Prognostic Analysis of Human Pluripotent Stem Cells Based on Their Morphological Portrait and Expression of Pluripotent Markers

The ability of human pluripotent stem cells for unlimited proliferation and self-renewal promotes their application in the fields of regenerative medicine. The morphological assessment of growing colonies and cells, as a non-invasive method, allows the best clones for further clinical applications to be safely selected. For this purpose, we analyzed seven morphological parameters of both colonies and cells extracted from the phase-contrast images of human embryonic stem cell line H9, control human induced pluripotent stem cell (hiPSC) line AD3, and hiPSC line HPCASRi002-A (CaSR) in various passages during their growth for 120 h. The morphological phenotype of each colony was classified using a visual analysis and associated with its potential for pluripotency and clonality maintenance, thus defining the colony phenotype as the control parameter. Using the analysis of variance for the morphological parameters of each line, we showed that selected parameters carried information about different cell lines and different phenotypes within each line. We demonstrated that a model of classification of colonies and cells by phenotype, built on the selected parameters as predictors, recognized the phenotype with an accuracy of 70–75%. In addition, we performed a qRT-PCR analysis of eleven pluripotency markers genes. By analyzing the variance of their expression in samples from different lines and with different phenotypes, we identified group-specific sets of genes that could be used as the most informative ones for the separation of the best clones. Our results indicated the fundamental possibility of constructing a morphological portrait of a colony informative for the automatic identification of the phenotype and for linking this portrait to the expression of pluripotency markers.     

Photoprocesses in Derivatives of 1,4- and 1,3-Diazadistyryldibenzenes

Photoprocesses in 1,4-diazadistyrylbenzene (1) and 1,3-diazadistyrylbenzene derivative (2) diperchlorates in MeCN were studied by absorption, luminescence, and kinetic laser spectroscopies. For compound 1, trans-cis-photoisomerization and intersystem crossing to a triplet state are observed. For compound 2, photoelectrocyclization is suggested. Quantum chemical calculations of diazadistyrylbenzene structures in the ground and excited states were carried out. The schemes for photoreactions were proposed.     

Synthesis,Structure, Spectral-Luminescent Properties,and Biological Activity of Chlorine-Substituted N-[2-(Phenyliminomethyl)phenyl]-4-methylbenzenesulfamide and Their Zinc(II) Complexes

New azomethine compounds of 2-(N-tosylamino)benzaldehyde or 5-chloro-2-(N-tosylamino)benzaldehyde and the corresponding chlorine-substituted anilines, zinc(II) complexes based on them have been synthesized. The structures of azomethines and their complexes were determined by elemental analysis, IR, ¹H NMR, X-ray spectroscopy, and X-ray diffraction. It is found that all ZnL₂ complexes have a tetrahedral structure according to XAFS and X-ray diffraction data. The photoluminescent properties of azomethines and zinc complexes in methylene chloride solution and in solid form have been studied. It is shown that the photoluminescence quantum yields of solid samples of the complexes are an order of magnitude higher compared to the solutions and range from 11.34% to 48.3%. The thermal properties of Zn(II) complexes were determined by thermal gravimetric analysis (TGA) and differential scanning calorimetry. The TGA curves of all the compounds suggest their high thermal stability up to temperatures higher than 290 °C. The electrochemical properties of all complexes were investigated by the cyclic voltammetry method. The multilayered devices ITO/PEDOT:PSS/NPD/Zn complex/ TPBI/LiF/Al with wide electroluminescence (EL) color range spanning the range from bluish-green (494 nm) to green (533 nm) and the high values of brightness, current and power efficiency were fabricated. The biological activity of azomethines and zinc complexes has been studied. In the case of complexes, the protistocidal activity of the zinc complex with azomethine of 5-chloro-2-(N-tosylamino)benzaldehyde with 4-chloroaniline was two times higher than the activity of the reference drug toltrazuril.     

Experimental Crossing Confirms Reproductive Isolation between Cryptic Species within Eulimnogammarus verrucosus (Crustacea: Amphipoda) from Lake Baikal

Ancient lakes are known speciation hotspots. One of the most speciose groups in the ancient Lake Baikal are gammaroid amphipods (Crustacea: Amphipoda: Gammaroidea). There are over 350 morphological species and subspecies of amphipods in Baikal, but the extent of cryptic variation is still unclear. One of the most common species in the littoral zone of the lake, Eulimnogammarus verrucosus (Gerstfeldt, 1858), was recently found to comprise at least three (pseudo)cryptic species based on molecular data. Here, we further explored these species by analyzing their mitogenome-based phylogeny, genome sizes with flow cytometry, and their reproductive compatibility. We found divergent times of millions of years and different genome sizes in the three species (6.1, 6.9 and 8 pg), further confirming their genetic separation. Experimental crossing of the western and southern species, which are morphologically indistinguishable and have adjacent ranges, showed their separation with a post-zygotic reproductive barrier, as hybrid embryos stopped developing roughly at the onset of gastrulation. Thus, the previously applied barcoding approach effectively indicated the separate biological species within E. verrucosus. These results provide new data for investigating genome evolution and highlight the need for precise tracking of the sample origin in any studies in this morphospecies.     

