Structural and functional diversity of snake sarafotoxins

International audienceSarafotoxins (SRTXs) are extracted solely from the venom of snakes belonging to the Atractaspis genus, a burrowing asp from Africa and Middle East. They share a high-sequence homology with endothelins, potent vasoconstrictors produced by mammalian endothelial cells and implicated in vascular tone regulation and cell growth. SRTXs and endothelins have a common core of 21 amino acids and two conserved disulfide bridges between cysteines +1/+15 and +3/+11 which constitute a unique and typical ``signature'' among natural bioactive peptides: Cys$_1$-XCys$_3$... Cys$_{11}$-X-X-X-Cys$_{15}$. SRTXs bind to endothelin receptors ET-A and ET-B situated on the membrane of numerous cells especially endothelial cells and smooth muscle cells. Recently long SRTXs have been discovered in the venom of A. microlepidota microlepidota and A. irregularis. These long SRTXs have three or four additional amino acids on their C-terminal domain. The affinity of long SRTXs for endothelin receptors is dramatically decreased compared to short SRTXs. Nevertheless, they still have a high toxicity as tested on mice. Combining invasive and echocardiographic procedures, recent experiments showed that short and long SRTXs have very different in vivo hemodynamic effects. Short SRTXs impair left ventricular function while long SRTXs impair right ventricular function and increase airway pressures with no effect on left ventricular function. Further experiments are needed to explain this discrepancy. The hypotheses of a new type of endothelin receptors (non-A non-B) cannot be discarded. Thus, the C-terminus extension seems to play a major role in defining the in vivo effect of these peptides

A singular view of DNA transactions

The genetic information of an organism is encoded in the base pair sequence of its DNA. Many specialized proteins are involved in organizing, preserving and processing the vast amounts of information on the DNA. In order to do this swiftly and correctly these proteins have to move quickly and accurately along and/or around the DNA constantly rearranging it. In order to elucidate these processes we perform single-molecule experiments on model systems such as bacterial gene regulators and repair proteins. The data we use to extract forces, energies and mechanochemistry driving these dynamic transactions. The results obtained from these model systems are then generalized and though to be applicable to many DNA-protein interactions

A low-cost, label-free DNA detection method in lab-on-chip format based on electrohydrodynamic instabilities, with application to long-range PCR

In order to evolve from a "chip in the lab" to a "lab on a chip" paradigm, there is still a strong demand for low-cost, portable detection technologies, notably for analytes at low concentrations. Here we report a new label-free DNA detection method with direct electronic read, and apply it to long-range PCR. This method uses a nonlinear electrohydrodynamic phenomenon: when subjected to high electric fields (typically above 100 V cm(-1)), suspensions of large polyelectrolytes, such as long DNA molecules, create "giant" dynamic concentration fluctuations. These fluctuations are associated with large conductivity inhomogeneities, and we use here a contact-mode local conductivity detector to detect these fluctuations. In order to decouple the detection electronics from the high voltage excitation one, an original "doubly symmetric" floating mode battery-operated detection scheme was developed. A wavelet analysis was then applied, to unravel from the chaotic character of the electohydrodynamic instabilities a scalar signal robustly reflecting the amplification of DNA. As a first proof of concept, we measured the products of the off-chip amplification of 10 kbp DNA from lambda phage DNA, achieving a sensitivity better than 100 fg DNA in the original 50 mu l sample. This corresponds to the amplification products of less than 100 initial copies of target DNA. The companion enabling technologies developed to implement this new concept, i.e. the doubly symmetric contact conductivity detection and wavelet analysis, may also find various other applications in lab-on-chips

Echocardiographic evaluation of the acute cardiovascular effects of an endothelin-like peptide extracted from the venom of Atractaspis irregularisAtractaspis\ irregularis

International audienceSarafotoxin-i3 from Atractaspis irregularis is a long sarafotoxin with an extended C terminus extension. Sarafotoxin-b from Atractaspis engaddensis is shorter by four amino acids. These peptides belong to the endothelin-like peptide family with a high sequence homology and similar three-dimensional structure. They act on endothelin receptors situated on the membrane of endothelial and smooth muscle cells. However, SRTX-i 3, despite a high toxicity, has a very low affinity for endothelin receptors compared to SRTX-b. The present work was carried out in order to compare the precise in vivo cardiovascular effect of SRTX-b and SRTX-i3. Male Wistar rats were anesthetized and mechanically ventilated. Doppler echocardiography was performed to measure left and right ventricular functions. The rats were divided into three groups that received intravenous injections of: saline, SRTX-b or SRTX-i3. All measurements were taken at baseline, at 1 min and at 6 min after injection. Both toxins impaired cardiac output. SRTX-b impaired left ventricular function, while SRTX-i3 increased airway pressures and led to acute right ventricular dilatation associated with a decreased tricuspid annulus peak systolic velocity. SRTX-b and SRTX-i3 appear to exert toxic effects via different mechanisms, SRTX-b impairs left ventricular function, while SRTX-i3 increases airway pressures and impairs right ventricular function

Respiratory effects of sarafotoxins from the venom of different AtractaspisAtractaspis genus snake species

