Robust Imaging of Speed of Sound Using Virtual Source Transmission.

Speed of sound (SoS) is a novel imaging biomarker for assessing the biomechanical characteristics of soft tissues. SoS imaging in the pulse-echo mode using conventional ultrasound (US) systems with hand-held transducers has the potential to enable new clinical uses. Recent work demonstrated that diverging waves (DWs) from a single element (SE) transmit to outperform plane-wave sequences. However, SE transmits have severely limited power and hence produce a low signal-to-noise ratio (SNR) in echo data. We herein propose Walsh-Hadamard (WH) coded and virtual-source (VS) transmit sequences for the improved SNR in SoS imaging. We additionally present an iterative method of estimating beamforming (BF) SoS in the medium, which otherwise confounds SoS reconstructions due to beamforming inaccuracies in the images used for reconstruction. Through numerical simulations, phantom experiments, and in vivo imaging data, we show that WH is not robust against motion, which is often unavoidable in clinical imaging scenarios. Our proposed VS sequence is shown to provide the highest SoS reconstruction performance, especially robust to motion artifacts. In phantom experiments, despite having a comparable SoS root-mean-square error (RMSE) of 17.5-18.0 m/s at rest, with a minor axial probe motion of ≈ 0.67 mm/s the RMSE for SE, WH, and VS already deteriorate to 20.2, 105.4, and 19.0 m/s, respectively, showing that WH produces unacceptable results, not robust to motion. In the clinical data, the high SNR and motion resilience of VS sequences are seen to yield superior contrast compared to SE and WH sequences.

Carotid Intima-Media Thickness Percentiles in Adolescence and Young Adulthood and Their Association With Obesity and Hypertensive Blood Pressure in a Population Cohort.

BACKGROUND: This study aimed to derive carotid intima media thickness (CIMT) percentiles from a population-based sample of adolescents and young adults using improved technology, standardization and quality control, and to investigate the association of CIMT with hypertensive blood pressure (BP) and obesity. METHODS: Four thousand seven hundred nine 14- to 28-year-old participants of the German KiGGS cohort 11-year follow-up, which was based on a nationwide population sample, had B-mode ultrasound CIMT measurement with semi-automated edge-detection and automatic ECG-gated real-time quality control. CIMT percentiles were estimated from far wall CIMT during 2 to 6 heart cycles using the GAMLSS statistical model. Hypertensive BP, overweight, obesity, and a risk score from added Z scores of triglycerides, total/HDL (high-density lipoprotein)-cholesterol ratio, and glycated hemoglobin were based on standardized measurements at baseline and follow-up. RESULTS: CIMT differed by sex at all ages, furthermore by age and height in a nonlinear fashion. Percentiles were estimated simultaneously by age and height. Hypertensive BP and obesity were associated cross-sectionally and longitudinally with a higher risk of CIMT ≥75th percentile in log-binomial regression models adjusted for age, sex, height, current smoking, and cardiovascular risk score. For CIMT ≥90th percentile, the relative risk effect estimates were consistently >1 but often had large confidence intervals including 1, largest adjusted relative risk 3.37 (95% CI, 1.41-8.04) for the combination of hypertensive BP and obesity at follow-up. CONCLUSIONS: Based on state-of-the-art measurements and statistical techniques, these population-based CIMT percentiles by sex, age and height add unbiased evidence for the association of subclinical atherosclerosis with hypertensive BP and obesity in the young.

Speed-of-sound imaging using diverging waves.

