Airway basal cells from human-induced pluripotent stem cells: a new frontier in cystic fibrosis research.

Human-induced airway basal cells (hiBCs) derived from human-induced pluripotent stem cells (hiPSCs) offer a promising cell model for studying lung diseases, regenerative medicine, and developing new gene therapy methods. We analyzed existing differentiation protocols and proposed our own protocol for obtaining hiBCs, which involves step-by-step differentiation of hiPSCs into definitive endoderm, anterior foregut endoderm, NKX2.1+ lung progenitors, and cultivation on basal cell medium with subsequent cell sorting using the surface marker CD271 (NGFR). We derived hiBCs from two healthy cell lines and three cell lines with cystic fibrosis (CF). The obtained hiBCs, expressing basal cell markers (NGFR, KRT5, and TP63), could differentiate into lung organoids (LOs). We demonstrated that LOs derived from hiBCs can assess cystic fibrosis transmembrane conductance regulator (CFTR) channel function using the forskolin-induced swelling (FIS) assay. We also carried out non-viral (electroporation) and viral (recombinant adeno-associated virus (rAAV)) serotypes 6 and 9 and recombinant adenovirus (rAdV) serotype 5 transgene delivery to hiBCs and showed that rAAV serotype 6 is most effective against hiBCs, potentially applicable for gene therapy research.

Evolution of Prime Editing Systems: Move Forward to the Treatment of Hereditary Diseases.

The development of gene therapy using genome editing tools recently became relevant. With the invention of programmable nucleases, it became possible to treat hereditary diseases due to introducing targeted double strand break in the genome followed by homology directed repair (HDR) or non-homologous end-joining (NHEJ) reparation. CRISPR-Cas9 is more efficient and easier to use in comparison with other programmable nucleases. To improve the efficiency and safety of this gene editing tool, various modifications CRISPR-Cas9 basis were created in recent years, such as prime editing - in this system, Cas9 nickase is fused with reverse transcriptase and guide RNA, which contains a desired correction. Prime editing demonstrates equal or higher correction efficiency as HDR-mediated editing and much less off-target effect due to inducing nick. There are several studies in which prime editing is used to correct mutations in which researchers reported little or no evidence of off-target effects. The system can also be used to functionally characterize disease variants. However, prime editing still has several limitations that could be further improved. The effectiveness of the method is not yet high enough to apply it in clinical trials. Delivery of prime editors is also a big challenge due to their size. In the present article, we observe the development of the platform, and discuss the candidate proteins for efficiency enhancing, main delivery methods and current applications of prime editing.

Unexpected extra exon skipping in the DYSF gene during restoring the reading frame by CRISPR/Cas9.

The DYSF gene encoding dysferlin protein is one of the largest and has many transcripts. Pathogenic variants in the gene can lead to various types of myopathies, which makes it a good object for studying the events occurring in it during genome editing by the CRISPR/Cas method. In this study, we evaluated the possibility of permanent skipping of exons 3-4, and 26-27 which deletion does not violate the reading frame and allows to eliminate truncated variants within exons. Editing was performed with simultaneous transfection of two sgRNA- and sa/spCas9-containing plasmids on HEK293T cell cultures and healthy donor myoblasts. Skipping of exons 3-4 was performed by destroying the splicing acceptor sites, and exons 26-27 by cuts in the flanking exons with the corresponding deletion in the DNA. Some unexpected results were obtained, when exons 26-27 were skipped, exon 30 was also absent in the transcript, although it is not alternatively spliced and is normally present in all transcripts. This event indicates that DNA changes near splicing sites can affect adjacent exons and the whole gene. However, this fact requires further study.

Generation of induced pluripotent stem cell line (RCMGi012-A) from fibroblasts of patient with mucopolysaccharidosis type VI.

Skin fibroblasts obtained from a 5-year-old girl with genetically proven (two heterozygous mutations in ARSB gene) and clinically manifested mucopolysaccharidosis type VI were successfully transformed into induced pluripotent stem cells by using Sendai virus-based reprogramming vectors including the four Yamanaka factors namely SOX2, OCT3/4, KLF4, and c-MYC. These iPSCs expressed pluripotency markers, had a normal karyotype and the potential to differentiate into three germ layers in spontaneous differentiation assay. The line may be used for cell differentiation and pharmacological investigations, and also may provide a model for development of a personalized treatment including drug screening and genome editing.

