Foam gratings as an alternative to customized acoustic lenses.

This article describes a method of manipulating acoustic fields using transmission through foam gratings. The approach is investigated with an analytical model, a numerical model simulating full wave ultrasound propagation through the gratings, and experimental measurements. A grating is demonstrated that mimics a conventional ultrasound lens, modulating the phase of transmitted ultrasound while maximizing the transmitted amplitude. The performance of a foam grating is compared to a lens made of polydimethylsiloxane or three-dimensional printed resin. Using two gratings, independent control of amplitude and phase is demonstrated, with increased insertion loss. The primary advantages of this technique over conventional lenses are very rapid manufacture (<30 min), high repeatability due to the simplicity of manufacture, and the ability to control the amplitude of the transmitted ultrasound. Potential applications include generation of complex ultrasound fields for patient specific treatments in ultrasound therapy.

Concussion, microvascular injury, and early tauopathy in young athletes after impact head injury and an impact concussion mouse model.

The mechanisms underpinning concussion, traumatic brain injury, and chronic traumatic encephalopathy, and the relationships between these disorders, are poorly understood. We examined post-mortem brains from teenage athletes in the acute-subacute period after mild closed-head impact injury and found astrocytosis, myelinated axonopathy, microvascular injury, perivascular neuroinflammation, and phosphorylated tau protein pathology. To investigate causal mechanisms, we developed a mouse model of lateral closed-head impact injury that uses momentum transfer to induce traumatic head acceleration. Unanaesthetized mice subjected to unilateral impact exhibited abrupt onset, transient course, and rapid resolution of a concussion-like syndrome characterized by altered arousal, contralateral hemiparesis, truncal ataxia, locomotor and balance impairments, and neurobehavioural deficits. Experimental impact injury was associated with axonopathy, blood-brain barrier disruption, astrocytosis, microgliosis (with activation of triggering receptor expressed on myeloid cells, TREM2), monocyte infiltration, and phosphorylated tauopathy in cerebral cortex ipsilateral and subjacent to impact. Phosphorylated tauopathy was detected in ipsilateral axons by 24 h, bilateral axons and soma by 2 weeks, and distant cortex bilaterally at 5.5 months post-injury. Impact pathologies co-localized with serum albumin extravasation in the brain that was diagnostically detectable in living mice by dynamic contrast-enhanced MRI. These pathologies were also accompanied by early, persistent, and bilateral impairment in axonal conduction velocity in the hippocampus and defective long-term potentiation of synaptic neurotransmission in the medial prefrontal cortex, brain regions distant from acute brain injury. Surprisingly, acute neurobehavioural deficits at the time of injury did not correlate with blood-brain barrier disruption, microgliosis, neuroinflammation, phosphorylated tauopathy, or electrophysiological dysfunction. Furthermore, concussion-like deficits were observed after impact injury, but not after blast exposure under experimental conditions matched for head kinematics. Computational modelling showed that impact injury generated focal point loading on the head and seven-fold greater peak shear stress in the brain compared to blast exposure. Moreover, intracerebral shear stress peaked before onset of gross head motion. By comparison, blast induced distributed force loading on the head and diffuse, lower magnitude shear stress in the brain. We conclude that force loading mechanics at the time of injury shape acute neurobehavioural responses, structural brain damage, and neuropathological sequelae triggered by neurotrauma. These results indicate that closed-head impact injuries, independent of concussive signs, can induce traumatic brain injury as well as early pathologies and functional sequelae associated with chronic traumatic encephalopathy. These results also shed light on the origins of concussion and relationship to traumatic brain injury and its aftermath.awx350media15713427811001.

Response of Single Cells to Shock Waves and Numerically Optimized Waveforms for Cancer Therapy.