PURPOSE: Due to its safe, low-cost, portable, and real-time nature, ultrasound is a prominent imaging method in computer-assisted interventions. However, typical B-mode ultrasound images have limited contrast and tissue differentiation capability for several clinical applications. METHODS: Recent introduction of imaging speed-of-sound (SoS) in soft tissues using conventional ultrasound systems and transducers has great potential in clinical translation providing additional imaging contrast, e.g., in intervention planning, navigation, and guidance applications. However, current pulse-echo SoS imaging methods relying on plane wave (PW) sequences are highly prone to aberration effects, therefore suboptimal in image quality. In this paper we propose using diverging waves (DW) for SoS imaging and study this comparatively to PW. RESULTS: We demonstrate wavefront aberration and its effects on the key step of displacement tracking in the SoS reconstruction pipeline, comparatively between PW and DW on a synthetic example. We then present the parameterization sensitivity of both approaches on a set of simulated phantoms. Analyzing SoS imaging performance comparatively indicates that using DW instead of PW, the reconstruction accuracy improves by over 20% in root-mean-square-error (RMSE) and by 42% in contrast-to-noise ratio (CNR). We then demonstrate SoS reconstructions with actual US acquisitions of a breast phantom. With our proposed DW, CNR for a high contrast tumor-representative inclusion is improved by 42%, while for a low contrast cyst-representative inclusion a 2.8-fold improvement is achieved. CONCLUSION: SoS imaging, so far only studied using a plane wave transmission scheme, can be made more reliable and accurate using DW. The high imaging contrast of DW-based SoS imaging will thus facilitate the clinical translation of the method and utilization in computer-assisted interventions such as ultrasound-guided biopsies, where B-Mode contrast is often to low to detect potential lesions.

Carotid IMT and Stiffness in the KiGGS 2 National Survey: Third-Generation Measurement, Quality Algorithms and Determinants of Completeness.

Carotid intima-media thickness (cIMT) and carotid stiffness (CS) are important markers of atherosclerotic risk in the young. We assessed a novel third-generation method for its applicability in large population-based epidemiologic studies to determine strengths, limitations, completeness and predictors of unsuccessful measurement. Four thousand seven hundred ninety-eight 14- to 31-y-old participants of the German KiGGS cohort, which is based on a nationally representative sample with 11-y follow-up, underwent B-mode ultrasound examinations of the left and right common carotid artery with semi-automatic edge detection and automatic electrocardiogram-gated real-time quality control based on a sophisticated snake algorithm and subpixel interpolation. Overall completeness was 98% for far wall cIMT and 89% for CS parameters. Plane-specific completeness varied from 92%-96% for far wall and from 64%-69% for near-wall cIMT. Obesity independently predicted unsuccessful cIMT and CS measurements with odds ratios of 12.67 (95% confidence interval: 5.50-29.19) and 7.30 (4.87-10.94) compared with non-overweight after adjustment for blood pressure, cholesterol, smoking, hazardous drinking, age, sex and sonographer. Inter- and intra-rater reliabilities of cIMT and CS parameters in a sample of 15 young adults were good or excellent. Third-generation cIMT and CS measurements in the young with semi-automatic edge-detection and automatic real-time quality control has been successfully standardized with high reliability and very high completeness in a national survey setting. This provides a strong methodological foundation for further validation of the predictive value of cIMT and CS for atherosclerotic risk in the young.

Frequency-dependent attenuation reconstruction with an acoustic reflector.

Attenuation of ultrasound waves varies with tissue composition, hence its estimation offers great potential for tissue characterization and diagnosis and staging of pathology. We recently proposed a method that allows to spatially reconstruct the distribution of the overall ultrasound attenuation in tissue based on computed tomography, using reflections from a passive acoustic reflector. This requires a standard ultrasound transducer operating in pulse-echo mode and a calibration protocol using water measurements, thus it can be implemented on conventional ultrasound systems with minor adaptations. Herein, we extend this method by additionally estimating and imaging the frequency-dependent nature of local ultrasound attenuation for the first time. Spatial distributions of attenuation coefficient and exponent are reconstructed, enabling an elaborate and expressive tissue-specific characterization. With simulations, we demonstrate that our proposed method yields a low reconstruction error of 0.04 dB/cm at 1 MHz for attenuation coefficient and 0.08 for the frequency exponent. With tissue-mimicking phantoms and ex-vivo bovine muscle samples, a high reconstruction contrast as well as reproducibility are demonstrated. Attenuation exponents of a gelatin-cellulose mixture and an ex-vivo bovine muscle sample were found to be, respectively, 1.4 and 0.5 on average, consistently from different images of their heterogeneous compositions. Such frequency-dependent parametrization could enable novel imaging and diagnostic techniques, as well as facilitate attenuation compensation of other ultrasound-based imaging techniques.

Arabidopsis thaliana FANCD2 Promotes Meiotic Crossover Formation.

