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.

COP9 signalosome component CSN-5 stabilizes PUF proteins FBF-1 and FBF-2 in Caenorhabditis elegans germline stem and progenitor cells.

RNA-binding proteins FBF-1 and FBF-2 (FBFs) are required for germline stem cell maintenance and the sperm/oocyte switch in Caenorhabditis elegans, although the mechanisms controlling FBF protein levels remain unknown. We identified an interaction between both FBFs and CSN-5), a component of the constitutive photomorphogenesis 9 (COP9) signalosome best known for its role in regulating protein degradation. Here, we find that the Mpr1/Pad1 N-terminal metalloprotease domain of CSN-5 interacts with the Pumilio and FBF RNA-binding domain of FBFs and the interaction is conserved for human homologs CSN5 and PUM1. The interaction between FBF-2 and CSN-5 can be detected in vivo by proximity ligation. csn-5 mutation results in the destabilization of FBF proteins, which may explain previously observed decrease in the numbers of germline stem and progenitor cells, and disruption of oogenesis. The loss of csn-5 does not decrease the levels of a related PUF protein PUF-3, and csn-5(lf) phenotype is not enhanced by fbf-1/2 knockdown, suggesting that the effect is specific to FBFs. The effect of csn-5 on oogenesis is largely independent of the COP9 signalosome and is cell autonomous. Surprisingly, the regulation of FBF protein levels involves a combination of COP9-dependent and COP9-independent mechanisms differentially affecting FBF-1 and FBF-2. This work supports a previously unappreciated role for CSN-5 in the stabilization of germline stem cell regulatory proteins FBF-1 and FBF-2.

Neural Network Corrections to Intermolecular Interaction Terms of a Molecular Force Field Capture Nuclear Quantum Effects in Calculations of Liquid Thermodynamic Properties.

We incorporate nuclear quantum effects (NQE) in condensed matter simulations by introducing short-range neural network (NN) corrections to the ab initio fitted molecular force field ARROW. Force field NN corrections are fitted to average interaction energies and forces of molecular dimers, which are simulated using the Path Integral Molecular Dynamics (PIMD) technique with restrained centroid positions. The NN-corrected force field allows reproduction of the NQE for computed liquid water and methane properties such as density, radial distribution function (RDF), heat of evaporation (HVAP), and solvation free energy. Accounting for NQE through molecular force field corrections circumvents the need for explicit computationally expensive PIMD simulations in accurate calculations of the properties of chemical and biological systems. The accuracy and locality of pairwise NN NQE corrections indicate that this approach could be applicable to complex heterogeneous systems, such as proteins.

Combining Force Fields and Neural Networks for an Accurate Representation of Bonded Interactions.

We present a formalism of a neural network encoding bonded interactions in molecules. This intramolecular encoding is consistent with the models of intermolecular interactions previously designed by this group. Variants of the encoding fed into a corresponding neural network may be used to economically improve the representation of torsional degrees of freedom in any force field. We test the accuracy of the reproduction of the ab initio potential energy surface on a set of conformations of two dipeptides, methyl-capped ALA and ASP, in several scenarios. The encoding, either alone or in conjunction with an analytical potential, improves agreement with ab initio energies that are on par with those of other neural network-based potentials. Using the encoding and neural nets in tandem with an analytical model places the agreements firmly within "chemical accuracy" of ±0.5 kcal/mol.

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.

Combining Force Fields and Neural Networks for an Accurate Representation of Chemically Diverse Molecular Interactions.

A key goal of molecular modeling is the accurate reproduction of the true quantum mechanical potential energy of arbitrary molecular ensembles with a tractable classical approximation. The challenges are that analytical expressions found in general purpose force fields struggle to faithfully represent the intermolecular quantum potential energy surface at close distances and in strong interaction regimes; that the more accurate neural network approximations do not capture crucial physics concepts, e.g., nonadditive inductive contributions and application of electric fields; and that the ultra-accurate narrowly targeted models have difficulty generalizing to the entire chemical space. We therefore designed a hybrid wide-coverage intermolecular interaction model consisting of an analytically polarizable force field combined with a short-range neural network correction for the total intermolecular interaction energy. Here, we describe the methodology and apply the model to accurately determine the properties of water, the free energy of solvation of neutral and charged molecules, and the binding free energy of ligands to proteins. The correction is subtyped for distinct chemical species to match the underlying force field, to segment and reduce the amount of quantum training data, and to increase accuracy and computational speed. For the systems considered, the hybrid ab initio parametrized Hamiltonian reproduces the two-body dimer quantum mechanics (QM) energies to within 0.03 kcal/mol and the nonadditive many-molecule contributions to within 2%. Simulations of molecular systems using this interaction model run at speeds of several nanoseconds per day.

