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Cutaneous melanoma is the most aggressive type of skin cancer. Although cutaneous melanoma accounts for a minority of all types of skin cancer, it causes the greatest number of skin cancer related deaths worldwide. Oxidative stress and redox homeostasis have been shown to be involved at each stage of a malignant melanocyte transformation, called melanomagenesis, as well as during drug resistance. Reactive oxygen species (ROS) play an important and diverse role that regulate many aspects of skin cell behaviors ranging from proliferation and stemness, to oxidative damage and cell death. On the other hand, antioxidants are associated with melanoma spread and metastasis. Overall, the contribution of redox homeostasis to melanoma development and progression is controversial and highly complex. The aim of this study is to examine the association between redox homeostasis and the melanomagenic process. To this purpose we are presenting what is currently known about the role of ROS in melanoma initiation and progression. In addition, we are discussing the role of antioxidant mechanisms during the spread of the disease and in cases of melanoma drug resistance. Although challenging, targeting redox homeostasis in melanoma progression remains to be a promising therapeutic approach, especially valid during melanoma drug resistance., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis are global challenges due to the limited number of effective drugs for treatment. Treatment with less than 4-5 effective drugs might lead to the further emergence of drug resistance and poor clinical outcomes. For better prediction of treatment outcomes, we compared drug-resistance profiles of consecutive clinical MDR Mycobacterium tuberculosis isolates from high- and low-burden settings. This was a retrospective cohort study. We analysed 225 and 229 MDR isolates from Moscow (Russia) and Taiwan, respectively, obtained between 2014 and 2015. Drug susceptibility testing was performed by the Bactec MGIT 960 automated system and the agar proportion method. Detection of resistance-associated mutations in the M. tuberculosis genome was carried out by an array and/or sequencing of selected loci. The principal differences between resistance profiles of MDR isolates in the two countries were the percentages of pre-XDR (40.9% vs. 14.8%) and XDR (34.7% vs. 1.7%) isolates, both of which were significantly higher in Moscow isolates. Forty-eight (33%) of 147 MDR and pre-XDR Russian isolates fall into a group with less than four effective drugs, which accounts for 40% (N = 120) of these isolates. The other 60% in this group were XDR strains (N = 72). Consequently, the average number of effective anti-tuberculosis drugs for MDR-TB treatment was lower for Russian isolates (3 vs. 7). Furthermore, a notable percentage (9%) of isolates resistant to kanamycin harboured mutations in the whiB7 locus, which was not detected by molecular tests targeting common mutations in the rrs and eis loci. We found that 98.2% and 45.9% of MDR isolates from Moscow and Taiwan, respectively, were resistant to streptomycin. Molecular tests for detecting resistance to drugs other than rifampicin, isoniazid, fluoroquinolones, and second-line injectable drugs are needed for individualized therapy. The conventional MDR treatment schemes most probably fail in these cases due to the limited number of effective drugs., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Objectives: To study the isolates with acquired resistance to bedaquiline and linezolid that were obtained from patients enrolled in a clinical study of a novel therapy regimen for drug-resistant TB in Moscow, Russia., Methods: Linezolid resistance was detected using MGIT 960 with a critical concentration of 1 mg/L. The MIC of bedaquiline was determined using the proportion method. To identify genetic determinants of resistance, sequencing of the mmpR ( Rv0678 ), atpE , atpC , pepQ , Rv1979c , rrl , rplC and rplD loci was performed., Results: A total of 85 isolates from 27 patients with acquired resistance to linezolid and reduced susceptibility to bedaquiline (MIC ≥0.06 mg/L) were tested. Most mutations associated with a high MIC of bedaquiline were found in the mmpR gene. We identified for the first time two patients whose clinical isolates had substitutions D28N and A63V in AtpE, which had previously been found only in in vitro -selected strains. Several patients had isolates with elevated MICs of bedaquiline prior to treatment; four of them also bore mutations in mmpR , indicating the presence of some hidden factors in bedaquiline resistance acquisition. The C154R substitution in ribosomal protein L3 was the most frequent in the linezolid-resistant strains. Mutations in the 23S rRNA gene (g2294a and g2814t) associated with linezolid resistance were also found in two isolates. Heteroresistance was identified in ∼40% of samples, which reflects the complex nature of resistance acquisition., Conclusions: The introduction of novel drugs into treatment must be accompanied by continuous phenotypic susceptibility testing and the analysis of genetic determinants of resistance., (© The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Ion homeostasis regulates critical physiological processes in the living cell. Intracellular chloride concentration not only contributes in setting the membrane potential of quiescent cells but it also plays a role in modulating the dynamic voltage changes during network activity. Dynamic chloride imaging demands new tools, allowing faster acquisition rates and correct accounting of concomitant pH changes. Joining a long-Stokes-shift red-fluorescent protein to a GFP variant with high sensitivity to pH and chloride, we obtained LSSmClopHensor, a genetically encoded fluorescent biosensor optimized for the simultaneous chloride and pH imaging and requiring only two excitation wavelengths (458 and 488 nm). LSSmClopHensor allowed us to monitor the dynamic changes of intracellular pH and chloride concentration during seizure like discharges in neocortical brain slices. Only cells with tightly controlled resting potential revealed a narrow distribution of chloride concentration peaking at about 5 and 8 mM, in neocortical neurons and SK-N-SH cells, respectively. We thus showed that LSSmClopHensor represents a new versatile tool for studying the dynamics of chloride and proton concentration in living systems.