Fanconi anemia (FA) is a human autosomal recessive disorder characterized by chromosomal instability, developmental pathologies, predisposition to cancer, and reduced fertility. So far, 19 genes have been implicated in FA, most of them involved in DNA repair. Some are conserved across higher eukaryotes, including plants. The Arabidopsis thaliana genome encodes a homolog of the Fanconi anemia D2 gene (FANCD2) whose function in DNA repair is not yet fully understood. Here, we provide evidence that AtFANCD2 is required for meiotic homologous recombination. Meiosis is a specialized cell division that ensures reduction of genomic content by half and DNA exchange between homologous chromosomes via crossovers (COs) prior to gamete formation. In plants, a mutation in AtFANCD2 results in a 14% reduction of CO numbers. Genetic analysis demonstrated that AtFANCD2 acts in parallel to both MUTS HOMOLOG4 (AtMSH4), known for its role in promoting interfering COs and MMS AND UV SENSITIVE81 (AtMUS81), known for its role in the formation of noninterfering COs. AtFANCD2 promotes noninterfering COs in a MUS81-independent manner and is therefore part of an uncharted meiotic CO-promoting mechanism, in addition to those described previously.

GMI1, a structural-maintenance-of-chromosomes-hinge domain-containing protein, is involved in somatic homologous recombination in Arabidopsis.

DNA double-strand breaks (DSBs) pose one of the most severe threats to genome integrity, potentially leading to cell death. After detection of a DSB, the DNA damage and repair response is initiated and the DSB is repaired by non-homologous end joining and/or homologous recombination. Many components of these processes are still unknown in Arabidopsis thaliana. In this work, we characterized γ-irradiation and mitomycin C induced 1 (GMI1), a member of the SMC-hinge domain-containing protein family. RT-PCR analysis and promoter-GUS fusion studies showed that γ-irradiation, the radio-mimetic drug bleocin, and the DNA cross-linking agent mitomycin C strongly enhance GMI1 expression particularly in meristematic tissues. The induction of GMI1 by γ-irradiation depends on the signalling kinase Ataxia telangiectasia-mutated (ATM) but not on ATM and Rad3-related (ATR). Epistasis analysis of single and double mutants demonstrated that ATM acts upstream of GMI1 while the atr gmi1-2 double mutant was more sensitive than the respective single mutants. Comet assay revealed a reduced rate of DNA double-strand break repair in gmi1 mutants during the early recovery phase after exposure to bleocin. Moreover, the rate of homologous recombination of a reporter construct was strongly reduced in gmi1 mutant plants upon exposure to bleocin or mitomycin C. GMI1 is the first member of its protein family known to be involved in DNA repair.

Cytogenetic map of common bean (Phaseolus vulgaris L.).

A cytogenetic map of common bean was built by in situ hybridization of 35 bacterial artificial chromosomes (BACs) selected with markers mapping to eight linkage groups, plus two plasmids for 5S and 45S ribosomal DNA and one bacteriophage. Together with three previously mapped chromosomes (chromosomes 3, 4, and 7), 43 anchoring points between the genetic map and the cytogenetic map of the species are now available. Furthermore, a subset of four BAC clones was proposed to identify the 11 chromosome pairs of the standard cultivar BAT93. Three of these BACs labelled more than a single chromosome pair, indicating the presence of repetitive DNA in their inserts. A repetitive distribution pattern was observed for most of the BACs; for 38% of them, highly repetitive pericentromeric or subtelomeric signals were observed. These distribution patterns corresponded to pericentromeric and subtelomeric heterochromatin blocks observed with other staining methods. Altogether, the results indicate that around half of the common bean genome is heterochromatic and that genes and repetitive sequences are intermingled in the euchromatin and heterochromatin of the species.

Cytogenetic mapping of common bean chromosomes reveals a less compartmentalized small-genome plant species.

