Footprint Sequences Research Articles

Direct CO2 mineralization and evolution behavior of fine CaCO3 from ammonia-induced phosphogypsum mineral carbonation

The mineral carbonation of phosphogypsum (PG) presents considerable potential for simultaneous CO2 sequestration and high-value utilization of PG. Variations in carbon footprints are crucial for enhancing carbonation efficiency and regulating product formation. This study explores the mechanism of direct CO2 mineralization by PG and the evolution of the resulting product. The results demonstrate that 97.14% of the CaSO4·2H2O in PG can be converted to calcite under conditions including a nitrogen–sulfur molar ratio of 3, a liquid–solid ratio of 6, a CO2 flowrate of 0.4 L/min, and a stirring rate of 800 rpm for 60 min. The carbonation rate exhibits significant variation with time, occurring in three distinct stages. Initially, CO2 reacts with ammonia to form (NH4)2CO3, releasing greater heat. This heat inhibits the dissolution of Ca2+ from PG, resulting in a lower carbonation efficiency, as only a limited amount of Ca2+ reacts with CO32– to preferentially form amorphous CaCO3 (ACC). As CO32– formation reaches equilibrium, the endothermic properties of the reaction between CaSO4 and (NH4)2CO3 promote the dissolution of Ca2+ from PG, further accelerating CO2 mineralization and resulting in significant increases in carbonation efficiency and ACC production. The ACC is rapidly transformed into calcite via a “dissolution-reprecipitation” mechanism. However, the carbonation rate of PG decreases as saturation is approached, mainly resulting from the aggregation of fine CaCO3 particles on the PG surface. As a result, the direct CO2 sequestration process by PG follows a carbon footprint sequence of “CO2–(NH4)2CO3–ACC–calcite”. This study provides significant insights into the mechanisms of direct CO2 mineralization and product evolution and offers a basis for optimizing carbonation processes enhancement and regulating product phases.

Calibrated ribosome profiling assesses the dynamics of ribosomal flux on transcripts

Ribosome profiling, which is based on deep sequencing of ribosome footprints, has served as a powerful tool for elucidating the regulatory mechanism of protein synthesis. However, the current method has substantial issues: contamination by rRNAs and the lack of appropriate methods to measure ribosome numbers in transcripts. Here, we overcome these hurdles through the development of “Ribo-FilterOut”, which is based on the separation of footprints from ribosome subunits by ultrafiltration, and “Ribo-Calibration”, which relies on external spike-ins of stoichiometrically defined mRNA-ribosome complexes. A combination of these approaches estimates the number of ribosomes on a transcript, the translation initiation rate, and the overall number of translation events before its decay, all in a genome-wide manner. Moreover, our method reveals the allocation of ribosomes under heat shock stress, during aging, and across cell types. Our strategy of modified ribosome profiling measures kinetic and stoichiometric parameters of cellular translation across the transcriptome.

A custom library construction method for super-resolution ribosome profiling in Arabidopsis

BackgroundRibosome profiling, also known as Ribo-seq, is a powerful technique to study genome-wide mRNA translation. It reveals the precise positions and quantification of ribosomes on mRNAs through deep sequencing of ribosome footprints. We previously optimized the resolution of this technique in plants. However, several key reagents in our original method have been discontinued, and thus, there is an urgent need to establish an alternative protocol.ResultsHere we describe a step-by-step protocol that combines our optimized ribosome footprinting in plants with available custom library construction methods established in yeast and bacteria. We tested this protocol in 7-day-old Arabidopsis seedlings and evaluated the quality of the sequencing data regarding ribosome footprint length, mapped genomic features, and the periodic properties corresponding to actively translating ribosomes through open resource bioinformatic tools. We successfully generated high-quality Ribo-seq data comparable with our original method.ConclusionsWe established a custom library construction method for super-resolution Ribo-seq in Arabidopsis. The experimental protocol and bioinformatic pipeline should be readily applicable to other plant tissues and species.

Normalized Ribo-Seq for Quantifying Absolute Global and Specific Changes in Translation.

