Role of lysines in cytochrome c – cardiolipin interaction

Cytochrome c undergoes structural variations during the apoptotic process; such changes have been related with modifications occurring in the protein when it forms a complex with cardiolipin, one of the phospholipids constituting the mitochondrial membrane. Although several studies have been performed to identify the site(s) of the protein involved in the cytochrome c/cardiolipin interaction, to date the location of this hosting region(s) remains unidentified and is a matter of debate. To gain a deeper insight into the reaction mechanism, we investigate the role that the Lys72, Lys73 and Lys79 residues play in the cytochrome c/cardiolipin interaction, as these side chains appear to be critical for cytochrome c/cardiolipin recognition. The Lys72Asn, Lys73Asn, Lys79Asn, Lys72/73Asn and Lys72/73/79Asn mutants of horse heart cytochrome c were produced and characterized by circular dichroism, UV-visible and resonance Raman spectroscopies, and the effects of the mutations on the interaction of the variants with cardiolipin have been investigated. The mutants are characterized by a subpopulation with non-native axial coordination, and are less stable than the wild type protein. Furthermore, the mutants lacking Lys72 and/or Lys79 do not bind cardiolipin and those lacking Lys73, although they form a complex with the phospholipid, do not show any peroxidase activity. These observations indicate that the Lys72, Lys73 and Lys79 residues stabilize the native axial Met80-Fe(III) coordination as well as the tertiary structure of cytochrome c. Moreover, while Lys72 and Lys79 are critical for cytochrome c/cardiolipin recognition, the simultaneous presence of Lys72, Lys73 and Lys79 is necessary for peroxidase activity of cardiolipin-bound cytochrome c

Probing a Complex of Cytochromecand Cardiolipin by Magnetic Circular Dichroism Spectroscopy: Implications for the Initial Events in Apoptosis

Oxidation of cardiolipin (CL) by its complex with cytochrome c (cyt c) plays a crucial role in triggering apoptosis. Through a combination of magnetic circular dichroism spectroscopy and potentiometric titrations, we show that both the ferric and ferrous forms of the heme group of a CL:cyt c complex exist as multiple conformers at a physiologically relevant pH of 7.4. For the ferric state, these conformers are His/Lys- and His/OH–-ligated. The ferrous state is predominantly high-spin and, most likely, His/–. Interconversion of the ferric and ferrous conformers is described by a single midpoint potential of -80 ± 9 mV vs SHE. These results suggest that CL oxidation in mitochondria could occur by the reaction of molecular oxygen with the ferrous CL:cyt c complex in addition to the well-described reaction of peroxides with the ferric form

A Microfluidic Platform for Cavitation-Enhanced Drug Delivery.

An endothelial-lined blood vessel model is obtained in a PDMS (Polydimethylsiloxane) microfluidic system, where vascular endothelial cells are grown under physiological shear stress, allowing -like maturation. This experimental model is employed for enhanced drug delivery studies, aimed at characterising the increase in endothelial permeability upon microbubble-enhanced ultrasound-induced (USMB) cavitation. We developed a multi-step protocol to couple the optical and the acoustic set-ups, thanks to a 3D-printed insonation chamber, provided with direct optical access and a support for the US transducer. Cavitation-induced interendothelial gap opening is then analysed using a customised code that quantifies gap area and the relative statistics. We show that exposure to US in presence of microbubbles significantly increases endothelial permeability and that tissue integrity completely recovers within 45 min upon insonation. This protocol, along with the versatility of the microfluidic platform, allows to quantitatively characterise cavitation-induced events for its potential employment in clinics

Design, fabrication, and characterization of a multimodal reconfigurable bioreactor for bone tissue engineering

In the past decades, bone tissue engineering developed and exploited many typologies of bioreactors, which, besides providing proper culture conditions, aimed at integrating those bio-physical stimulations that cells experience in vivo, to promote osteogenic differentiation. Nevertheless, the highly challenging combination and deployment of many stimulation systems into a single bioreactor led to the generation of several unimodal bioreactors, investigating one or at mostly two of the required biophysical stimuli. These systems miss the physiological mimicry of bone cells environment, and often produced contrasting results, thus making the knowledge of bone mechanotransduction fragmented and often inconsistent. To overcome this issue, in this study we developed a perfusion and electroactive-vibrational reconfigurable stimulation bioreactor to investigate the differentiation of SaOS-2 bone-derived cells, hosting a piezoelectric nanocomposite membrane as cell culture substrate. This multimodal perfusion bioreactor is designed based on a numerical (finite element) model aimed at assessing the possibility to induce membrane nano-scaled vibrations (with ~12 nm amplitude at a frequency of 939 kHz) during perfusion (featuring 1.46 dyn cm−2 wall shear stress), large enough for inducing a physiologically-relevant electric output (in the order of 10 mV on average) on the membrane surface. This study explored the effects of different stimuli individually, enabling to switch on one stimulation at a time, and then to combine them to induce a faster bone matrix deposition rate. Biological results demonstrate that the multimodal configuration is the most effective in inducing SaOS-2 cell differentiation, leading to 20-fold higher collagen deposition compared to static cultures, and to 1.6- and 1.2-fold higher deposition than the perfused- or vibrated-only cultures. These promising results can provide tissue engineering scientists with a comprehensive and biomimetic stimulation platform for a better understanding of mechanotransduction phenomena beyond cells differentiation

Reversible Cavitation-Induced Junctional Opening in an Artificial Endothelial Layer.

Targeting pharmaceuticals through the endothelial barrier is crucial for drug delivery. In this context, cavitation-assisted permeation shows promise for effective and reversible opening of intercellular junctions. A vessel-on-a-chip is exploited to investigate and quantify the effect of ultrasound-excited microbubbles-stable cavitation-on endothelial integrity. In the vessel-on-a-chip, the endothelial cells form a complete lumen under physiological shear stress, resulting in intercellular junctions that exhibit barrier functionality. Immunofluorescence microscopy is exploited to monitor vascular integrity following vascular endothelial cadherin staining. It is shown that microbubbles amplify the ultrasound effect, leading to the formation of interendothelial gaps that cause barrier permeabilization. The total gap area significantly increases with pressure amplitude compared to the control. Gap opening is fully reversible with gap area distribution returning to the control levels 45 min after insonication. The proposed integrated platform allows for precise and repeatable in vitro measurements of cavitation-enhanced endothelium permeability and shows potential for validating irradiation protocols for in vivo applications

Aberrant signaling in T-cell acute lymphoblastic leukemia: biological and therapeutic implications

T-cell acute lymphoblastic leukemia (T-ALL) is a biologically heterogeneous disease with respect to phenotype, gene expression profile and activation of particular intracellular signaling pathways. Despite very significant improvements, current therapeutic regimens still fail to cure a portion of the patients and frequently implicate the use of aggressive protocols with long-term side effects. In this review, we focused on how deregulation of critical signaling pathways, in particular Notch, PI3K/Akt, MAPK, Jak/STAT and TGF-beta, may contribute to T-ALL. Identifying the alterations that affect intracellular pathways that regulate cell cycle and apoptosis is essential to understanding the biology of this malignancy, to define more effective markers for the correct stratification of patients into appropriate therapeutic regimens and to identify novel targets for the development of specific, less detrimental therapies for T-ALL