Synthesis, Structural Versatility, Magnetic Properties, and I- Adsorption in a Series of Cobalt(II) Metal-Organic Frameworks with a Charge-Neutral Aliphatic (O,O)-Donor Bridge.

Four new metal-organic frameworks based on cobalt(II) salts and 1,4-diazabicyclo[2.2.2]octane N,N'-dioxide (odabco) were obtained. Their crystallographic formulae are [Co3(odabco)2(OAc)6] (1, OAc- = acetate), [Co(H2O)2(HCOO)2]·odabco (2), [Co2(H2O)(NO3)(odabco)5](NO3)3·3.65H2O (3), and [Co2(DMF)2(odabco)4](NO3)4·3H2O (4; DMF = N,N-dimethylformamide). Crystal structures of 1-4 were determined by single-crystal X-ray crystallography. Coordination polymer 1 comprises binuclear and mononuclear metal-acetate blocks alternating within uncharged one-dimensional chains, in which odabco acts as a bridging ligand. A layered Co(II) formate 2 contains odabco only as guest molecules located in the interlayer space. Layered compound 3 and three-dimensional 4 have cationic coordination frameworks with 26% and 34% specific void volumes, respectively, unveiling high structural diversity of Co(II)-odabco MOFs based on quite a rare aliphatic moiety. Magnetization measurements were performed for 1, 3, and 4 and the obtained data were interpreted on the basis of their crystal structures. A strong (J/kB~100 K) antiferromagnetic coupling was found within binuclear metal blocks in 1. Ion exchange experiments revealed a considerable iodide uptake by 3 resulting in an up to 75% guest nitrate substitution within the voids of a coordination framework, found by capillary zone electrophoresis data and confirmed by single-crystal XRD. A preservation of 3 crystallinity during the exchange allowed for the guest I- positions within a new adduct with the formula [Co2(H2O)(NO3)(odabco)5]I2(NO3)·1.85H2O (3-I) to be successfully determined and the odabco aliphatic core to be revealed as a main adsorption center for quite large and easily polarizable iodide anions. In summary, this work presents a comprehensive study for a series of 1,4-diazabicyclo[2.2.2]octane N,N'-dioxide-based MOFs of cobalt(II) within the framework of magnetic properties and reports the first example of anion exchange in odabco-based coordination networks, supported by direct X-ray structural data. The reported results unveil promising applications of such frameworks bearing ligands with an aliphatic core in the diverse structural design of selective adsorbents and other types of functional materials.

Synthesis, Structure, and Magnetic and Biological Properties of Copper(II) Complexes with 1,3,4-Thiadiazole Derivatives.

New coordination compounds of copper(II) with 2,5-bis(ethylthio)-1,3,4-thiadiazole (L1) and 2,5-bis(pyridylmethylthio)-1,3,4-thiadiazole (L2) with compositions Cu(L1)2Br2, Cu(L1)(C2N3)2, Cu(L2)Cl2, and Cu(L2)Br2 were prepared. The complexes were identified and studied by CHN analysis, infrared (IR) spectroscopy, powder X-Ray diffraction (XRD), and static magnetic susceptibility. The crystal structures of Cu(II) complexes with L1 were determined. The structures of the coordination core of complexes Cu(L2)Cl2 and Cu(L2)Br2 were determined by Extended X-ray absorption fine structure (EXAFS) spectroscopy. Magnetization measurements have revealed various magnetic states in the studied complexes, ranging from an almost ideal paramagnet in Cu(L1)2Br2 to alternating-exchange antiferromagnetic chains in Cu(L1)(C2N3)2, where double dicyanamide bridges provide an unusually strong exchange interaction (J1/kB ≈ -23.5 K; J2/kB ≈ -20.2 K) between Cu(II) ions. The cytotoxic activity of copper(II) complexes with L2 was estimated on the human cell lines of breast adenocarcinoma (MCF-7) and hepatocellular carcinoma (HepG2).

Iridium Complexes with BIAN-Type Ligands: Synthesis, Structure and Redox Chemistry.