Ribosome profiling (Ribo-Seq) is a highly sensitive method to quantify ribosome occupancies along individual mRNAs on a genome-wide scale. Hereby, ribosome-protected fragments (= footprints) are generated by nuclease digestion, isolated, and sequenced together with the corresponding randomly fragmented input samples, to determine ribosome densities (RD). For library preparation, equal amounts of total RNA are used. Subsequently, all transcript fragments are subjected to linker ligation, cDNA synthesis, and PCR amplification. Importantly, the number of reads obtained for every transcript in input and footprint samples during sequencing depends on sequencing depth and library size, as well as the relative abundance of the transcript in the sample. However, the information pertaining to the absolute amount of input and footprint sequences is lost during sample preparation, hence ruling out any conclusion whether translation is generally suppressed or activated in one condition over the other. Therefore, the RD fold-changes determined for individual genes do not reflect absolute regulation, but have to be interpreted as relative to bulk mRNA translation. Here, we modified the original ribosome profiling protocol that was first established by Ingolia et al. (2009), by adding small amounts of yeast lysate to the mammalian lysates of interest as a spike-in. This allows us to not only detect changes in the RD of specific transcripts relative to each other, but also to simultaneously measure global differences in RD (normalized ribosome density values) between samples. Graphic abstract: Global changes in translation efficiency can be detected with polysome profiling, where the proportion of polysomal ribosomes is interpreted as a proxy for ribosome density (RD) on bulk mRNA. Ribo-Seq measures changes in RD of specific mRNAs relative to bulk mRNA. The addition of a yeast-lysate, as a spike-in for normalization of read counts, allows for an absolute measurement of changes in RD.

Analysis of Ribosome Profiling Data.

Ribosome profiling methods are based on high-throughput sequencing of ribosome-protected mRNA footprints and allow to study in detail translational changes. Bioinformatic and statistical tools are necessary to analyze sequencing data. Here, we describe our developed methods for a fast and reliable quality control of ribosome profiling data, to efficiently visualize ribosome positions and to estimate ribosome speed in an unbiased way. The methodology described here is applicable to several genetic and environmental conditions including stress and are based on the R package RiboVIEW and calculation of quantitative estimates of local and global translation speed, based on a biophysical model of translation dynamics.

Construction of High-Quality Rice Ribosome Footprint Library.

High-throughput sequencing of ribosome footprints precisely maps and quantifies in vivo mRNA translation. The ribosome footprint sequencing has undergone continuing development since its original report. Here we provide a detailed protocol for construction of high-quality ribosome footprint library of rice. Rice total polysomes are isolated with a modified low ionic polysome extraction buffer. After nuclease digestion, rice ribosome footprints are extracted using SDS method followed by column purification. High-quality rice ribosome footprint library with peak reads of approximately 28-nucleotide (nt) length and strong 3-nt periodicity is constructed via key steps including rRNA depletion, end repair, 3’ adapter ligation, reverse transcription, circularization, PCR enrichment and several rounds of purification. Biological significance of rice ribosome footprint library is further revealed by the comparison of transcriptomic and translatomic responses to salt stress and the utilization for novel open reading frame (ORF) identification. This improved protocol for rice ribosome footprint library construction will facilitate the global comprehension and quantitative measurement of dynamic translation in rice.

RiboVIEW: a computational framework for visualization, quality control and statistical analysis of ribosome profiling data.

Recently, newly developed ribosome profiling methods based on high-throughput sequencing of ribosome-protected mRNA footprints allow to study genome-wide translational changes in detail. However, computational analysis of the sequencing data still represents a bottleneck for many laboratories. Further, specific pipelines for quality control and statistical analysis of ribosome profiling data, providing high levels of both accuracy and confidence, are currently lacking. In this study, we describe automated bioinformatic and statistical diagnoses to perform robust quality control of ribosome profiling data (RiboQC), to efficiently visualize ribosome positions and to estimate ribosome speed (RiboMine) in an unbiased way. We present an R pipeline to setup and undertake the analyses that offers the user an HTML page to scan own data regarding the following aspects: periodicity, ligation and digestion of footprints; reproducibility and batch effects of replicates; drug-related artifacts; unbiased codon enrichment including variability between mRNAs, for A, P and E sites; mining of some causal or confounding factors. We expect our pipeline to allow an optimal use of the wealth of information provided by ribosome profiling experiments.