Shock waves are used clinically for breaking kidney stones and treating musculoskeletal indications. The mechanisms by which shock waves interact with tissue are still not well understood. Here, ultra-high-speed imaging was used to visualize the deformation of individual cells embedded in a tissue-mimicking phantom when subject to shock-wave exposure from a clinical source. Three kidney epithelial cell lines were considered to represent normal healthy (human renal epithelial), cancer (CAKI-2), and virus-transformed (HK-2) cells. The experimental results showed that during the compressive phase of the shock waves, there was a small (<2%) decrease in the projected cell area, but during the tensile phase, there was a relatively large (∼10%) increase in the projected cell area. The experimental observations were captured by a numerical model with a constitutive material framework consisting of an equation of state for the volumetric response and hyper-viscoelasticity for the deviatoric response. To model the volumetric cell response, it was necessary to change from a higher bulk modulus during the compression to a lower bulk modulus during the tensile shock loading. It was discovered that cancer cells showed a smaller deformation but faster response to the shock-wave tensile phase compared to their noncancerous counterparts. Cell viability experiments, however, showed that cancer cells suffered more damage than other cell types. These data suggest that the cell response to shock waves is specific to the type of cell and waveforms that could be tailored to an application. For example, the model predicts that a shock wave with a tensile stress of 4.59 MPa would increase cell membrane permeability for cancer cells with minimal impact on normal cells.

On measuring the acoustic state changes in lipid membranes using fluorescent probes.

Ultrasound is increasingly being used to modulate the properties of biological membranes for applications in drug delivery and neuromodulation. While various studies have investigated the mechanical aspects of the interaction such as acoustic absorption and membrane deformation, it is not clear how these effects transduce into biological functions, for example, changes in the permeability or the enzymatic activity of the membrane. A critical aspect of the activity of an enzyme is the thermal fluctuations of its solvation or hydration shell. Thermal fluctuations are also known to be directly related to membrane permeability. Here solvation shell changes of lipid membranes subject to an acoustic impulse were investigated using a fluorescence probe, Laurdan. Laurdan was embedded in multi-lamellar lipid vesicles in water, which were exposed to broadband pressure impulses of the order of 1 MPa peak amplitude and 10 µs pulse duration. An instrument was developed to monitor changes in the emission spectrum of the dye at two wavelengths with sub-microsecond temporal resolution. The experiments show that changes in the emission spectrum, and hence the fluctuations of the solvation shell, are related to the changes in the thermodynamic state of the membrane and correlated with the compression and rarefaction of the incident sound wave. The results suggest that acoustic fields affect the state of a lipid membrane and therefore can potentially modulate the kinetics of channels and enzymes embedded in the membrane.

Spatial filters suppress ripple artifacts in the computation of acoustic fields with the angular spectrum method.

The angular spectrum method (ASM) is an effective tool for propagating wave fields between parallel planes through decomposition of the field into a series of independent plane waves. One source of error is interference from mirror sources introduced through the inherent periodicity of the fast Fourier transform (FFT) used to implement this method numerically. Here, spatial filters attenuate waves propagating at large angles, which are sensitive to mirror sources. Simulations show that this suppresses the ripple artifact whilst preserving the accuracy of the ASM-computed fields. To achieve comparable performance without filtering requires up to a 13.5-fold increase in computation time.

Promoting improved social support and quality of life with the CenteringPregnancy® group model of prenatal care.

This prospective cohort study compared women participating in CenteringPregnancy® group prenatal care (N = 120) with those in standard individual care (N = 221) to determine if participation in Centering was associated with improvements in perceived social support and quality of life, with concomitant decreases in screens of postpartum depression and improvements in breastfeeding rates. Participants completed surveys at the onset of prenatal care, in the late third trimester and in the postpartum period. Centering participants had higher scores of perceived social support from friends after participating in group care (p < 0.05) with associated improvements in quality of life in the psychological and relational domains (p < 0.05) compared to standard care participants who showed higher scores of perceived support from family (p < 0.05) but did not show concomitant improvements in quality of life. This did not translate to any significant difference in scores on postpartum depression screens but was associated with improvements in breastfeeding continuation rates among Centering participants in the postpartum period. This study indicates that Centering care is associated with improved perceptions of peer social support with associated improvements in quality of life and higher rates of continued breastfeeding.

Tracking kidney stones in a homogeneous medium using a trilateration approach.

Shock wave lithotripsy is a non-invasive procedure by which kidney stones are fragmented by thousands of shock waves. Currently, many shock waves are delivered to the body that do not impact the stone, but do result in tissue trauma. This motivates developing a monitoring system to locate kidney stones, with the goal of gating shock waves not aligned with the stone, and hence, reducing renal trauma during lithotripsy. The system consists of a circular array housing twenty-two 0.5 MHz transducers that can be mounted on a clinical lithotripter. It was deployed in a water tank and tested with two stone models made from gypsum cement and a stone model fragment. An algorithm consisting of threshold detection, automatic rejection of weak signals, and triangulation was developed to determine the location of stones. The results show that within ±15 mm of the focus of the lithotripter, the accuracy was better than 4 mm in the lateral directions and 2 mm in the axial direction. Using off-the-shelf hardware, the algorithm can calculate stone positions every 1 s allowing for real-time tracking during lithotripsy.