Replication stress causes delayed mitotic entry and chromosome 12 fragility at the ANKS1B large neuronal gene in human induced pluripotent stem cells.

Substantial background level of replication stress is a feature of embryonic and induced pluripotent stem cells (iPSCs), which can predispose to numerical and structural chromosomal instability, including recurrent aberrations of chromosome 12. In differentiated cells, replication stress-sensitive genomic regions, including common fragile sites, are widely mapped through mitotic chromosome break induction by mild aphidicolin treatment, an inhibitor of replicative polymerases. IPSCs exhibit lower apoptotic threshold and higher repair capacity hindering fragile site mapping. Caffeine potentiates genotoxic effects and abrogates G2/M checkpoint delay induced by chemical and physical mutagens. Using 5-ethynyl-2'-deoxyuridine (EdU) for replication labeling, we characterized the mitotic entry dynamics of asynchronous iPSCs exposed to aphidicolin and/or caffeine. Under the adjusted timing of replication stress exposure accounting revealed cell cycle delay, higher metaphase chromosome breakage rate was observed in iPSCs compared to primary lymphocytes. Using differential chromosome staining and subsequent locus-specific fluorescent in situ hybridization, we mapped the FRA12L fragile site spanning the large neuronal ANKS1B gene at 12q23.1, which may contribute to recurrent chromosome 12 missegregation and rearrangements in iPSCs. Publicly available data on the ANKS1B genetic alterations and their possible functional impact are reviewed. Our study provides the first evidence of common fragile site induction in iPSCs and reveals potential somatic instability of a clinically relevant gene during early human development and in vitro cell expansion.

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. .

Tripartite interactions between filamentous Pf4 bacteriophage, Pseudomonas aeruginosa, and bacterivorous nematodes.

The opportunistic pathogen Pseudomonas aeruginosa PAO1 is infected by the filamentous bacteriophage Pf4. Pf4 virions promote biofilm formation, protect bacteria from antibiotics, and modulate animal immune responses in ways that promote infection. Furthermore, strains cured of their Pf4 infection (ΔPf4) are less virulent in animal models of infection. Consistently, we find that strain ΔPf4 is less virulent in a Caenorhabditis elegans nematode infection model. However, our data indicate that PQS quorum sensing is activated and production of the pigment pyocyanin, a potent virulence factor, is enhanced in strain ΔPf4. The reduced virulence of ΔPf4 despite high levels of pyocyanin production may be explained by our finding that C. elegans mutants unable to sense bacterial pigments through the aryl hydrocarbon receptor are more susceptible to ΔPf4 infection compared to wild-type C. elegans. Collectively, our data support a model where suppression of quorum-regulated virulence factors by Pf4 allows P. aeruginosa to evade detection by innate host immune responses.

Disruption of C. elegans embryonic P granules upon dlc-1(RNAi) is not associated with P granule component loss.

Dynein light chain (DLC-1) is a light chain component of the dynein motor complex, it functions as an allosteric regulator of multi-subunit protein complexes and promotes P granule integrity in the C. elegans embryo. P granules are RNA-protein complexes located in the C. elegans germline that are important for RNA regulation and fertility. To further study the role of DLC-1 during C. elegans embryogenesis we performed quantitative tandem mass tag mass spectrometry on embryos after dlc-1 knock down. The amount of core P granule components and nucleoporin proteins did not change after dlc-1(RNAi). These results show that DLC-1 does not help regulate P granule protein levels and support the model that DLC-1 facilitates phase separation of P granule components in vivo .

Protein-Ligand Binding Free-Energy Calculations with ARROW─A Purely First-Principles Parameterized Polarizable Force Field.