Abstract 4344: Integrative functional proteogenomics for unannotated or uncharacterized proteins in cancer

Abstract Introduction: Numerous transcripts annotated as long noncoding RNAs play a central role in cancer biology. For many, their noncoding status is merely a presumption. Genome-wide sequencing of ribosomal footprints has nominated thousands of unstudied open reading frames (ORFs) within lncRNAs, representing an expansion of the proteome. Here, we investigate previously unstudied proteins in cancer cell biology. Methods: Ribosome profiling data was analyzed with RibORF. 96 hours after infection with lentivirus for selected ORFs, L1000 expression profiling was performed on 4 cell lines. A CRISPR library was screened across 8 cells lines with sgRNA sequencing on days 0, 7 and 21 post-infection. Results: We analyzed ribosomal profiling data for 14 cell lines (~320 million sequencing reads). We nominated 28530 non-canonical ORFs within annotated protein-coding genes, 6697 ORFs in annotated lncRNAs, and 1252 ORFs in pseudogenes. For further study, we selected 553 candidate ORFs that exhibited compelling features, including DNA conservation, translational efficiency, protein domain, among others. We validated protein expression for 260 of 553 ORFs (47%): 89 (16%) had supporting peptides in deep-coverage proteomics datasets; 233 (42%) expressed protein after ectopic expression of individual V5-tagged cDNAs; 10 of 30 tested untagged ORFs expressed protein by biochemical in vitro translation. Ectopic overexpression followed by RNA profiling revealed 259 cDNAs that caused cellular transcriptional changes in at least one of four cancer cell lines (A549, HA1E, A375, MCF7). 137 of the 259 (49%) were validated proteins. As controls, we generated methionine-mutant constructs: 65 of 71 mutant cDNA experiments were unable to cause similar expression changes. We used a CRISPR library to identify novel ORF dependencies in 8 Cas9-derivatized cancer cell lines (MCF7, A549, A375, PC3, HEPG2, HELA, HA1E, HT29). For 42 ORFs, ≥ 2 targeting sgRNAs produced ≤ -1 log fold depletion in ≥ 1 cell lines. These ORFs were re-tested with a second sgRNA library. Next, we investigated compelling candidates more deeply with immunoprecipitation with mass spectrometry. For the cancer outlier transcript LINC01314, which encodes a highly conserved 59 amino acid protein harboring a cortexin domain (pfam domain cl12620), we found interactions with IMMT, SAMM50, and CHCHD3, members of a mitochondrial complex. Another example is LINC00116, which encodes a highly conserved 56 amino acid protein that binds the importin-nuclear pore complex. Conclusion: We establish a framework to discover, validate, and characterize unstudied proteins. About half of tested ORFs generated a detectable protein, and of these, half impacted cellular transcription. We discover novel gene dependencies, and are elucidating mechanisms for several ORFs. Together, our work is the first large-scale attempt to study the role of unannotated proteins in cancer cell biology. Citation Format: John R. Prensner, Oana Enache, Zhe Ji, Karsten Krug, Karl R. Clauser, Xiaoping Yang, Federica Piccioni, David E. Root, Todd R. Golub. Integrative functional proteogenomics for unannotated or uncharacterized proteins in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4344.

Ribosome Profiling of Synechocystis Reveals Altered Ribosome Allocation at Carbon Starvation

Ribosome profiling accesses the translational step of gene expression via deep sequencing of ribosome-protected mRNA footprints. Pairing of ribosome profiling and transcriptomics data provides a translational efficiency for each gene. Here, the translatome and transcriptome of the model cyanobacterium Synechocystis were compared under carbon-replete and carbon starvation conditions. The latter may be experienced when cyanobacteria are cultivated in poorly mixed bioreactors or engineered to be product-secreting cell factories. A small fraction of genes (<200), including stress response genes, showed changes in translational efficiency during carbon starvation, indicating condition-dependent translation-level regulation. We observed ribosome occupancy in untranslated regions, possibly due to an alternative translation initiation mechanism in Synechocystis. The higher proportion of ribosomes residing in untranslated regions during carbon starvation may be a mechanism to quickly inactivate superfluous ribosomes. This work provides the first ribosome profiling data for cyanobacteria and reveals new regulation strategies for coping with nutrient limitation.

The GWIPS‐viz Browser

GWIPS-viz is a publicly available browser that provides Genome Wide Information on Protein Synthesis through the visualization of ribosome profiling data. Ribosome profiling (Ribo-seq) is a high-throughput technique which isolates fragments of messenger RNA that are protected by the ribosome. The alignment of the ribosome-protected fragments or footprint sequences to the corresponding reference genome and their visualization using GWIPS-viz allows for unique insights into the genome loci that are expressed as potentially translated RNA. The GWIPS-viz browser hosts both Ribo-seq data and corresponding mRNA-seq data from publicly available studies across a number of genomes, avoiding the need for computational processing on the user side. Since its initial publication in 2014, over 1885 tracks have been produced across 24 genomes. This unit describes the navigation of the GWIPS-viz genome browser, the uploading of custom tracks, and the downloading of the Ribo-seq/mRNA-seq alignment data. © 2018 by John Wiley & Sons, Inc.

