Kinetic Analysis of the Mitochondrial Permeability Transition

The permeability transition process in rat liver mitochondria was studied by following the swelling consequent to external solute equilibration through the membrane pore. The kinetics of the transition was analyzed according to a model based on the assumptions that the transition rate follows a first-order process and that the solute diffusion rate strongly depends on the pore conformation. Three kinetic parameters, easily calculated from absorbance measurements during mitochondrial swelling, were used to determine whether changes of the swelling rate are due to changes of (i) the transition rate, (ii) the amount of permeabilized mitochondria, (iii) the mean pore dimension, or (iv) the number of pores per mitochondrion. The model was tested for transitions induced either by phenylarsine oxide (PhAsO) or by Ca2+ and Pi. Under both conditions, only a definite fraction of mitochondria was permeabilized, and the transition always followed a first-order reaction, indicating that mitochondria behaved as a homogeneous population. However, the equilibration of external solutes was rapid only in a fraction of permeabilized mitochondria and slow in the remaining fraction, due to restricted solute diffusion through narrower pores. With 0.2 mM PhAsO as the inducer, the fraction of permeabilized mitochondria was about 0.8. Sucrose diffusion was rapid only in 15% of this fraction (half-time less than 1 s) and restricted in the remaining 85% (half-time of about 60 s). Increasing PhAsO concentrations increased the number of pores per mitochondrion and the rate constant of the permeability transition, but not the mean pore diameter and the fraction of permeabilized mitochondria. With 0.28 microM free [Ca2+] and 1 mM Pi as the inducers, the fraction of permeabilized mitochondria was about 0.43. Sucrose diffusion was rapid in 60% of this fraction and restricted in the remaining 40% (half-time of about 9 s). The permeabilization process started above a threshold- free [Ca2+] of 0.15 microM, and the rate constant and the fraction of permeabilized mitochondria reached a maximum at about 0.2 microM, while both parameters were inhibited at higher free [Ca2+]

Physiological effectors modify voltage sensing by the cyclosporin A-sensitive permeability transition pore of mitochondria.

This paper reports an investigation on the modulation of the mitochondrial permeability transition pore (MTP) by the membrane potential. Energized rat liver mitochondria loaded with a small Ca2+ pulse in sucrose medium supplemented with phosphate favor a high MTP "closed" probability because of the high membrane potential and therefore maintain a low permeability to sucrose. Upon depolarization by the addition of fully uncoupling concentrations of carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) mitochondria favor a high MTP "open" probability and rapidly undergo a process of osmotic swelling following sucrose diffusion toward the matrix. A titration with FCCP reveals that discrete subpopulations of mitochondria with different gating potentials for MTP opening may exist, since increasing concentrations of FCCP increase the fraction of mitochondria undergoing osmotic swelling. We show that physiological effectors (Ca2+, Mg2+, ADP, palmitate) modify pore opening in a mitochondrial population by shifting the fraction of mitochondria with a functionally open pore at any given membrane potential. Many inducers and inhibitors may therefore affect the pore directly through an effect on the MTP voltage sensing rather than indirectly through an effect on the membrane potential. Thus, many effectors may induce pore opening by shifting the MTP gating potential to higher levels, whereas many inhibitors may induce pore closure by shifting the MTP gating potential to lower levels

Designing multimodal interactive systems using EyesWeb XMI

This paper introduces the EyesWeb XMI platform (for eXtended Multimodal Interaction) as a tool for fast prototyping of multimodal systems, including interconnection of multiple smart devices, e.g., smartphones. EyesWeb is endowed with a visual programming language enabling users to compose modules into applications. Modules are collected in several libraries and include support of many input devices (e.g., video, audio, motion capture, accelerometers, and physiological sensors), output devices (e.g., video, audio, 2D and 3D graphics), and synchronized multimodal data processing. Specific libraries are devoted to real-time analysis of nonverbal expressive motor and social behavior. The EyesWeb platform encompasses further tools such EyesWeb Mobile supporting the development of customized Graphical User Interfaces for specific classes of users. The paper will review the EyesWeb platform and its components, starting from its historical origins, and with a particular focus on the Human-Computer Interaction aspects

CARE-compliant stereotactic radiotherapy of urothelial nodal metastases: A case report

The aim of the present study was to report the case of a 58-year-old male patient with ureteral carcinoma who underwent ureteroileostomy treatment. At 2 years following surgery, six lymph node metastases (LNMs) were detected in the patient's para-aortic and pelvic regions using F-18-labeled fluoro-2-deoxyglucose (FDG) positron emission tomography (PET)/CT. All LNMs were treated using stereotactic body radiotherapy (SBRT; 35-40 Gy/5 fractions). At 3 months after radiotherapy, F-18-FDG-PET/CT examination revealed a complete radiological and metabolic response of all targeted treatment sites in the patient. In the 2 years following radiotherapy, another three same-dose SBRT treatments were performed on single or multiple LNMs, which were all detected in the abdomen and pelvis of the patient. Overall, a total of 11 LNMs were targeted in the patient and all exhibited complete radiological and metabolic response following treatment. The only treatment side effect reported by the patient was a slight and temporary loss of appetite. In patients with lymph node oligometastases there are two options for radiotherapy: i) Irradiation focusing on LNMs alone; and ii) prophylactic irradiation of the entire lymph node area combined with a boost on macroscopic lesions. In the patient discussed in the present study, the choice of irradiation focusing on LNMs alone made it possible to postpone systemic therapies and instead use an optimally tolerated treatment. The treatment outcome in this patient indicated that there was no radioresistance of urothelial LNMs

Tumor growth rate to assess therapy response to immune-based combinations for metastatic renal cell carcinoma