A series of iridium complexes with bis(diisopropylphenyl)iminoacenaphtene (dpp-bian) ligands, [Ir(cod)(dpp-bian)Cl] (1), [Ir(cod)(NO)(dpp-bian)](BF4)2 (2) and [Ir(cod)(dpp-bian)](BF4) (3), were prepared and characterized by spectroscopic techniques, elemental analysis, X-ray diffraction analysis and cyclic voltammetry (CV). The structures of 1-3 feature a square planar backbone consisting of two C = C π-bonds of 1,5-cyclooctadiene (cod) and two nitrogen atoms of dpp-bian supplemented with a chloride ion (for 1) or a NO group (for 2) to complete a square-pyramidal geometry. In the nitrosyl complex 2, the Ir-N-O group has a bent geometry (the angle is 125°). The CV data for 1 and 3 show two reversible waves between 0 and -1.6 V (vs. Ag/AgCl). Reversible oxidation was also found at E1/2 = 0.60 V for 1. Magnetochemical measurements for 2 in a range from 1.77 to 300 K revealed an increase in the magnetic moment with increasing temperature up to 1.2 µB (at 300 K). Nitrosyl complex 2 is unstable in solution and loses its NO group to yield [Ir(cod)(dpp-bian)](BF4) (3). A paramagnetic complex, [Ir(cod)(dpp-bian)](BF4)2 (4), was also detected in the solution of 2 as a result of its decomposition. The EPR spectrum of 4 in CH2Cl2 is described by the spin Hamiltonian H = gßHS with S = 1/2 and gxx = gyy = 2.393 and gzz = 1.88, which are characteristic of the low-spin 5d7-Ir(II) state. DFT calculations were carried out in order to rationalize the experimental results.

Generation of induced pluripotent stem cell line (RCMGi009-A) from urine cells of patient with fibrodysplasia ossificans progressiva.

Urine cells obtained from a 14-year-old man with genetically proven (ACVR1: c.6176G > A) and clinically manifested fibrodysplasia ossificans progressiva were successfully transformed into induced pluripotent stem cells by using Sendai virus-based reprogramming vectors including the four Yamanaka factors such as OCT3/4, SOX2, KLF4, and c-MYC. These iPSCs expressed pluripotency markers, exhibited the potential to differentiate into three germ layers in spontaneous differentiation assay and had a normal karyotype. The iPSC line may provide a model for development of a personalized treatment including genome editing and drug screening, may be used for disease modelling, cell differentiation and pharmacological investigations. .

Semiconducting Electronic Structure of the Ferromagnetic Spinel HgCr_{2}Se_{4} Revealed by Soft-X-Ray Angle-Resolved Photoemission Spectroscopy.

We study the electronic structure of the ferromagnetic spinel HgCr_{2}Se_{4} by soft-x-ray angle-resolved photoemission spectroscopy (SX-ARPES) and first-principles calculations. While a theoretical study has predicted that this material is a magnetic Weyl semimetal, SX-ARPES measurements give direct evidence for a semiconducting state in the ferromagnetic phase. Band calculations based on the density functional theory with hybrid functionals reproduce the experimentally determined band gap value, and the calculated band dispersion matches well with ARPES experiments. We conclude that the theoretical prediction of a Weyl semimetal state in HgCr_{2}Se_{4} underestimates the band gap, and this material is a ferromagnetic semiconductor.

Bridging Gaps in HDR Improvement: The Role of MAD2L2, SCAI, and SCR7.

This study aimed to enhance homology-directed repair (HDR) efficiency in CRISPR/Cas-mediated genome editing by targeting three key factors regulating the balance between HDR and non-homologous end joining (NHEJ): MAD2L2, SCAI, and Ligase IV. In order to achieve this, a cellular model using mutated eGFP was designed to monitor HDR events. Results showed that MAD2L2 knockdown and SCR7 treatment significantly improved HDR efficiency during Cas9-mediated HDR repair of the mutated eGFP gene in the HEK293T cell line. Fusion protein Cas9-SCAI did not improve HDR. This study is the first to demonstrate that MAD2L2 knockdown during CRISPR-mediated gene editing in HEK293T cells can increase precise correction by up to 10.2 times. The study also confirmed a moderate but consistent effect of SCR7, an inhibitor of Ligase IV, which increased HDR by 1.7 times. These findings provide valuable insights into improving HDR-based genome editing efficiency.

Airway and Lung Organoids from Human-Induced Pluripotent Stem Cells Can Be Used to Assess CFTR Conductance.