Protein-ligand binding free-energy calculations using molecular dynamics (MD) simulations have emerged as a powerful tool for in silico drug design. Here, we present results obtained with the ARROW force field (FF)─a multipolar polarizable and physics-based model with all parameters fitted entirely to high-level ab initio quantum mechanical (QM) calculations. ARROW has already proven its ability to determine solvation free energy of arbitrary neutral compounds with unprecedented accuracy. The ARROW FF parameterization is now extended to include coverage of all amino acids including charged groups, allowing molecular simulations of a series of protein-ligand systems and prediction of their relative binding free energies. We ensure adequate sampling by applying a novel technique that is based on coupling the Hamiltonian Replica exchange (HREX) with a conformation reservoir generated via potential softening and nonequilibrium MD. ARROW provides predictions with near chemical accuracy (mean absolute error of ∼0.5 kcal/mol) for two of the three protein systems studied here (MCL1 and Thrombin). The third protein system (CDK2) reveals the difficulty in accurately describing dimer interaction energies involving polar and charged species. Overall, for all of the three protein systems studied here, ARROW FF predicts relative binding free energies of ligands with a similar accuracy level as leading nonpolarizable force fields.

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.

Generation of induced pluripotent stem cell line (RCMGi008-A) from human skin fibroblasts of a cystic fibrosis patient with compound heterozygous F508del/CFTRdele2.3 mutations in CFTR gene.

Skin fibroblasts obtained from a 20-year-old woman with clinically manifested and genetically proven (F508del/CFTRdele2.3) cystic fibrosis were successfully transformed into induced pluripotent stem cells (iPSCs) by using Sendai virus-based reprogramming vectors including the four Yamanaka factors, OCT3/4, SOX2, KLF4, and c-MYC. The iPSCs showed a normal karyotype, expressed pluripotency markers and exhibited the potential to differentiate into three germ layers in spontaneous differentiation assay. This iPSC line may be used for development of a personalized treatment including genome editing, disease modelling, cell differentiation and organoid formation, pharmacological investigations and drug screening.

DLC-1 facilitates germ granule assembly in Caenorhabditis elegans embryo.

Germ granules are cytoplasmic assemblies of RNA-binding proteins (RBPs) required for germ cell development and fertility. During the first four cell divisions of the Caenorhabditis elegans zygote, regulated assembly of germ (P) granules leads to their selective segregation to the future germ cell. Here we investigate the role of DLC-1, a hub protein implicated in stabilization and function of diverse protein complexes, in maintaining P granule integrity. We find that DLC-1 directly interacts with several core P granule proteins, predominantly during embryogenesis. The loss of dlc-1 disrupts assembly of P granule components into phase-separated organelles in the embryos, regardless of whether or not DLC-1 directly interacts with these proteins. Finally, we infer that P granule dispersal in the absence of dlc-1 is likely independent of DLC-1's function as a subunit of the dynein motor and does not result from a loss of cell polarity.

Accurate determination of solvation free energies of neutral organic compounds from first principles.

The main goal of molecular simulation is to accurately predict experimental observables of molecular systems. Another long-standing goal is to devise models for arbitrary neutral organic molecules with little or no reliance on experimental data. While separately these goals have been met to various degrees, for an arbitrary system of molecules they have not been achieved simultaneously. For biophysical ensembles that exist at room temperature and pressure, and where the entropic contributions are on par with interaction strengths, it is the free energies that are both most important and most difficult to predict. We compute the free energies of solvation for a diverse set of neutral organic compounds using a polarizable force field fitted entirely to ab initio calculations. The mean absolute errors (MAE) of hydration, cyclohexane solvation, and corresponding partition coefficients are 0.2 kcal/mol, 0.3 kcal/mol and 0.22 log units, i.e. within chemical accuracy. The model (ARROW FF) is multipolar, polarizable, and its accompanying simulation stack includes nuclear quantum effects (NQE). The simulation tools' computational efficiency is on a par with current state-of-the-art packages. The construction of a wide-coverage molecular modelling toolset from first principles, together with its excellent predictive ability in the liquid phase is a major advance in biomolecular simulation.

Transfer of correlations from photons to electron excitations and currents induced in semiconductor quantum wells by non-classical twisted light.