Background: Radiological response assessment is becoming challenging with novel immune-based combinations for metastatic renal cell carcinoma (mRCC). RECIST criteria appear not exhaustively adequate to capture the kinetics of treatment response, which is better reflected by tumor growth rate (TGR). We explored TGR changes during first-line treatments and its association with clinical outcomes in mRCC. Research design and methods: We retrospectively evaluated TGR in untreated patients undergoing pembrolizumab/axitinib (P/A) or tyrosine-kinase inhibitors (TKI). TGR was calculated at the first (TGR1, after 3 months) and the second (TGR2, after 6 months) evaluation, thus assessing the TGR2-TGR1 difference. Results: Thirty-three patients were included (P/A n = 15, TKIs n = 18). Volumes firstly decreased more rapidly with TKIs, and then more slowly. Volumes initially remained stable with P/A, quickly decreasing until the second evaluation. TGR1 was related to progression-free survival (PFS; p = 0.023) and overall survival (p = 0.046) with P/A. TGR2 was correlated with PFS in all patients (p = 0.025). Patients with higher velocity volume reduction appeared to have improved survival benefits than patients with lower velocity considering both treatments, but especially with P/A. Conclusion: Combining immunotherapy with TKIs has an important role in enhancing the rapidity of tumor shrinkage. A rapid disease volume reduction correlates with better OS and PFS

1,2,4-Triazolo[1,5-a]pyrimidines as a Novel Class of Inhibitors of the HIV-1 Reverse Transcriptase-Associated Ribonuclease H Activity

Despite great efforts have been made in the prevention and therapy of human immunodeficiency virus (HIV-1) infection, however the difficulty to eradicate latent viral reservoirs together with the emergence of multi-drug-resistant strains require the search for innovative agents, possibly exploiting novel mechanisms of action. In this context, the HIV-1 reverse transcriptase (RT)-associated ribonuclease H (RNase H), which is one of the few HIV-1 encoded enzymatic function still not targeted by any current drug, can be considered as an appealing target. In this work, we repurposed in-house anti-influenza derivatives based on the 1,2,4-triazolo[1,5-a]-pyrimidine (TZP) scaffold for their ability to inhibit HIV-1 RNase H function. Based on the results, a successive multi-step structural exploration around the TZP core was performed leading to identify catechol derivatives that inhibited RNase H in the low micromolar range without showing RT-associated polymerase inhibitory activity. The antiviral evaluation of the compounds in the MT4 cells showed any activity against HIV-1 (IIIB strain). Molecular modelling and mutagenesis analysis suggested key interactions with an unexplored allosteric site providing insights for the future optimization of this class of RNase H inhibitors

PD-1 blockade therapy in renal cell carcinoma: current studies and future promises

RCC is considered an immunogenic tumor with a prominent dysfunctional immune cell infiltrate, unable to control tumor growth. Evasion of immune surveillance, a process defined immune-editing, leads to malignant progression. The striking improvement of knowledge in immunology has led to the identification of immune checkpoints (such as CTLA-4 and PD-1), whose blockage enhances the antitumor immunity. The interaction between PD-1, an inducible inhibitory receptor expressed on lymphocytes and DCs, and PD-L1 ligand, expressed by tumor cells, results in a down-regulation of the T-cell response. Therefore, the PD-1/PD-L1 axis inhibition by targeted-antibodies, increasing the T-cell proliferation and cytotoxicity, represents a promising mechanism to stimulate the anti-tumor activity of the immune system, improving the outcomes of cancer patients. Several PD-1 and PD-L1 inhibitors have been evaluated in different tumor types, showing promising results. The interesting correlation between lymphocytes PD-1 expression and RCC advanced stage, grade and prognosis, as well as the selective PD-L1 expression by RCC tumor cells and its potential association with worse clinical outcomes, have led to the development of new anti PD-1/PD-L1 agents, alone or in combination with anti-angiogenic drugs or other immunotherapeutic approaches, for the treatment of RCC. In this review we discuss the role of PD-1/PD-L1 in RCC, focusing on the biological rationale, current clinical studies and promising therapeutic perspectives to target the PD-1 pathway

Structural Modifications of the Quinolin-4-yloxy Core to Obtain New Staphylococcus aureus NorA Inhibitors

: Tackling antimicrobial resistance (AMR) represents a social responsibility aimed at renewing the antimicrobial armamentarium and identifying novel therapeutical approaches. Among the possible strategies, efflux pumps inhibition offers the advantage to contrast the resistance against all drugs which can be extruded. Efflux pump inhibitors (EPIs) are molecules devoid of any antimicrobial activity, but synergizing with pumps-substrate antibiotics. Herein, we performed an in silico scaffold hopping approach starting from quinolin-4-yloxy-based Staphylococcus aureus NorA EPIs by using previously built pharmacophore models for NorA inhibition activity. Four scaffolds were identified, synthesized, and modified with appropriate substituents to obtain new compounds, that were evaluated for their ability to inhibit NorA and synergize with the fluoroquinolone ciprofloxacin against resistant S. aureus strains. The two quinoline-4-carboxamide derivatives 3a and 3b showed the best results being synergic (4-fold MIC reduction) with ciprofloxacin at concentrations as low as 3.13 and 1.56 µg/mL, respectively, which were nontoxic for human THP-1 and A549 cells. The NorA inhibition was confirmed by SA-1199B ethidium bromide efflux and checkerboard assays against the isogenic pair SA-K2378 (norA++)/SA-K1902 (norA-). These in vitro results indicate the two compounds as valuable structures for designing novel S. aureus NorA inhibitors to be used in association with fluoroquinolones