Simulation of nonlinear propagation of biomedical ultrasound using pzflex and the Khokhlov-Zabolotskaya-Kuznetsov Texas code.

Nonlinear acoustics plays an important role in both diagnostic and therapeutic applications of biomedical ultrasound and a number of research and commercial software packages are available. In this manuscript, predictions of two solvers available in a commercial software package, pzflex, one using the finite-element-method (FEM) and the other a pseudo-spectral method, spectralflex, are compared with measurements and the Khokhlov-Zabolotskaya-Kuznetsov (KZK) Texas code (a finite-difference time-domain algorithm). The pzflex methods solve the continuity equation, momentum equation and equation of state where they account for nonlinearity to second order whereas the KZK code solves a nonlinear wave equation with a paraxial approximation for diffraction. Measurements of the field from a single element 3.3 MHz focused transducer were compared with the simulations and there was good agreement for the fundamental frequency and the harmonics; however the FEM pzflex solver incurred a high computational cost to achieve equivalent accuracy. In addition, pzflex results exhibited non-physical oscillations in the spatial distribution of harmonics when the amplitudes were relatively low. It was found that spectralflex was able to accurately capture the nonlinear fields at reasonable computational cost. These results emphasize the need to benchmark nonlinear simulations before using codes as predictive tools.

Monodisperse Micro-Oil Droplets Stabilized by Polymerizable Phospholipid Coatings as Potential Drug Carriers.

There is a critical need to formulate stable micron-sized oil droplets as hydrophobic drug carriers for efficient drug encapsulation, long-term storage, and sustained drug release. Microfluidic methods were developed to maximize the stability of micron-sized, oil-in-water (o/w) emulsions for potential use in drug delivery, using doxorubicin-loaded triacetin oil as a model hydrophobic drug formulation. Initial experiments examined multiple flow conditions for the dispersed (oil) and continuous (liposome aqueous) phases in a microfluidic device to establish the parameters that influenced droplet size. These data were fit to a mathematical model from the literature and indicate that the droplet sizes formed are controlled by the ratio of flow rates and the height of the device channel, rather than the orifice size. Next, we investigated effects of o/w emulsion production methods on the stability of the droplets. The stability of o/w emulsion produced by microfluidic flow-focusing techniques was found to be much greater (5 h vs 1 h) than for emulsions produced by mechanical agitation (vortexing). The increased droplet stability was attributed to the uniform size and lipid distribution of droplets generated by flow-focusing. In contrast, vortexed populations consisted of a wide size distribution that resulted in a higher prevalence of Ostwald ripening. Finally, the effects of shell polymerization on stability were investigated by comparing oil droplets encapsulated by a photopolymerizable diacetylene lipid shell to those with a nonpolymerizable lipid shell. Shell polymerization was found to significantly enhance stability against dissolution for flow-focused oil droplets but did not significantly affect the stability of vortexed droplets. Overall, results of these experiments show that flow-focusing is a promising technique for generating tunable, stable, monodisperse oil droplet emulsions, with potential applications for controlled delivery of hydrophobic drug formulations.

Stereotypical alterations in cortical patterning are associated with maternal illness-induced placental dysfunction.

We have previously shown in mice that cytokine-mediated damage to the placenta can temporarily limit the flow of nutrients and oxygen to the fetus. The placental vulnerability is pronounced before embryonic day 11, when even mild immune challenge results in fetal loss. As gestation progresses, the placenta becomes increasingly resilient to maternal inflammation, but there is a narrow window in gestation when the placenta is still vulnerable to immune challenge yet resistant enough to allow for fetal survival. This gestational window correlates with early cortical neurogenesis in the fetal brain. Here, we show that maternal illness during this period selectively alters the abundance and laminar positioning of neuronal subtypes influenced by the Tbr1, Satb2, and Ctip2/Fezf2 patterning axis. The disturbances also lead to a laminar imbalance in the proportions of projection neurons and interneurons in the adult and are sufficient to cause changes in social behavior and cognition. These data illustrate how the timing of an illness-related placental vulnerability causes developmental alterations in neuroanatomical systems and behaviors that are relevant to autism spectrum disorders.