Cytogenetic maps of common bean chromosomes 3, 4 and 7 were constructed by fluorescence in-situ hybridization (FISH) of BAC and a few other genomic clones. Although all clones were selected with genetically mapped markers, mostly with single-copy RFLPs, a large subset of BACs, from 13 different genomic regions, contained repetitive sequences, as concluded from the regional distribution patterns of multiple FISH signals on chromosomes: pericentromeric, subtelomeric and dispersed. Pericentromeric repeats were present in all 11 chromosome pairs with different intensities, whereas subtelomeric repeats were present in several chromosome ends, but with different signal intensities depending on the BAC, suggesting that the terminal heterochromatin fraction of this species may be composed of different repeats. The correlation of genetic and physical distances along the three studied chromosomes was obtained for 23 clones. This correlation suggests suppression of recombination around extended pericentromeric regions in a similar way to that previously reported for plant species with larger genomes. These results indicate that a relatively small plant genome may also possess a large proportion of repeats interspersed with single-copy sequences in regions other than the pericentromeric heterochromatin and, nevertheless, exhibit lower recombination around the pericentromeric fraction of the genome.

Arabidopsis SMG7 protein is required for exit from meiosis.

Meiosis consists of two nuclear divisions that are separated by a short interkinesis. Here we show that the SMG7 protein, which plays an evolutionarily conserved role in nonsense-mediated RNA decay (NMD) in animals and yeast, is essential for the progression from anaphase to telophase in the second meiotic division in Arabidopsis. Arabidopsis SMG7 is an essential gene, the disruption of which causes embryonic lethality. Plants carrying a hypomorphic smg7 mutation exhibit an elevated level of transcripts containing premature stop codons. This suggests that the role of SMG7 in NMD is conserved in plants. Furthermore, hypomorphic smg7 alleles render mutant plants sterile by causing an unusual cell-cycle arrest in anaphase II that is characterized by delayed chromosome decondensation and aberrant rearrangement of the meiotic spindle. The smg7 phenotype was mimicked by exposing meiocytes to the proteasome inhibitor MG115. Together, these data indicate that SMG7 counteracts cyclin-dependent kinase (CDK) activity at the end of meiosis, and reveal a novel link between SMG7 and regulation of the meiotic cell cycle.

Complex rearrangements are involved in Cephalanthera (Orchidaceae) chromosome evolution.

The genus Cephalanthera is an excellent plant group for karyotype evolution studies because it exhibits a dysploid series and bimodal karyotypes. With the aim of understanding their chromosomal and phylogenetic relationships, rRNA genes and the Arabidopsis-type telomeric sequence were mapped by fluorescence in-situ hybridization (FISH), and the rDNA intergenic spacer (ITS) was sequenced for the first time in three European species: C. longifolia (2n = 4x = 32), C. damasonium (2n = 4x = 36) and C. rubra (2n = 4x = 44). One 45S and three 5S rDNA sites are observed in C. longifolia, one 45S and two 5S sites in C. damasonium, and two 45S and one 5S site in C. rubra. Telomeric signals were observed at every chromosome end in all three species and C. damasonium also displays interstitial signals on three chromosome pairs. In agreement with chromosome data, molecular analyses support C. longifolia and C. damasonium as closely related taxa, while C. rubra stands apart. Possible pathways of karyotype evolution are discussed in reference to a previous hypothesis. The results indicate that complex chromosomal rearrangements, possibly involving Robertsonian fusions and fissions, loss of telomeric repeats, gain or loss of rDNA sites and other heterochromatic sequences and inversions, may have contributed to generating the present-day karyotypes.

The nuclear envelope protein Matefin/SUN-1 is required for homologous pairing in C. elegans meiosis.

We identify a highly specific mutation (jf18) in the Caenorhabditis elegans nuclear envelope protein matefin MTF-1/SUN-1 that provides direct evidence for active involvement of the nuclear envelope in homologous chromosome pairing in C. elegans meiosis. The reorganization of chromatin in early meiosis is disrupted in mtf-1/sun-1(jf18) gonads, concomitant with the absence of presynaptic homolog alignment. Synapsis is established precociously and nonhomologously. Wild-type leptotene/zygotene nuclei show patch-like aggregations of the ZYG-12 protein, which fail to develop in mtf-1/sun-1(jf18) mutants. These patches remarkably colocalize with a component of the cis-acting chromosomal pairing center (HIM-8) rather than the centrosome. Our data on this mtf-1/sun-1 allele challenge the previously postulated role of the centrosome/spindle organizing center in chromosome pairing, and clearly support a role for MTF-1/SUN-1 in meiotic chromosome reorganization and in homolog recognition, possibly by mediating local aggregation of the ZYG-12 protein in meiotic nuclei.

