66 amino acids at the N terminus, could restore all three, including the chloroplastic CuZnSOD activities in the Atccs mutant. The extra 66 amino acids were shown to direct the import of AtCCS into chloroplasts. Our results indicated that one AtCCS gene was responsible for the activation of all three types of CuZnSOD activity. In addition, a truncated AtCCS, containing only the central and C-terminal domains without the ATX1-like domain failed to restore any CuZnSOD activity in the Atccs mutant. This result indicates that the ATX1-like domain is essential for the copper chaperone function of AtCCS in planta.

Registration of MR and CT images of the liver: comparison of voxel similarity and surface based registration algorithms.

The purpose of this work was to determine the feasibility and efficacy of retrospective registration of MR and CT images of the liver. The open-source ITK Insight Software package developed by the National Library of Medicine (USA) contains a multi-resolution, voxel-similarity-based registration algorithm which we selected as our baseline registration method. For comparison we implemented a multi-scale surface fitting technique based on the head-and-hat algorithm. Registration accuracy was assessed using the mean displacement of automatically selected point landmarks. The ITK voxel-similarity-based registration algorithm performed better than the surface-based approach with mean misregistration in the range of 7.7-8.4 mm for CT-CT registration, 8.2 mm for MR-MR registration, and 14.0-18.9 mm for MR-CT registration compared to mean misregistration from the surface-based technique in the range of 9.6-11.1 mm for CT-CT registration, 9.2-12.4 mm for MR-MR registration, and 15.2-19.0 mm for MR-CT registration.