Ribosome profiling reveals changes in translational status of soybean transcripts during immature cotyledon development.

To understand translational capacity on a genome-wide scale across three developmental stages of immature soybean seed cotyledons, ribosome profiling was performed in combination with RNA sequencing and cluster analysis. Transcripts representing 216 unique genes demonstrated a higher level of translational activity in at least one stage by exhibiting higher translational efficiencies (TEs) in which there were relatively more ribosome footprint sequence reads mapping to the transcript than were present in the control total RNA sample. The majority of these transcripts were more translationally active at the early stage of seed development and included 12 unique serine or cysteine proteases and 16 2S albumin and low molecular weight cysteine-rich proteins that may serve as substrates for turnover and mobilization early in seed development. It would appear that the serine proteases and 2S albumins play a vital role in the early stages. In contrast, our investigation of profiles of 19 genes encoding high abundance seed storage proteins, such as glycinins, beta-conglycinins, lectin, and Kunitz trypsin inhibitors, showed that they all had similar patterns in which the TE values started at low levels and increased approximately 2 to 6-fold during development. The highest levels of these seed protein transcripts were found at the mid-developmental stage, whereas the highest ribosome footprint levels of only up to 1.6 TE were found at the late developmental stage. These experimental findings suggest that the major seed storage protein coding genes are primarily regulated at the transcriptional level during normal soybean cotyledon development. Finally, our analyses also identified a total of 370 unique gene models that showed very low TE values including over 48 genes encoding ribosomal family proteins and 95 gene models that are related to energy and photosynthetic functions, many of which have homology to the chloroplast genome. Additionally, we showed that genes of the chloroplast were relatively translationally inactive during seed development.

The extent of ribosome queuing in budding yeast.

Ribosome queuing is a fundamental phenomenon suggested to be related to topics such as genome evolution, synthetic biology, gene expression regulation, intracellular biophysics, and more. However, this phenomenon hasn't been quantified yet at a genomic level. Nevertheless, methodologies for studying translation (e.g. ribosome footprints) are usually calibrated to capture only single ribosome protected footprints (mRPFs) and thus limited in their ability to detect ribosome queuing. On the other hand, most of the models in the field assume and analyze a certain level of queuing. Here we present an experimental-computational approach for studying ribosome queuing based on sequencing of RNA footprints extracted from pairs of ribosomes (dRPFs) using a modified ribosome profiling protocol. We combine our approach with traditional ribosome profiling to generate a detailed profile of ribosome traffic. The data are analyzed using computational models of translation dynamics. The approach was implemented on the Saccharomyces cerevisiae transcriptome. Our data shows that ribosome queuing is more frequent than previously thought: the measured ratio of ribosomes within dRPFs to mRPFs is 0.2–0.35, suggesting that at least one to five translating ribosomes is in a traffic jam; these queued ribosomes cannot be captured by traditional methods. We found that specific regions are enriched with queued ribosomes, such as the 5’-end of ORFs, and regions upstream to mRPF peaks, among others. While queuing is related to higher density of ribosomes on the transcript (characteristic of highly translated genes), we report cases where traffic jams are relatively more severe in lowly expressed genes and possibly even selected for. In addition, our analysis demonstrates that higher adaptation of the coding region to the intracellular tRNA levels is associated with lower queuing levels. Our analysis also suggests that the Saccharomyces cerevisiae transcriptome undergoes selection for eliminating traffic jams. Thus, our proposed approach is an essential tool for high resolution analysis of ribosome traffic during mRNA translation and understanding its evolution.

Super-resolution ribosome profiling reveals unannotated translation events in Arabidopsis

Deep sequencing of ribosome footprints (ribosome profiling) maps and quantifies mRNA translation. Because ribosomes decode mRNA every 3 nt, the periodic property of ribosome footprints could be used to identify novel translated ORFs. However, due to the limited resolution of existing methods, the 3-nt periodicity is observed mostly in a global analysis, but not in individual transcripts. Here, we report a protocol applied to Arabidopsis that maps over 90% of the footprints to the main reading frame and thus offers super-resolution profiles for individual transcripts to precisely define translated regions. The resulting data not only support many annotated and predicted noncanonical translation events but also uncover small ORFs in annotated noncoding RNAs and pseudogenes. A substantial number of these unannotated ORFs are evolutionarily conserved, and some produce stable proteins. Thus, our study provides a valuable resource for plant genomics and an efficient optimization strategy for ribosome profiling in other organisms.