Airway and lung organoids derived from human-induced pluripotent stem cells (hiPSCs) are current models for personalized drug screening, cell-cell interaction studies, and lung disease research. We analyzed the existing differentiation protocols and identified the optimal conditions for obtaining organoids. In this article, we describe a step-by-step protocol for differentiating hiPSCs into airway and lung organoids. We obtained airway and lung organoids from a healthy donor and from five donors with cystic fibrosis. Analysis of the cellular composition of airway and lung organoids showed that airway organoids contain proximal lung epithelial cells, while lung organoids contain both proximal and distal lung epithelial cells. Forskolin-induced swelling of organoids derived from a healthy donor showed that lung organoids, as well as airway organoids, contain functional epithelial cells and swell after 24 h exposure to forskolin, which makes it a suitable model for analyzing the cystic fibrosis transmembrane conductance regulator (CFTR) channel conductance in vitro. Thus, our results demonstrate the feasibility of generating and characterizing airway and lung organoids from hiPSCs, which can be used for a variety of future applications.

A Series of Metal-Organic Frameworks with 2,2'-Bipyridyl Derivatives: Synthesis vs. Structure Relationships, Adsorption, and Magnetic Studies.

Five new metal-organic frameworks based on Mn(II) and 2,2'-bithiophen-5,5'-dicarboxylate (btdc2-) with various chelating N-donor ligands (2,2'-bipyridyl = bpy; 5,5'-dimethyl-2,2'-bipyridyl = 5,5'-dmbpy; 4,4'-dimethyl-2,2'-bipyridyl = 4,4'-dmbpy) [Mn3(btdc)3(bpy)2]·4DMF, 1; [Mn3(btdc)3(5,5'-dmbpy)2]·5DMF, 2; [Mn(btdc)(4,4;-dmbpy)], 3; [Mn2(btdc)2(bpy)(dmf)]·0.5DMF, 4; [Mn2(btdc)2(5,5'-dmbpy)(dmf)]·DMF, 5 (dmf, DMF = N,N-dimethylformamide) have been synthesized, and their crystal structure has been established using single-crystal X-ray diffraction analysis (XRD). The chemical and phase purities of Compounds 1-3 have been confirmed via powder X-ray diffraction, thermogravimetric, and chemical analyses as well as IR spectroscopy. The influence of the bulkiness of the chelating N-donor ligand on the dimensionality and structure of the coordination polymer has been analyzed, and the decrease in the framework dimensionality, as well as the secondary building unit's nuclearity and connectivity, has been observed for bulkier ligands. For three-dimensional (3D) coordination polymer 1, the textural and gas adsorption properties have been studied, revealing noticeable ideal adsorbed solution theory (IAST) CO2/N2 and CO2/CO selectivity factors (31.0 at 273 K and 19.1 at 298 K and 25.7 at 273 K and 17.0 at 298 K, respectively, for the equimolar composition and the total pressure of 1 bar). Moreover, significant adsorption selectivity for binary C2-C1 hydrocarbons mixtures (33.4 and 24.9 for C2H6/CH4, 24.8 and 17.7 for C2H4/CH4, 29.3 and 19.1 for C2H2/CH4 at 273 K and 298 K, respectively, for the equimolar composition and the total pressure of 1 bar) has been observed, making it possible to separate on 1 natural, shale, and associated petroleum gas into valuable individual components. The ability of Compound 1 to separate benzene and cyclohexane in a vapor phase has also been analyzed based on the adsorption isotherms of individual components measured at 298 K. The preferable adsorption of C6H6 over C6H12 by 1 at high vapor pressures (VB/VCH = 1.36) can be explained by the existence of multiple van der Waals interactions between guest benzene molecules and the metal-organic host revealed by the XRD analysis of 1 immersed in pure benzene for several days (1≅2C6H6). Interestingly, at low vapor pressures, an inversed behavior of 1 with preferable adsorption of C6H12 over C6H6 (KCH/KB = 6.33) was observed; this is a very rare phenomenon. Moreover, magnetic properties (the temperature-dependent molar magnetic susceptibility, χp(T) and effective magnetic moments, µeff(T), as well as the field-dependent magnetization, M(H)) have been studied for Compounds 1-3, revealing paramagnetic behavior consistent with their crystal structure.