The Arabidopsis thaliana MND1 homologue plays a key role in meiotic homologous pairing, synapsis and recombination.

Mnd1 has recently been identified in yeast as a key player in meiotic recombination. Here we describe the identification and functional characterisation of the Arabidopsis homologue, AtMND1, which is essential for male and female meiosis and thus for plant fertility. Although axial elements are formed normally, sister chromatid cohesion is established and recombination initiation appears to be unaffected in mutant plants, chromosomes do not synapse. During meiotic progression, a mass of entangled chromosomes, interconnected by chromatin bridges, and severe chromosome fragmentation are observed. These defects depend on the presence of SPO11-1, a protein that initiates recombination by catalysing DNA double-strand break (DSB) formation. Furthermore, we demonstrate that the AtMND1 protein interacts with AHP2, the Arabidopsis protein closely related to budding yeast Hop2. These data demonstrate that AtMND1 plays a key role in homologous synapsis and in DSB repair during meiotic recombination.

Extensive ribosomal DNA amplification during Andean common bean (Phaseolus vulgaris L.) evolution.

The extent of 5S and 45S ribosomal DNA (rDNA) variation was investigated in wild and domesticated common beans (Phaseolus vulgaris) chosen to represent the known genetic diversity of the species. 5S and 45S rDNA probes were localized on mitotic chromosomes of 37 accessions by fluorescent in situ hybridization (FISH). The two 5S rDNA loci were largely conserved within the species, whereas a high variation in the number of 45S rDNA loci and changes in position of loci and number of repeats per locus were observed. Domesticated accessions from the Mesoamerican gene pool frequently had three 45S rDNA loci per haploid genome, and rarely four. Domesticated accessions from Andean gene pool, particularly from the race Peru, showed six, seven, eight or nine loci, but seven loci were found in all three races of this gene pool. Between three and eight loci were observed in accessions resulting from crosses between Andean and Mesoamerican genotypes. The presence of two to eight 45S rDNA loci in wild common beans from different geographic locations indicates that the 45S rDNA amplification observed in the Andean lineage took place before domestication. Our data suggest that ectopic recombination between terminal chromosomal regions might be the mechanism responsible for this variation.

PhyloDome--visualization of taxonomic distributions of domains occurring in eukaryote protein sequence sets.

The analysis of taxonomic distribution and lineage-specific variation of domains and domain combinations is an important step in the assessment of their functional roles and potential interoperability. In the study of eukaryote sequence sets with many multi-domain proteins, it can become laborious to evaluate the phylogenetic context of the many occurring domains and their mutual relationships. PhyloDome is an answer to that problem. It provides a fast overview on the taxonomic spreading and potential interrelation of domains that are either given as a list of names and PFAM/SMART accessions or derived from a user-defined set of sequences. This taxonomic distribution analysis can be helpful in protein function and interaction assignment as the comparative study of potential Hedgehog pathway members in C.elegans shows. An implementation of PhyloDome is accessible for public use as a WWW-Service at http://mendel.imp.univie.ac.at/phylodome/. Software components are available on request.

Targeted gene knockout reveals a role in meiotic recombination for ZHP-3, a Zip3-related protein in Caenorhabditis elegans.

The meiotically expressed Zip3 protein is found conserved from Saccharomyces cerevisiae to humans. In baker's yeast, Zip3p has been implicated in synaptonemal complex (SC) formation, while little is known about the protein's function in multicellular organisms. We report here the successful targeted gene disruption of zhp-3 (K02B12.8), the ZIP3 homolog in the nematode Caenorhabditis elegans. Homozygous zhp-3 knockout worms show normal homologue pairing and SC formation. Also, the timing of appearance and the nuclear localization of the recombination protein Rad-51 seem normal in these animals, suggesting proper initiation of meiotic recombination by DNA double-strand breaks. However, the occurrence of univalents during diplotene indicates that C. elegans ZHP-3 protein is essential for reciprocal recombination between homologous chromosomes and thus chiasma formation. In the absence of ZHP-3, reciprocal recombination is abolished and double-strand breaks seem to be repaired via alternative pathways, leading to achiasmatic chromosomes and the occurrence of univalents during meiosis I. Green fluorescent protein-tagged C. elegans ZHP-3 forms lines between synapsed chromosomes and requires the SC for its proper localization.