Ribosomal profiling adds new coding sequences to the proteome.

Next generation sequencing (NGS) has enabled an in-depth look into genes, transcripts and their translation at the genomic scale. The application of NGS sequencing of ribosome footprints (Ribo-Seq) reveals translation with single nucleotide (nt) resolution, through the deep sequencing of ribosome-bound fragments (RBFs). Some results of Ribo-Seq challenge our understanding of the protein-coding potential of the genome. Earlier bioinformatic approaches had shown the presence of hundreds of thousands of putative small ORFs (smORFs) in eukaryotic genomes, but they had been largely ignored due to their large numbers and difficulty in determining their translation and function. Ribo-Seq has revealed that hundreds of putative smORFs within previously assumed long non-coding RNAs (lncRNAs) and UTRs of canonical mRNAs are associated with ribosomes, appearing to be translated. Here we review some of the approaches used to define translation within Ribo-Seq experiments and the challenges in defining translation of these novel smORFs in lncRNAs and UTRs. We also look at some of the bioinformatic and biochemical approaches used to independently corroborate these exciting new findings and elucidate real translation events.

Evaluación estacional de la riqueza y abundancia de especies de mamíferos en la Reserva Biológica Municipal “Mário Viana”, Mato Grosso, Brasil

We evaluated seasonal species presence and richness, and abundance of medium and large sized mammalian terrestrial fauna in the "Mário Viana" Municipal Biological Reserve, Nova Xavantina, Mato Grosso, Brazil. During 2001, two monthly visits were made to an established transect, 2,820 m in length. Records of 22 mammal species were obtained and individual footprint sequences quantified for seasonal calculation of species richness and relative abundance index (x footprints/km traveled). All 22 species occurred during the rainy season, but only 18 during the dry season. Pseudalopex vetulus (Lund, 1842) (hoary fox), Eira barbara (Linnaeus, 1758) (tayra), Puma concolor (Linnaeus, 1771) (cougar) and Hydrochaeris hydrochaeris (Linnaeus, 1766) (capybara) were only registered during the rainy season. The species diversity estimated using the Jackknife procedure in the dry season (19.83, CI = 2.73) was smaller than in the rainy season (25.67, CI = 3.43). Among the 18 species common in the two seasons, only four presented significantly different abundance indexes: Dasypus novemcinctus Linnaeus, 1758 (nine-banded armadillo), Euphractus sexcinctus (Linnaeus, 1758) (six-banded armadillo), Dasyprocta azarae Lichtenstein, 1823 (Azara's Agouti) and Tapirus terrestris (Linnaeus, 1758) (tapir). On the other hand, Priodontes maximus (Kerr, 1792) (giant armadillo) and Leopardus pardalis (Linnaeus, 1758) (ocelot) had identical abundance index over the two seasons. Distribution of species abundance in the sampled area followed the expected pattern for communities in equilibrium, especially in the rainy season, suggesting that the environment still maintains good characteristics for mammal conservation. The present study shows that the reserve, although only 470 ha in size, plays an important role for conservation of mastofauna of the area as a refuge in an environment full of anthropic influence (mainly cattle breeding in exotic pasture).

Trials and Tribulations with VH Replacement

VH replacement (VHR) is a type of antibody gene rearrangement in which an upstream heavy chain variable gene segment (VH) invades a pre-existing rearrangement (VDJ). In this Hypothesis and Theory article, we begin by reviewing the mechanism of VHR, its developmental timing and its potential biological consequences. Then we explore the hypothesis that specific sequence motifs called footprints reflect VHR versus other processes. We provide a compilation of footprint sequences from different regions of the antibody heavy chain, and include data from the literature and from a high throughput sequencing experiment to evaluate the significance of footprint sequences. We conclude by discussing the difficulties of attributing footprints to VHR.