International audienceSarafotoxins (SRTX) are endothelin-like peptides extracted from the venom of snakes belonging to the Atractaspididae family. A recent in vivo study on anesthetized and ventilated animals showed that sarafotoxin-b (SRTX-b), extracted from the venom of Atractaspis engaddensis, decreases cardiac output by inducing left ventricular dysfunction while sarafotoxin-m (SRTX-m), extracted from the venom of Atractaspis microlepidota microlepidota, induces right ventricular dysfunction with increased airway pressure. The aim of the present experimental study was to compare the respiratory effects of SRTX-m and SRTX-b. Male Wistar rats were anesthetized, tracheotomized and mechanically ventilated. They received either a 1 LD$_{50}$ IV bolus of SRTX-b (n = 5) or 1 LD50 of SRTX-m (n = 5). The low-frequency forced oscillation technique was used to measure respiratory impedance. Airway resistance (Raw), parenchymal damping (G) and elastance (H) were determined from impedance data, before and 5 min after SRTX injection. SRTX-m and SRTX-b injections induced acute hypoxia and metabolic acidosis with an increased anion gap. Both toxins markedly increased Raw, G and H, but with a much greater effect of SRTX-b on H, which may have been due to pulmonary edema in addition to bronchoconstriction. Therefore, despite their structural analogy, these two toxins exert different effects on respiratory function. These results emphasize the role of the C-terminal extension in the in vivo effect of these toxins

Hemodynamic impact of molecular adsorbent recirculating system in refractory vasoplegic shock due to calcium channel blocker poisoning

International audienceObjective: To report the hemodynamic effect of to the molecular adsorbent recirculating system (MARS (TM)) therapy for patients in refractory vasoplegic shock due to calcium channel blocker (CCB) poisoning Methods: We report a retrospective cohort of patients who were hospitalized for CCB poisoning with refractory vasoplegic shock and treated by MARS therapy, at Amiens Hospital University, from January 2010 to December 2019. Improvement in hemodynamic was assessed by dynamic changes in mean arterial pressure (MAP) and norepinephrine levels over a 24-h period after MARS therapy. Cardiac function was assessed by transthoracic echocardiography. Results: MARS therapy was performed on seven patients for CCB poisoning. CCB poisoning included nicardipine (n = 3, 43%) amlodipine (n = 3, 43%), and verapamil (n = 1, 14%). The median time to start MARS therapy was 24 [14-27] h after drug ingestion and 6 [2-9] h after ICU admission. Cardiac output was preserved for all patients. MAP values improved from 56 [43-58] to 65 [61-78] 16 mmHg (p = 0.005). Norepinephrine dose significantly decreased from 3.2 [0.8-10] mu g/kg/min to 1.2 [0.1-1.9] mu g/kg/min (p = 0.008) and lactate level decreased from 3.2 [2.4-3.4] mmol/l(-1) to 1.6 [0.9-2.2] mmol/l(-1) (p = 0.008). The median length of ICU stay was 4 (2-7) days and hospital stay was 4 (4-16) days. No complication related to the MARS therapy were reported. No patient died and all were discharged from the hospital. Conclusion: We reported the largest case-series of MARS therapy for refractory vasoplegic shock due to CCB poisoning. We observed that MARS therapy was associated with an improvement of hemodynamic parameters

A microfluidic fluidized bed to capture, amplify and detect bacteria from raw samples

International audienceBacterial contamination and subsequent infections are a major threat to human health. An early detection in the food chain, clinics or the environment, is key to limit this threat. We present a new concept to develop low-cost hand-held devices for the ultra-sensitive and specific detection of bacteria in a one-step process of 2–8 h, directly from complex raw samples. This approach is based on a novel microfluidic magnetic fluidized bed. It reaches a 4 CFU (colony forming unit) sensitivity with high quantification accuracy in a large dynamic range of 100–107CFU/mL. The versatility of the approach was demonstrated with the detection of different bacteria strains, among which Salmonella Typhimurium and E. coli O157:H15. Additionally, the method is sensitive to infectious bacteria only, a criterion requested by main applications and currently requiring additional culture steps of one to several days

Nature-inspired active micro-fluidic mixing using artificial cilia

Creating good mixing flows in lab-on-chip systems poses a special challenge, due to the impossibility to generate turbulence. We propose a novel concept for active mixing, that is inspired by nature, namely micro-organisms that swim through a liquid by beating microscopic hairs, cilia, covering their surface. We have fabricated artificial cilia, based on electro-statically actuated polymer structures, and have integrated these in a micro-fluidic channel. Flow visualization experiments show that our cilia can generate very efficient mixing. Since the artificial cilia can be actively controlled using an electrical signal, they have exciting applications in micro-fluidic devices

In vitro bone metastasis dwelling in a 3D bioengineered niche

International audienceBone is the most frequent metastasis site for breast cancer. As well as dramatically increasing disease burden, bone metastases are also an indicator of poor prognosis. One of the main challenges in investigating bone metastasis in breast cancer is engineering in vitro models that replicate the features of in vivo bone environments. Such in vitro models ideally enable the biology of the metastatic cells to mimic their in vivo behavior as closely as possible. Here, taking benefit of cutting-edge technologies both in microfabrication and cancer cell biology, we have developed an in vitro breast cancer bone-metastasis model. To do so we first 3D printed a bone scaffold that reproduces the trabecular architecture and that can be conditioned with osteoblast-like cells, a collagen matrix, and mineralized calcium. We thus demonstrated that this device offers an adequate soil to seed primary breast cancer bone metastatic cells. In particular, patient-derived xenografts being considered as a better approach than cell lines to achieve clinically relevant results, we demonstrate the ability of this biomimetic bone niche model to host patient-derived xenografted metastatic breast cancer cells. These patient-derived xenograft cells show a long-term survival in the bone model and maintain their cycling propensity, and exhibit the same modulated drug response as in vivo. This experimental system enables access to the idiosyncratic features of the bone microenvironment and cancer bone metastasis, which has implications for drug testing

VEGF (Vascular Endothelial Growth Factor) Functionalized Magnetic Beads in a Microfluidic Device to Improve the Angiogenic Balance in Preeclampsia

International audienc