Rheological properties of porcine organs: measurements and fractional viscoelastic model.

The rheological properties of porcine heart, kidney, liver and brain were measured using dynamic oscillatory shear tests over a range of frequencies and shear strains. Frequency sweep tests were performed from 0.1 Hz to a maximum of 9.5 Hz at a shear strain of 0.1%, and strain sweep tests were carried out from 0.01% to 10% at 1 Hz. The effect of pre-compression of samples up to 10% axial strain was considered. The experimental measurements were fit to a Semi-Fractional Kelvin Voight (S-FKV) model. The model was then used to predict the stress relaxation in response to a step strain of 0.1%. The prediction was compared to experimental relaxation data for the porcine organ samples, and the results agreed to within 30%. In conclusion, this study measured the rheological properties of porcine organs and used a fractional viscoelastic model to describe the response in frequency and time domain.

Biomechanical Modelling of Porcine Kidney.

In this study, the viscoelastic properties of porcine kidney in the upper, middle and lower poles were investigated using oscillatory shear tests. The viscoelastic properties were extracted in the form of the storage modulus and loss modulus in the frequency and time domain. Measurements were taken as a function of frequency from 0.1 Hz to 6.5 Hz at a shear strain amplitude of 0.01 and as function of strain amplitude from 0.001 to 0.1 at a frequency of 1 Hz. Measurements were also taken in the time domain in response to a step shear strain. Both the frequency and time domain data were fitted to a conventional Standard Linear Solid (SLS) model and a semi-fractional Kelvin-Voigt (SFKV) model with a comparable number of parameters. The SFKV model fitted the frequency and time domain data with a correlation coefficient of 0.99. Although the SLS model well fitted the time domain data and the storage modulus data in the frequency domain, it was not able to capture the variation in loss modulus with frequency with a correlation coefficient of 0.53. A five parameter Maxwell-Wiechert model was able to capture the frequency dependence in storage modulus and loss modulus better than the SLS model with a correlation of 0.85.

Agarose as a Tissue Mimic for the Porcine Heart, Kidney, and Liver: Measurements and a Springpot Model.

Agarose gels are often used as a tissue mimic. The goal of this work was to determine the appropriate agarose concentrations that result in mechanical properties that match three different porcine organs. Strain tests were carried out with an amplitude varying from 0.01% to 10% at a frequency of 1 Hz on a range of agarose concentrations and porcine organs. Frequency sweep tests were performed from 0.1 Hz to a maximum of 9.5 Hz at a shear strain amplitude of 0.1% for agarose and porcine organs. In agarose samples, the effect of pre-compression of the samples up to 10% axial strain was considered during frequency sweep tests. The experimental measurements from agarose samples were fit to a fractional order viscoelastic (springpot) model. The model was then used to predict stress relaxation in response to a step strain of 0.1%. The prediction was compared to experimental relaxation data, and the results agreed within 12%. The agarose concentrations (by mass) that gave the best fit were 0.25% for the liver, 0.3% for the kidney, and 0.4% for the heart. At a frequency of 0.1 Hz and a shear strain of 0.1%, the agarose concentrations that best matched the shear storage modulus of the porcine organs were 0.4% agarose for the heart, 0.3% agarose for the kidney, and 0.25% agarose for the liver.

Ph. Eur. testing for histamine and depressor substances using guinea-pigs and cats: the end of an era. Strategy for removal of animal tests for histamine and depressor substances and their vestiges from the Ph. Eur.