3D imaging of genetically-engineered fluorescent tumors enables quantitative monitoring of tumor growth/regression, metastatic processes, including during anticancer therapy in real-time.Fluorescent tumor models for 3D imaging require stable expression of genetically encoded fluorescent proteins and maintenance of the properties of tumor cell line including growth rate, morphology, and immunophenotype.In this chapter, the protocol for 3D imaging of tumors expressing red fluorescent protein are described in detail.

The genetically encoded FRET-pair was developed on the basis of terbium-binding peptide and red fluorescent protein DsRed2. To study fluorescence resonance energy transfer within FRET-pair, the gene-engineered construction was obtained, where sequences of terbium-binding peptide and red fluorescent protein DsRed2 were fused in single reading frame. The expression of this construction in strain E. coli BL21 (DE3) was studied and conditions of synthesis, isolation and purification of recombinant protein were optimized. The hydrodynamic radius of hybrid protein was determined by the method of dynamic light scattering. Energy transfer between sensitized terbium and red fluorescent protein was confirmed by the methods of phosporescent spectroscopy. The obtained FRET-pair can be used both for studies in vitro and as a reporter in living cells.

A fluorescence diffuse tomography (FDT) setup for monitoring tumor growth in small animals has been created. In this setup an animal is scanned in the transilluminative configuration by a single source and detector pair. To remove stray light in the detection system, we used a combination of interferometric and absorption filters. To reduce the scanning time, an experimental animal was scanned using the following algorithm: (1) large-step scanning to obtain a general view of the animal (source and detector move synchronously); (2) selection of the fluorescing region; and (3) small-step scanning of the selected region and different relative shifts between the source and detector to obtain sufficient information for 3D reconstruction. We created a reconstruction algorithm based on the Holder norm to estimate the fluorophore distribution. This algorithm converges to the solution with a minimum number of fluorescing zones. The use of tumor cell lines transfected with fluorescent proteins allowed us to conduct intravital monitoring studies. Cell lines of human melanomas Mel-P, Mel-Ibr, Mel-Kor, and human embryonic kidney HEK293 Phoenix were transfected with DsRed-Express and Turbo-RFP genes. The emission of red fluorescent proteins (RFPs) in the long-wave optical range permits detection of deep-seated tumors. In vivo experiments were conducted immediately after subcutaneous injection of fluorescing cells into small animals.