Halogen Bonding Channels for Magnetic Exchange in Cu(II) Complexes with 2,5-Di(methylthio)-1,3,4-thiadiazole.

Copper(II) complexes with 2,5-bis(methylthio)-1,3,4-thiadiazole (tda) formulated as [Cu(tda)n X2 ] (n=2, X=Cl- , Br- , C2 N3 - ; n= 1, X=C2 N3 - ) have been isolated and fully characterized. The crystal structures of all compounds have been determined using single-crystal X-ray diffraction (SCXRD). A study of the magnetic susceptibility in the range 1.77-300 K has shown that magnetic properties of the [Cu(tda)2 Cl2 ] and [Cu(tda)2 Br2 ] complexes match those of 1D chains of antiferromagnetically-coupled Cu2+ ions. The intrachain interaction J in [Cu(tda)2 Cl2 ] turns out to be ∼1.2 times weaker than in its bromide analogue. In its turn, [Cu(tda)2 (C2 N3 )2 ] exhibits J being an order of magnitude smaller and of the opposite ferromagnetic sign. Halogen bonding (HB) between adjacent complexes is much stronger than the H-bonds or π-π interactions between tda ligands according to the DFT calculations.

Methods for CRISPR-Cas as Ribonucleoprotein Complex Delivery In Vivo.

The efficient delivery of CRISPR-Cas components is still a key and unsolved problem. CRISPR-Cas delivery in the form of a Cas protein+sgRNA (ribonucleoprotein complex, RNP complex), has proven to be extremely effective, since it allows to increase on-target activity, while reducing nonspecific activity. The key point for in vivo genome editing is the direct delivery of artificial nucleases and donor DNA molecules into the somatic cells of an adult organism. At the same time, control of the dose of artificial nucleases is impossible, which affects the efficiency of genome editing in the affected cells. Poor delivery efficiency and low editing efficacy reduce the overall potency of the in vivo genome editing process. Here we review how this problem is currently being solved in scientific works and what types of in vivo delivery methods of Cas9/sgRNA RNPs have been developed.

Spectral Photoluminescent Parameters of Barley Seeds (Hordéum vulgáre) Infected with Fusarium ssp.

We needed effective and sustainable technologies for better microbiological control of crops, including Fusarium. However, photoluminescent UV-Vis methods are potential for diagnosing plant diseases with Fusarium. It has not been sufficiently studied despite the application of these methods for other biological researches. The excitation spectrum of the seeds during infection shifts to the shorter wavelength and a new maximum appears in the region λ ≈ 232 nm. The photoluminescence of infected seeds increases with excitation by radiation of wavelengths λe,1 = 232 nm, λe,2 = 362 nm and λe,3 = 424 nm by 1.33-3.14 times, and λe,3 = 424 nm-decreases by 1.1 times. Statistical moments µ3 and µ4 , asymmetry and kurtosis change only with short-wave excitation. When analyzing the decomposition of the frequency spectrum into Gaussian curves, the most informative ratio is the ratio of right-handed and left-handed Gaussians under excitation λe,2 = 362 nm and λe,3 = 424 nm. The ratios of their maxima change during infection by 1.36-3.2 times, and for excitation by radiation λe,2 , the frequency boundaries of Gaussians change. The results of measurements and calculations provide a basis for the development of a method and device for photoluminescence diagnostics of fusarium seeds in UV-Vis ranges.

Structure and Properties of Heterometallics Based on Lanthanides and Transition Metals with Methoxy-ß-Diketonates.