For more than 50 years, in vivo assays have been used for testing pharmaceutical product safety due to their assumed ability to broadly detect potential unidentified contaminants. As part of these in vivo tests, the animal tests for depressor substances and histamine have been described in the European Pharmacopoeia since its first edition in 1977. Both tests measure the effect of histamine and histamine-like substances, using guinea-pigs and cats respectively. In 2024, the Histamine (2.6.10) general chapter is referenced in the Production section of four monographs and 10 monographs have variations of a sentence on designing the manufacturing process to eliminate or minimise substances lowering blood pressure in this same section, without referencing the chapter. The Depressor substances (2.6.11) chapter is referenced only in the Histamine (2.6.10) chapter as a next step if the histamine test is invalid. A historical search was performed and it has shown that the tests for histamine and for depressor substances were introduced by different groups of experts in an inconsistent way at different times, and for different reasons, leading to non-harmonised approaches across monographs. The control of histamine and other depressor substances has been the subject of numerous debates where their use was questioned. During these discussions, reports on positive cases or batches failing the test for histamine or depressor substances were anecdotal. In addition, in vivo tests can be considered non-specific, very variable, time-consuming, costly and ethically doubtful. More importantly, the majority of in vivo methods originate from a time when good manufacturing practice was not widely used and formal method validation requirements were not yet established. In view of the above, the removal of all references to animal tests for histamine or depressor substances from all texts still referring to them is proposed. Since the sentences in the Production section referring to the control of "substances lowering blood pressure", "vasoactive substances" or "hypotensive substances" appeared as remainders of the animal test without further guarantee of safety, it will also be proposed to remove all these sentences from the concerned monographs. Ultimately, the suppression of general chapters 2.6.10 and 2.6.11 from the Ph. Eur. is envisaged. Independently from the above, it is also envisaged to elaborate a new general chapter Histamine in active substances (2.5.47) to include physicochemical or immunochemical methods enabling the detection of histamine. This new text would aim at supporting manufacturers in their histamine control strategy following the inclusion of precaution statements in the general monograph on Products of fermentation (1468); these statements had been added in Ph. Eur. Supplements 9.6 and 10.4, following adverse events related to a GMP issue with gentamicin sulfate. This strategy has been endorsed by the European Pharmacopoeia Commission at its 177th Session in November 2023. The concerned monographs would be a subject of public consultation in Pharmeuropa 36.2 (April 2024).

Quantitative evaluation of anti-biofilm cavitation activity seeded from microbubbles or protein cavitation nuclei by passive acoustic mapping.

OBJECTIVE: Bacterial biofilms represent a major challenge for effective antibiotic therapy as they confer physical and functional changes that protect bacteria from their surrounding environment. In this work, focused ultrasound in combination with cavitation nuclei was used to disrupt biofilms of Staphylococcus aureus and Pseudomonas aeruginosa, both of which are on the World Health Organization's priority list for new antimicrobial research. Approach: Single species biofilms were exposed to ultrasound (0.5 MHz centre frequency, 0.5-1.5 MPa peak rarefactional pressure, 200 cycle pulses, 5 Hz repetition frequency, 30 s duration), in the presence of two different types of cavitation nuclei. Quantitative passive acoustic mapping (PAM) was used to monitor cavitation emissions during treatment using a calibrated linear array. Main Results: It was observed that the cumulative energy of acoustic emissions during treatment was positively correlated with biofilm disruption, with differences between bacterial species attributed to differences in biofilm morphology. PCaN provided increased biofilm reduction compared to microbubbles due in large part to their persistence over the duration of ultrasound exposure. There was also good correlation between the spatial distribution of cavitation as characterized by PAM and the extent of biofilm disruption observed with microscopy. Significance: Collectively, the results from this work indicate the potential broad applicability of cavitation for eliminating biofilms of priority pathogens and the opportunity presented by Passive Acoustic Mapping for real-time monitoring of antimicrobial processes.

Single-shot measurements of the acoustic field of an electrohydraulic lithotripter using a hydrophone array.

Piezopolymer-based hydrophone arrays consisting of 20 elements were fabricated and tested for use in measuring the acoustic field from a shock-wave lithotripter. The arrays were fabricated from piezopolymer films and were mounted in a housing to allow submersion into water. The motivation was to use the array to determine how the shot-to-shot variability of the spark discharge in an electrohydraulic lithotripter affects the resulting focused acoustic field. It was found that the dominant effect of shot-to-shot variability was to laterally shift the location of the focus by up to 5 mm from the nominal acoustic axis of the lithotripter. The effect was more pronounced when the spark discharge was initiated with higher voltages. The lateral beamwidth of individual, instantaneous shock waves were observed to range from 1.5 mm to 24 mm. Due to the spatial variation of the acoustic field, the average of instantaneous beamwidths were observed to be 1 to 2 mm narrower than beamwidths determined from traditional single-point measurements that average the pressure measured at each location before computing beamwidth.

Immunocontraception of captive exotic species: V. Prolonged antibody titers in Dall sheep (Ovis dalli dalli) and domestic goats (Capra hircus) immunized with porcine zona pellucida.