In this paper the excitations of collective electronic modes and currents induced in nanostructured semiconductor systems by two-mode quantum light with non-zero orbital angular momenta are investigated. Transfer of photon correlations to the excitations and currents induced in the semiconductor system is demonstrated. Birth of correlated electrons arising in the conduction band of the nanostructure due to the interaction with correlated photons of quantum light is found. Azimuthal and radial spatial distributions of the entangled electrons are established. The obtained results make possible to register the correlated electrons experimentally and to implement quantum information and nanoelectronics circuits in nanosystems using the found azimuthal and radial electron entanglement.

Behavior of implanted Xe, Kr and Ar in nanodiamonds and thin graphene stacks: experiment and modeling.

Implantation and subsequent behaviour of heavy noble gases (Ar, Kr, and Xe) in few-layer graphene sheets and in nanodiamonds are studied both using computational methods and experimentally using X-ray absorption spectroscopy. X-ray absorption spectroscopy provides substantial support for Xe-vacancy (Xe-V) defects as main sites for Xe in nanodiamonds. It is shown that noble gases in thin graphene stacks distort the layers, forming bulges. The energy of an ion placed in between flat graphene sheets is notably lower than that in domains with high curvature. However, if the ion is trapped in the curved domain, considerable additional energy is required to displace it. This phenomenon is likely responsible for strong binding of noble gases implanted into disordered carbonaceous phase in meteorites (the Q-component).

Heavy alcohol drinking and subclinical echocardiographic abnormalities of structure and function.

OBJECTIVE: The aim of the study is to assess changes in heart structure and function associated with heavy alcohol use by comparing echocardiographic indices in a population-based sample to those in patients admitted to an inpatient facility with severe alcohol problems. METHODS AND RESULTS: We used data from the Know Your Heart study (2015-2017) which is a cross-sectional study that recruited 2479 participants aged 35-69 years from the general population of the city of Arkhangelsk in Northwest Russia and 278 patients from the Arkhangelsk Regional Psychiatric Hospital with a primary diagnosis related to chronic alcohol use (narcology clinic subsample). The drinking patterns of the population-based sample were characterised in detail. We used regression models controlling for age, sex, smoking, education and waist to hip ratio to evaluate the differences in echocardiographic indices in participants with different drinking patterns. The means of left ventricular end-diastolic diameter and indexed left atrial systolic diameter were increased among heavy drinkers (narcology clinic subsample), while mean left ventricular ejection fraction was decreased in this group compared with the population-based sample. In contrast, the harmful and hazardous drinkers in the population-based sample did not differ from non-problem drinkers with respect to echocardiographic indices of systolic and diastolic function. CONCLUSIONS: Extremely heavy drinking is associated with a specific set of structural and functional abnormalities of the heart that may be regarded as precursors of alcohol-related dilated cardiomyopathy.

Derivation of iPSC line (RCMGi002-A) from dermal fibroblasts of a cystic fibrosis female patient with homozygous F508del mutation.

Cystic fibrosis is one of the most common inherited diseases caused by mutations in CFTR gene, of which F508del is the most frequent. Currently, the possibility of cell therapy including genome editing is widely discussed. We generated induced pluripotent stem cells from fibroblasts obtained from a 22-year-old woman with clinically manifested and genetically proven disease by using non-viral, non-integrating RNA reprogramming vector that contains five reprogramming factors: OCT4, KLF4, SOX2, GLIS1, and c-MYC. The established cell line can express endogenous pluripotency markers, possesses a normal karyotype, and has the ability to differentiate into three germ layers in spontaneous differentiation assay.

Generation of two induced pluripotent stem cell lines (RCMGi004-A and -B) from human skin fibroblasts of a cystic fibrosis patient with compound heterozygous F508del/W1282X mutations in CFTR gene.

Skin fibroblasts obtained from a 28-year-old man with clinically manifested and genetically proven (F508del/W1282X) cystic fibrosis were successfully transformed into induced pluripotent stem cells (iPSCs) by using non-viral, non-integrating, self-replicating RNA reprogramming vectorthat contains five reprogramming factors: OCT4, KLF4, SOX2, GLIS1, and c-MYC as well as a puromycin-resistance gene. Two iPSC lines showed a normal karyotype, expressed pluripotency markers and exhibited the potential to differentiate into three germ layers in spontaneous differentiation assay. These iPSC lines may be subsequently used for development of a personalized etiotropic treatment,disease modelling, cell differentiation and organoid formation, pharmacological investigations and drug screening.