Research on Incomplete Transaction Footprints in Networked Software

In networked software, interactive behaviors of software entities generate lots of behavioral footprints, some of them may lose tokens or tokens are useless. The paper studies the constructing process of State Transition Model (STM) in which the process of incomplete transactions are satisfied with Markov property, it is pointed that the STM are originally extracted from behavior log files generated by the runtime behaviors of networked software. The contributions of this paper are to mark the partly tokenized behavior footprints in the STM through Maximum Flow (MF) algorithm, then find the original source for each behavior footprint. The experiment results indicate that the maximum flow algorithm can accurately turn the partly tokenized behavior into complete footprint sequences.

Reverted glutathione <i>S</i>-transferase-like genes that influence flower color intensity of carnation (<i>Dianthus caryophyllus</i> L.) originated from excision of a transposable element

A glutathione S-transferase-like gene, DcGSTF2, is responsible for carnation (Dianthus caryophyllus L.) flower color intensity. Two defective genes, DcGSTF2mu with a nonsense mutation and DcGSTF2-dTac1 containing a transposable element dTac1, have been characterized in detail in this report. dTac1 is an active element that produces reverted functional genes by excision of the element. A pale-pink cultivar ‘Daisy’ carries both defective genes, whereas a spontaneous deep-colored mutant ‘Daisy-VPR’ lost the element from DcGSTF2-dTac1. This finding confirmed that dTac1 is active and that the resulting reverted gene, DcGSTF2rev1, missing the element is responsible for this color change. Crosses between the pale-colored cultivar ‘06-LA’ and a deep-colored cultivar ‘Spectrum’ produced segregating progeny. Only the deep-colored progeny had DcGSTF2rev2 derived from the ‘Spectrum’ parent, whereas progeny with pale-colored flowers had defective forms from both parents, DcGSTF2mu and DcGSTF2-dTac1. Thus, DcGSTF2rev2 had functional activity and likely originated from excision of dTac1 since there was a footprint sequence at the vacated site of the dTac1 insertion. Characterizing the DcGSTF2 genes in several cultivars revealed that the two functional genes, DcGSTF2rev1 and DcGSTF2rev2, have been used for some time in carnation breeding with the latter in use for more than half a century.

An Improved Method for Transaction Footprints Stripping with Bigraph System

Multiple parallel transactions in new-type distributed software environment result in that the events produced by every transaction are randomly ranked. If the tokens of these events are incomplete or unavailable, it is difficult for software system to distinguish these events to actually belong to which transaction, corresponding transaction analysis and prediction can’t be executed. In this paper, the problem of stripping events with incomplete tokens is transferred into maximum-weight perfect matching of bigraph system. If the transition time among these events is independently and identically distributed, all possible states (events) are separated into multiple cutsets, every cutset composes a bigraph system. The maximum-weight perfect matching is used to finish respective matching, and then the results of independent matching of multiple bigraph systems are spliced to gain the most possible footprint sequences produced by multiple transactions, which is convenient for subsequent analysis and prediction. For implementing quick stripping for transaction footprints, the paper presents rank-maximal matching algorithm to improve matching efficiency. Simulation experiment confirms that the method presented in this paper can effectively implement transaction footprint stripping with incomplete tokens. Compared to other methods, the rank-maximal matching algorithm has higher matching efficiency and lower time cost.

Max CAPR: High‐resolution 3D contrast‐enhanced MR angiography with acquisition times under 5 seconds

High temporal and spatial resolution is desired in imaging of vascular abnormalities having short arterial-to-venous transit times. Methods that exploit temporal correlation to reduce the observed frame time demonstrate temporal blurring, obfuscating bolus dynamics. Previously, a Cartesian acquisition with projection reconstruction-like (CAPR) sampling method has been demonstrated for three-dimensional contrast-enhanced angiographic imaging of the lower legs using two-dimensional sensitivity-encoding acceleration and partial Fourier acceleration, providing 1mm isotropic resolution of the calves, with 4.9-sec frame time and 17.6-sec temporal footprint. In this work, the CAPR acquisition is further undersampled to provide a net acceleration approaching 40 by eliminating all view sharing. The tradeoff of frame time and temporal footprint in view sharing is presented and characterized in phantom experiments. It is shown that the resultant 4.9-sec acquisition time, three-dimensional images sets have sufficient spatial and temporal resolution to clearly portray arterial and venous phases of contrast passage. It is further hypothesized that these short temporal footprint sequences provide diagnostic quality images. This is tested and shown in a series of nine contrast-enhanced MR angiography patient studies performed with the new method.