Ultraharmonic VUS Imaging of Microvascularization

The coronary arteries are major blood vessels branching along the heart surface to convey nutrients and oxygen carried in the blood to the heart muscle cells. In turn, the heart ensures the perpetual transportation of blood throughout the other organs of the circulatory system. Coronary arteries comprise the right and left coronary artery. Both originate from the root of the aorta. The left main coronary artery (LCA) gives off the left circumflex artery (LCX) and continues its descent as the left anterior descending coronary artery (LAD). The LCX runs around the left border of the heart to reach the posterior surface where it supplies the left ventricle muscle. The LAD and its branches supply two thirds of the anterior heart surface. The right coronary artery (RCA) descends along the border of the right atrium and the right ventricle to supply the right ventricle muscle. A simplified representation of the coronary artery anatomy is represented in Figure 1

Monitoring the complex benthic habitat on semi-dark underwater marine caves using photogrammetry-based 3D reconstructions

Marine caves are dark environments considered a priority habitat for conservation included in the EU Habitats Directive (H8330). They harbor fragile benthic communities and represent a major reservoir of marine biodiversity. However, there is a lack of knowledge of these habitats due to the difficulties of creating detailed benthic maps and characterizing the biodiversity, structure, and dynamics of their communities. The uniqueness of marine caves fosters their popularity among recreational divers, who can cause disturbances through abrasion of the biota, resuspension of sediment, and accumulation of exhaled air bubbles in the caves' ceilings. This study aims to build a monitoring framework to characterize the structure and temporal dynamics of this complex habitat using Structurefrom- Motion (SfM) photogrammetry. SfM is a novel, non-invasive technique that allows a major advancement in the monitoring of changes in the cave’s community assemblages. This method relies on images acquired by 4K video footage to build fine-scaled 3D digital models of the substrate using overlapping imagery. For this study, we combined SfM photogrammetry and photo quadrats extracted from the video recordings. We evaluate the effectiveness of this methodology in a marine cave highly frequented by divers, located in Illa de l’Aire (Balearic Islands, Spain), and carried out two surveys before and after the diving season (2019-2021). As a result, we found a loss of 25 colonies of bryozoans with fragile skeletons, like Schizoretepora sp., and 8 individual sponges with globose morphotypes. Our results indicate that this methodology enables accurate and efficient monitoring of benthic communities in underwater caves that allow us to better understand their dynamics and, therefore, to develop the need management measures

Negative DNA supercoiling induces genome-wide Cas9 off-target activity

CRISPR-Cas9 is a powerful gene-editing technology; however, off-target activity remains an important consideration for therapeutic applications. We have previously shown that force-stretching DNA induces off-target activity and hypothesized that distortions of the DNA topology in vivo, such as negative DNA supercoiling, could reduce Cas9 specificity. Using single-molecule optical-tweezers, we demonstrate that negative supercoiling λ-DNA induces sequence-specific Cas9 off-target binding at multiple sites, even at low forces. Using an adapted CIRCLE-seq approach, we detect over 10,000 negative-supercoiling-induced Cas9 off-target double-strand breaks genome-wide caused by increased mismatch tolerance. We further demonstrate in vivo that directed local DNA distortion increases off-target activity in cells and that induced off-target events can be detected during Cas9 genome editing. These data demonstrate that Cas9 off-target activity is regulated by DNA topology in vitro and in vivo, suggesting that cellular processes, such as transcription and replication, could induce off-target activity at previously overlooked sites

MAPPING COMMUNITY INTEREST HABITATS IN THE COLUMBRETES ARCHIPELAGO, AN EXTRAORDINARY HOT SPOT OF BIODIVERSITY

The Columbretes Archipelago and their submerged surroundings are part of an unusual, Pleistocene volcanic field located in the Western Mediterranean designated as a Site of Community Importance (SCI) of the Natura 2000 Network. In the present study, 4 benthic habitats of community interest (1110, 1170, 1180 and 8330) have been identified by analyzing several sources of information. Generalized additive models (GAMs) have been used to model the potential distribution of reefs (1170) and maërl beds (1110). Our results highlight the diversity and extent of these habitats and allow comparisons to other marine SCIs of Spain. This can be attributed to the variability of the environment of this site. The Columbretes Islands combine a relatively shallow environment with volcanic structures, hydrothermalism with active degassing, current-driven sedimentary lobes and the influence of inland flows. Understanding high biodiversity spots is crucial as they offer natural laboratories to describe how ecosystems respond to the effects of global change. The knowledge obtained will be of paramount importance for the conservation of species and habitats. Furthermore, it will establish a baseline for future monitoring and assist in the development of effective management plans

Imaging microvasculature with contrast-enhanced ultraharmonic ultrasound

Atherosclerotic plaque neovascularization was shown to be one of the strongest predictors of future cardiovascular events. Yet, the clinical tools for coronary wall microvasculature detection invivo are lacking. Here we report an ultrasound pulse sequence capable of detecting microvasculature invisible in conventional intracoronary imaging. The method combines intravascular ultrasound with an ultrasound contrast agent, i.e., a suspension of microscopic vascular acoustic resonators that are small enough to penetrate the capillary bed after intravenous administration. The pulse sequence relies on brief chirp excitations to extract ultraharmonic echoes specific to the ultrasound contrast agent. We implemented the pulse sequence on an intravascular ultrasound probe and successfully imaged the microvasculature of a 6 days old chicken embryo respiratory organ. The feasibility of microvasculature imaging with intravascular ultrasound sets the stage for a translation of the method to studies of intra-plaque neovascularization detection in humans

Contrast-Enhanced Intravascular Ultrasound Pulse Sequences for Bandwidth-Limited Transducers

We demonstrate two methods for vasa vasorum imaging using contrast-enhanced intravascular ultrasound, which can be performed using commercial catheters. Plaque neovascularization was recognized as an independent marker of coronary artery plaque vulnerability. IVUS-based methods to image the microvessels available to date require high bandwidth (-6 dB relative frequency bandwidth >70%), which are not routinely available commercially. We explored the potential of ultraharmonic imaging and chirp reversal imaging for vasa vasorum imaging. In vitro recordings were performed on a tissue-mimicking phantom using a commercial ultrasound contrast agent and a transducer with a center frequency of 34 MHz and a -6 dB relative bandwidth of 56%. Acoustic peak pressures <500 kPa were used. A tissue-mimicking phantom with channels down to 200 μm in diameter was successfully imaged by the two contrast detection sequences while the smallest channel stayed invisible in conventional intravascular ultrasound images. Ultraharmonic imaging provided the best contrast agent detection

Negative DNA supercoiling induces genome-wide Cas9 off-target activity.

CRISPR-Cas9 is a powerful gene-editing technology; however, off-target activity remains an important consideration for therapeutic applications. We have previously shown that force-stretching DNA induces off-target activity and hypothesized that distortions of the DNA topology in vivo, such as negative DNA supercoiling, could reduce Cas9 specificity. Using single-molecule optical-tweezers, we demonstrate that negative supercoiling λ-DNA induces sequence-specific Cas9 off-target binding at multiple sites, even at low forces. Using an adapted CIRCLE-seq approach, we detect over 10,000 negative-supercoiling-induced Cas9 off-target double-strand breaks genome-wide caused by increased mismatch tolerance. We further demonstrate in vivo that directed local DNA distortion increases off-target activity in cells and that induced off-target events can be detected during Cas9 genome editing. These data demonstrate that Cas9 off-target activity is regulated by DNA topology in vitro and in vivo, suggesting that cellular processes, such as transcription and replication, could induce off-target activity at previously overlooked sites