Partial Reduction in BRCA1 Gene Dose Modulates DNA Replication Stress Level and Thereby Contributes to Sensitivity or Resistance

BRCA1 is a well-known breast cancer risk gene, involved in DNA damage repair via homologous recombination (HR) and replication fork protection. Therapy resistance was linked to loss and amplification of the BRCA1 gene causing inferior survival of breast cancer patients. Most studies have focused on the analysis of complete loss or mutations in functional domains of BRCA1. How mutations in non-functional domains contribute to resistance mechanisms remains elusive and was the focus of this study. Therefore, clones of the breast cancer cell line MCF7 with indels in BRCA1 exon 9 and 14 were generated using CRISPR/Cas9. Clones with successful introduced BRCA1 mutations were evaluated regarding their capacity to perform HR, how they handle DNA replication stress (RS), and the consequences on the sensitivity to MMC, PARP1 inhibition, and ionizing radiation. Unexpectedly, BRCA1 mutations resulted in both increased sensitivity and resistance to exogenous DNA damage, despite a reduction of HR capacity in all clones. Resistance was associated with improved DNA double-strand break repair and reduction in replication stress (RS). Lower RS was accompanied by increased activation and interaction of proteins essential for the S phase-specific DNA damage response consisting of HR proteins, FANCD2, and CHK1.     

Molecular Characteristics,Receptor Specificity,and Pathogenicity of Avian Influenza Viruses Isolated from Wild Ducks in Russia

Avian influenza viruses (AIV) of wild ducks are known to be able to sporadically infect domestic birds and spread along poultry. Regular surveillance of AIV in the wild is needed to prepare for potential outbreaks. During long-year monitoring, 46 strains of AIV were isolated from gulls and mallards in Moscow ponds and completely sequenced. Amino acid positions that affect the pathogenicity of influenza viruses in different hosts were tested. The binding affinity of the virus for receptors analogs typical for different hosts and the pathogenicity of viruses for mice and chickens were investigated. Moscow isolates did not contain well-known markers of pathogenicity and/or adaptation to mammals, so as a polybasic cleavage site in HA, substitutions of 226Q and 228G amino acids in the receptor-binding region of HA, and substitutions of 627E and 701D amino acids in the PB2. The PDZ-domain ligand in the NS protein of all studied viruses contains the ESEV or ESEI sequence. Although several viruses had the N66S substitution in the PB1-F2 protein, all Moscow isolates were apathogenic for both mice and chickens. This demonstrates that the phenotypic manifestation of pathogenicity factors is not absolute but depends on the genome context.     

Temperature Rise Inside Shear Bands in a Simple Model Glass

One of the key factors, which hampers the application of metallic glasses as structural components, is the localization of deformation in narrow bands of a few tens up to one hundred nanometers thickness, the so-called shear bands. Processes, which occur inside shear bands are of central importance for the question whether a catastrophic failure of the material is unavoidable or can be circumvented or, at least, delayed. Via molecular dynamics simulations, this study addresses one of these processes, namely the local temperature rise due to viscous heat generation. The major contribution to energy dissipation is traced back to the plastic work performed by shear stress during steady deformation. Zones of largest strain contribute the most to this process and coincide with high-temperature domains (hottest spots) inside the sample. Magnitude of temperature rise can reach a few percent of the sample’s glass transition temperature. Consequences of these observations are discussed in the context of the current research in the field.     

Unpredicted Aberrant Splicing Products Identified in Postmortem Sudden Cardiac Death Samples

Molecular screening for pathogenic mutations in sudden cardiac death (SCD)-related genes is common practice for SCD cases. However, test results may lead to uncertainty because of the identification of variants of unknown significance (VUS) occurring in up to 70% of total identified variants due to a lack of experimental studies. Genetic variants affecting potential splice site variants are among the most difficult to interpret. The aim of this study was to examine rare intronic variants identified in the exonic flanking sequence to meet two main objectives: first, to validate that canonical intronic variants produce aberrant splicing; second, to determine whether rare intronic variants predicted as VUS may affect the splicing product. To achieve these objectives, 28 heart samples of cases of SCD carrying rare intronic variants were studied. Samples were analyzed using 85 SCD genes in custom panel sequencing. Our results showed that rare intronic variants affecting the most canonical splice sites displayed in 100% of cases that they would affect the splicing product, possibly causing aberrant isoforms. However, 25% of these cases (1/4) showed normal splicing, contradicting the in silico results. On the contrary, in silico results predicted an effect in 0% of cases, and experimental results showed >20% (3/14) unpredicted aberrant splicing. Thus, deep intron variants are likely predicted to not have an effect, which, based on our results, might be an underestimation of their effect and, therefore, of their pathogenicity classification and family members’ follow-up.