The possibility of obtaining volatile polynuclear heterometallic complexes containing lanthanides and transition metals bound by methoxy-ß-diketonates was studied. New compounds were prepared by cocrystallization of monometallic complexes from organic solvents. Ln(tmhd)3 were used as initial monometallic complexes (Ln = La, Pr, Sm, Gd, Tb, Dy, Lu; tmhd = 2,2,6,6-tetramethylheptane-3,5-dionate) in combination with TML2 in various ratios (TM = Cu, Co, Ni, Mn; L: L1 = 1,1,1-trifluoro-5,5-dimethoxypentane-2,4-dionate, L2 = 1,1,1-trifluoro-5,5-dimethoxy-hexane-2,4-dionate, L3 = 1,1,1-trifluoro-5-methoxy-5-methylhexane-2,4-dionate). Heterometallic complexes of the composition [(LnL2tmhd)2TM(tmhd)2] were isolated for light lanthanides Ln= La, Pr, Sm, Gd, and L= L1 or L2. By single crystal XRD, it has been established that heterometallic compounds containing La, Pr, Cu, Co, and Ni are isostructural linear coordination polymers of alternating mononuclear transition metal complexes and binuclear heteroleptic lanthanide complexes, connected by donor-acceptor interactions between oxygen atoms of the methoxy groups and transition metal atoms. A comparison of powder XRD patterns has shown that all heterometallic complexes obtained are isostructural. Havier lanthanides Ln = Tb, Dy, Lu did not form heterometallics. Instead, homometallic complexes Ln(L3)3 were identified for Ln = Dy, Lu as well as for Ln = La. The thermal properties of the complexes were investigated by TG-DTA and vacuum sublimation tests. The heterometallic complexes were found to be not volatile and decomposed under heating to produce inorganic composites of TM oxides and Ln fluorides. In contrast, Ln(L3)3 is volatile and may be sublimed in a vacuum. Results of magnetic measurements are discussed for several heterometallic and homometallic complexes.

Generation of two induced pluripotent stem cell lines (RCMGi005-A/B) from human skin fibroblasts of a cystic fibrosis patient with homozygous F508del mutation in CFTR gene.

Induced pluripotent stem cells (iPSCs) was successfully generated from skin fibroblast obtained from patient with cystic fibrosis by using non-integrating, viral CytoTune™-iPS 2.0 Sendai Reprogramming Kit, which contain three vectors preparation: polycistronic Klf4-Oct3/4-Sox2, cMyc, and Klf4. Created iPSC lines showed a normal karyotype, expressed pluripotency markers and demonstrated the potential to differentiate into three germ layers in spontaneous differentiation assay.

Lung organoids: current strategies for generation and transplantation.

Lung diseases occupy a leading position in human morbidity and are the third leading cause of death. Often the chronic forms of these diseases do not respond to therapy, so that lung transplantation is the only treatment option. The development of cellular and biotechnologies offers a new solution-the use of lung organoids for transplantation in such patients. Here, we review types of lung organoids, methods of their production and characterization, and experimental works on transplantation in vivo. These results show the promise of work in this direction. Despite the current problems associated with a low degree of cell engraftment, immune response, and insufficient differentiation, we are confident that organoid transplantation will find it is clinical application.

Dysferlinopathies: Clinical and genetic variability.

Dysferlinopathies are a clinically heterogeneous group of diseases caused by mutations in the DYSF gene encoding the dysferlin protein. Dysferlin is mostly expressed in muscle tissues and is localized in the sarcolemma, where it performs its main function of resealing and maintaining of the integrity of the cell membrane. At least four forms of dysferlinopathies have been described: Miyoshi myopathy, limb-girdle muscular dystrophy type 2B, distal myopathy with anterior tibial onset, and isolated hyperCKemia. Here we review the clinical features of different forms of dysferlinopathies and attempt to identify genotype-phenotype correlations. Because of the great clinical variability and rarety of the disease and mutations little is known, how different phenotypes develop as a result of different mutations. However, missense mutations seem to induce more severe disease than LoF, which is typical for many muscle dystrophies. The role of several specific mutations and possible gene modifiers is also discussed in the paper.

Complex Diagnostics of Non-Specific Intellectual Developmental Disorder.

Intellectual development disorder (IDD) is characterized by a general deficit in intellectual and adaptive functioning. In recent years, there has been a growing interest in studying the genetic structure of IDD. Of particular difficulty are patients with non-specific IDD, for whom it is impossible to establish a clinical diagnosis without complex genetic diagnostics. We examined 198 patients with non-specific IDD from 171 families using whole-exome sequencing and chromosome microarray analysis. Hereditary forms of IDD account for at least 35.7% of non-specific IDD, of which 26.9% are monogenic forms. Variants in the genes associated with the BAF (SWI/SNF) complex were the most frequently identified. We were unable to identify phenotypic features that would allow differential diagnosis of monogenic and microstructural chromosomal rearrangements in non-specific IDD at the stage of clinical examination, but due to its higher efficiency, exome sequencing should be the diagnostic method of the highest priority study after the standard examination of patients with NIDD in Russia.