Mre11 deficiency in Arabidopsis is associated with chromosomal instability in somatic cells and Spo11-dependent genome fragmentation during meiosis.

The Mre11/Rad50/Nbs1 complex is involved in many aspects of chromosome metabolism. Aberrant function of the complex is associated with defects in the DNA checkpoint, double-strand break repair, meiosis, and telomere maintenance. In this article, we report the consequences of Mre11 dysfunction for the stability of mitotic and meiotic chromosomes in Arabidopsis thaliana. Although plants homozygous for a T-DNA insertion in a conserved region of the MRE11 gene are viable, they exhibit growth defects and are infertile. Analysis of mitotic chromosomes prepared from the mutant plants revealed abundant dicentric chromosomes and chromosomal fragments. Fluorescence in situ hybridization showed that anaphase bridges are often formed by homologous chromosome arms. The frequency of chromosome fusions was not reduced in mre11 ku70 double mutants, suggesting that plants possess DNA end-joining activities independent of the Ku70/80 and Mre11 complexes. Cytogenetic examination of pollen mother cells revealed massive chromosome fragmentation and the absence of synapsis in the initial stages of meiosis. The fragmentation was substantially suppressed in mre11 spo11-1 double mutants, indicating that Mre11 is required for repair but not for the induction of Spo11-dependent meiotic DNA breaks in Arabidopsis.

Chromosome termini of the monocot plant Othocallis siberica are maintained by telomerase, which specifically synthesises vertebrate-type telomere sequences.

Lack of Arabidopsis-type T3AG3 telomere sequences has recently been reported for the majority of investigated taxa of the monocot order Asparagales. In order to investigate this phenomenon in more detail, we conducted extensive cytogenetic and molecular analyses of the telomeres in Othocallis siberica, a member of this order. Terminal restriction fragment analysis together with Bal31 exonuclease assay showed that chromosome termini in O. siberica are formed by long stretches (more than 10 kbp) of vertebrate-type T2AG3 repeats. In addition, telomerase activity specifically synthesising (T2AG3)n sequence was detected in O. siberica protein extracts by telomerase repeat amplification protocol (TRAP). Fluorescence in situ hybridisation (FISH) revealed the presence of the vertebrate-type T2AG3 telomere sequences at all chromosome termini and at a few additional regions of O. siberica chromosomes, whereas Arabidopsis-type T3AG3 DNA and peptide nucleic acid (PNA) probes did not hybridise to chromosomes of Othocallis, except for polymorphic blocks in chromosomes 2 (interstitial) and 4 (terminal). These interstitial/terminal regions are apparently composed of large blocks of (T2AG3)n and (T3AG3)n DNA and represent a unique example of interspersion of two types of telomeric repeats within one genome. This may be a reflection of the recent evolutionary switch from Arabidopsis- to vertebrate-type telomeric repeats in this plant group.

Karyotype analysis in Hyacinthella dalmatica (Hyacinthaceae) reveals vertebrate-type telomere repeats at the chromosome ends.

Chromosome analysis of three different populations of Hyacinthella dalmatica (Lallem.) Trinajstic, an endemic species of the coastal region of southeastern Europe, showed a unique chromosome number, 2n = 2x = 20, and bimodal karyotype with one large and nine smaller pairs of chromosomes. Staining with fluorochromes CMA3 (chromomycin A3) and DAPI (4,6-diamidino-2-phenylindole) revealed heterochromatic regions associated with NORs, centromeres, and several interstitial heterochromatic bands on the longest chromosome pair. Double-target FISH with two ribosomal DNA probes revealed one locus of 5S rRNA genes in the pericentromeric region of chromosome pair 3 and one locus of 18S-5.8S-26S rRNA genes on the short arm of chromosome pair 4 in all plants and populations analyzed. Southern hybridization analysis and FISH experiments demonstrated that the distal ends of H. dalmatica chromosomes contain the vertebrate telomere (5'-TTAGGG-3') repeat type rather than the Arabidopsis (5'-TTTAGGG-3') heptamer, and so suggest that this Asparagales species along with Aloe and Othocallis contains the vertebrate-type telomere repeat.