Native porcine zona pellucida (PZP) immunocontraception has been used to inhibit fertility in more than 80 species of ungulates, although the duration of contraception efficacy varies among species in both Perissodactyla and Artiodactyla. This study examined anti-PZP antibody titers in Dall sheep and domestic goats at the Milwaukee County Zoo, and also Himalayan tahr and Armenian Mouflon sheep at the San Diego Zoo Safari Park, and, for comparison, Altai wapiti, lowland wisent, Javan banteng, and southern pudu at the San Diego Zoo Safari Park, all were given a primer dose and booster dose of PZP. Of the San Diego Zoo Safari Park animals, the 4 comparison species demonstrated the typical 1-yr pattern of anti-PZP antibodies, whereas the Armenian sheep and Himalayan tahr showed prolonged (2-3 yr) antibody responses after a single primer and booster dose. The Dall sheep and domestic goats had significantly longer durations of antibody titers (3 yr) from a single year's treatment (primer plus booster). Analysis of the data indicates that Armenian sheep, Himalayan tahr, Dall sheep, and domestic goats have prolonged responses, and are more sensitive to PZP in that they produce a protracted antibody response.

Informal investigation on the added value of a potential certification system for the qualification of raw materials for the production of ATMPs.

The manufacture of advanced therapy medicinal products (ATMPs) is critically impacted by the quality of the raw materials (RMs) used. Following the need expressed by stakeholders to establish a certification scheme for biological RMs used in the manufacture of ATMPs, the European Pharmacopoeia (Ph. Eur.) Cell Therapy Products Working Party (CTP WP) conducted an informal investigation with the aim of issuing a technical opinion on the feasibility of such a certification scheme. Seven RM Drug Master Files were reviewed for compliance of the RM with Ph. Eur. general chapter 5.2.12. Raw materials of biological origin for the production of cell-based and gene therapy medicinal products by members of the CTP WP who were representatives of competent authorities and experienced in the evaluation of RMs for ATMPs. This article presents the results of these case studies, including the potential benefits and concerns identified by the experts. It was concluded that it would be technically feasible, albeit challenging, to set up a certification system for RMs of biological origin against chapter 5.2.12. Although the establishment of such a scheme is currently perceived by some CTP WP members as premature, it could potentially be beneficial for all stakeholders (RM manufacturers, ATMP manufacturers and assessors).

Tunable diacetylene polymerized shell microbubbles as ultrasound contrast agents.

Monodisperse gas microbubbles, encapsulated with a shell of photopolymerizable diacetylene lipids and phospholipids, were produced by microfluidic flow focusing, for use as ultrasound contrast agents. The stability of the polymerized shell microbubbles against both aggregation and gas dissolution under physiological conditions was studied. Polyethylene glycol (PEG) 5000, which was attached to the diacetylene lipids, was predicted by molecular theory to provide more steric hindrance against aggregation than PEG 2000, and this was confirmed experimentally. The polymerized shell microbubbles were found to have higher shell-resistance than nonpolymerizable shell microbubbles and commercially available microbubbles (Vevo MicroMarker). The acoustic stability under 7.5 MHz ultrasound insonation was significantly greater than that for the two comparison microbubbles. The acoustic stability was tunable by varying the amount of diacetylene lipid. Thus, our polymerized shell microbubbles are a promising platform for ultrasound contrast agents.

Swarm of terminal 35S in Cheirolophus (Asteraceae, Centaureinae).

Island radiation constitutes a playground for species diversification, which has long fascinated researchers and still does today. Because only a small subset of taxa within the pool of island colonizers is concerned by this process, the question is raised on whether some factors could make a taxon prone to radiate. Cheirolophus is the only genus of Centaureinae subtribe to have experienced a radiation in the Canary Islands. Cytogenetic characterization through FISH of 5S and 35S ribosomal RNA genes in eight Cheirolophus species from continent and Canary Islands revealed an unusually high number of 35S predominantly at terminal position, together with a single interstitial 5S rDNA locus in all the studied taxa. Such an abundance of 35S rDNA signals is unique among Centaureinae and predates Cheirolophus arrival in Canary Islands. The possible link of the rDNA profile with radiation process is discussed through a comparison with two other case studies, the closely related Rhaponticum group and the genus Centaurea.