Genetically modified mice as a tool for the study of human diseases.

Modeling a human disease is an essential part of biomedical research. The recent advances in the field of molecular genetics made it possible to obtain genetically modified animals for the study of various diseases. Not only monogenic disorders but also chromosomal and multifactorial disorders can be mimicked in lab animals due to genetic modification. Even human infectious diseases can be studied in genetically modified animals. An animal model of a disease enables the tracking of its pathogenesis and, more importantly, to test new therapies. In the first part of this paper, we review the most common DNA modification technologies and provide key ideas on specific technology choices according to the task at hand. In the second part, we focus on the application of genetically modified mice in studying human diseases.

Noncovalent Self-Assembly of Single-Layer MoS2 Nanosheets and Zinc Porphyrin into Stable SURMOF Nanohybrids with Multimodal Photocatalytic Properties.

A noncovalent integration of nanosheets of molybdenum disulfide (MoS2) and the zinc porphyrin complex Zn(II) 5,10,15,20-tetrakis(4-carboxyphenyl)porphine (ZnTCPP) through coordination bonding with metal clusters of zinc acetate (Zn[OAc]2) was applied for synthesis of stable hybrid nanomaterial avoiding surface prefunctionalization. The X-ray powder diffraction in combination with the BET nitrogen adsorption method confirms formation of a ZnTCPP-based surface-attached metal-organic framework (SURMOF) with micropores of 1.63 nm on the MoS2 nanosheets. Fluorescence spectroscopy confirmed Forster resonance energy transfer (FRET) between MoS2 and ZnTCPP without contact quenching. Fluorescent trapping with terephthalic acid for hydroxyl radicals and Sensor Green for singlet oxygen was applied for studying the pathways of photodegradation of model organic pollutant 1,5-dihydroxynaphthalene (DHN) in the presence of SURMOF/MoS2. Visible light initiates sensitization through the excitation of ZnTCPP generating singlet oxygen, whereas UV-light promotes either aerobic FRET-mediated "Z scheme" or anaerobic "Type II heterojunction" mechanisms. Owing to its multimodal photochemistry, the SURMOF/MoS2 hybrid showed comparatively high photocatalytic activity in UV-assisted degradation of DHN (keffUV = 4.0 × 10-2 min-1) as well as the antibacterial activity confirmed by E. coli survival test under visible light. Noncovalent self-assembly utilizing coordination bonding in SURMOFs as supramolecular adhesive to avoid surface premodification provides a basis for new types of multicomponent nanosystems with switchable functionalities by combining different 2D materials and chromophores in one hybrid structure.

Antimicrobial activity of photosensitizers: arrangement in bacterial membrane matters.

Porphyrins are well-known photosensitizers (PSs) for antibacterial photodynamic therapy (aPDT), which is still an underestimated antibiotic-free method to kill bacteria, viruses, and fungi. In the present work, we developed a comprehensive tool for predicting the structure and assessment of the photodynamic efficacy of PS molecules for their application in aPDT. We checked it on a series of water-soluble phosphorus(V) porphyrin molecules with OH or ethoxy axial ligands and phenyl/pyridyl peripheral substituents. First, we used biophysical approaches to show the effect of PSs on membrane structure and their photodynamic activity in the lipid environment. Second, we developed a force field for studying phosphorus(V) porphyrins and performed all-atom molecular dynamics simulations of their interactions with bacterial lipid membranes. Finally, we obtained the structure-activity relationship for the antimicrobial activity of PSs and tested our predictions on two models of Gram-negative bacteria, Escherichia coli and Acinetobacter baumannii. Our approach allowed us to propose a new PS molecule, whose MIC50 values after an extremely low light dose of 5 J/cm2 (5.0 ± 0.4 µg/mL for E. coli and 4.9 ± 0.8 µg/mL for A. baumannii) exceeded those for common antibiotics, making it a prospective antimicrobial agent.

Limitations of Tamoxifen Application for In Vivo Genome Editing Using Cre/ERT2 System.

Inducible Cre-dependent systems are frequently used to produce both conditional knockouts and transgenic mice with regulated expression of the gene of interest. Induction can be achieved by doxycycline-dependent transcription of the wild type gene or OH-tamoxifen-dependent nuclear translocation of the chimeric Cre/ERT2 protein. However, both of these activation strategies have some limitations. We analyzed the efficiency of knockout in different tissues and found out that it correlates with the concentration of the hydroxytamoxifen and endoxifen-the active metabolites of tamoxifen-measured by LC-MS in these tissues. We also describe two cases of Cdk8floxed/floxed/Rosa-Cre-ERT2 mice tamoxifen-induced knockout limitations. In the first case, the standard scheme of tamoxifen administration does not lead to complete knockout formation in the brain or in the uterus. Tamoxifen metabolite measurements in multiple tissues were performed and it has been shown that low recombinase activity in the brain is due to the low levels of tamoxifen active metabolites. Increase of tamoxifen dosage (1.5 fold) and duration of activation (from 5 to 7 days) allowed us to significantly improve the knockout rate in the brain, but not in the uterus. In the second case, knockout induction during embryonic development was impossible due to the negative effect of tamoxifen on gestation. Although DNA editing in the embryos was achieved in some cases, the treatment led to different complications of the pregnancy in wild-type female mice. We propose to use doxycycline-induced Cre systems in such models.

Gene Transcription as a Therapeutic Target in Leukemia.

Blood malignancies often arise from undifferentiated hematopoietic stem cells or partially differentiated stem-like cells. A tight balance of multipotency and differentiation, cell division, and quiescence underlying normal hematopoiesis requires a special program governed by the transcriptional machinery. Acquisition of drug resistance by tumor cells also involves reprogramming of their transcriptional landscape. Limiting tumor cell plasticity by disabling reprogramming of the gene transcription is a promising strategy for improvement of treatment outcomes. Herein, we review the molecular mechanisms of action of transcription-targeted drugs in hematological malignancies (largely in leukemia) with particular respect to the results of clinical trials.