TransCRISPR-sgRNA design tool for CRISPR/Cas9 experiments targeting specific sequence motifs.

Eukaryotic genomes contain several types of recurrent sequence motifs, e.g. transcription factor motifs, miRNA binding sites, repetitive elements. CRISPR/Cas9 can facilitate identification and study of crucial motifs. We present transCRISPR, the first online tool dedicated to search for sequence motifs in the user-provided genomic regions and design optimal sgRNAs targeting them. Users can obtain sgRNAs for chosen motifs, for up to tens of thousands of target regions in 30 genomes, either for the Cas9 or dCas9 system. TransCRISPR provides user-friendly tables and visualizations, summarizing features of identified motifs and designed sgRNAs such as genomic localization, quality scores, closest transcription start sites and others. Experimental validation of sgRNAs for MYC binding sites designed with transCRISPR confirmed efficient disruption of the targeted motifs and effect on expression of MYC-regulated genes. TransCRISPR is available from https://transcrispr.igcz.poznan.pl/transcrispr/.

Core regions in immunoglobulin heavy chain enhancers essential for survival of non-Hodgkin lymphoma cells are identified by a CRISPR interference screen.

Chromosomal translocations in non-Hodgkin lymphoma (NHL) result in activation of oncogenes by placing them under the regulation of immunoglobulin heavy chain (IGH) super-enhancers. Aberrant expression of translocated oncogenes induced by enhancer activity can contribute to lymphomagenesis. The role of the IGH enhancers in normal B-cell development is well established, but knowledge regarding the precise mechanisms of their involvement in control of the translocated oncogenes is limited. The goal of this project was to define the critical regions in the IGH regulatory elements and identify enhancer RNAs (eRNA). We designed a sgRNA library densely covering the IGH enhancers and performed tiling CRISPR interference screens in three NHL cell lines. This revealed three regions crucial for NHL cell growth. With chromatin-enriched RNA-Seq we showed transcription from the core enhancer regions and subsequently validated expression of the eRNAs in a panel of NHL cell lines and tissue samples. Inhibition of the essential IGH enhancer regions decreased expression of eRNAs and translocated oncogenes in several NHL cell lines. The observed expression and growth patterns were consistent with the breakpoints in the IGH locus. Moreover, targeting the Eµ enhancer resulted in loss of B-cell receptor expression. In a Burkitt lymphoma cell line, MYC overexpression partially rescued the phenotype induced by IGH enhancer inhibition. Our results indicated the most critical regions in the IGH enhancers and provided new insights into the current understanding of the role of IGH enhancers in B-cell NHL. As such, this study forms a basis for development of potential therapeutic approaches.

In vivo secondary structural analysis of Influenza A virus genomic RNA.

Influenza A virus (IAV) is a respiratory virus that causes epidemics and pandemics. Knowledge of IAV RNA secondary structure in vivo is crucial for a better understanding of virus biology. Moreover, it is a fundament for the development of new RNA-targeting antivirals. Chemical RNA mapping using selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) coupled with Mutational Profiling (MaP) allows for the thorough examination of secondary structures in low-abundance RNAs in their biological context. So far, the method has been used for analyzing the RNA secondary structures of several viruses including SARS-CoV-2 in virio and in cellulo. Here, we used SHAPE-MaP and dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) for genome-wide secondary structure analysis of viral RNA (vRNA) of the pandemic influenza A/California/04/2009 (H1N1) strain in both in virio and in cellulo environments. Experimental data allowed the prediction of the secondary structures of all eight vRNA segments in virio and, for the first time, the structures of vRNA5, 7, and 8 in cellulo. We conducted a comprehensive structural analysis of the proposed vRNA structures to reveal the motifs predicted with the highest accuracy. We also performed a base-pairs conservation analysis of the predicted vRNA structures and revealed many highly conserved vRNA motifs among the IAVs. The structural motifs presented herein are potential candidates for new IAV antiviral strategies.

Electronic and Excitonic Properties of MSi2 Z4 Monolayers.

MA2 Z4 monolayers form a new class of hexagonal non-centrosymmetric materials hosting extraordinary spin-valley physics. While only two compounds (MoSi2 N4 and WSi2 N4 ) are recently synthesized, theory predicts interesting (opto)electronic properties of a whole new family of such two-dimensional (2D) materials. Here, the chemical trends of band gaps and spin-orbit splittings of bands in selected MSi2 Z4 (M = Mo, W; Z = N, P, As, Sb) compounds are studied from first-principles. Effective Bethe-Salpeter-equation-based calculations reveal high exciton binding energies. Evolution of excitonic energies under external magnetic field is predicted by providing their effective g-factors and diamagnetic coefficients, which can be directly compared to experimental values. In particular, large positive g-factors are predicted for excitons involving higher conduction bands. In view of these predictions, MSi2 Z4 monolayers yield a new platform to study excitons and are attractive for optoelectronic devices, also in the form of heterostructures. In addition, a spin-orbit induced bands inversion is observed in the heaviest studied compound, WSi2 Sb4 , a hallmark of its topological nature.

Strain-Induced Exciton Hybridization in WS_{2} Monolayers Unveiled by Zeeman-Splitting Measurements.

Mechanical deformations and ensuing strain are routinely exploited to tune the band gap energy and to enhance the functionalities of two-dimensional crystals. In this Letter, we show that strain leads also to a strong modification of the exciton magnetic moment in WS_{2} monolayers. Zeeman-splitting measurements under magnetic fields up to 28.5 T were performed on single, one-layer-thick WS_{2} microbubbles. The strain of the bubbles causes a hybridization of k-space direct and indirect excitons resulting in a sizable decrease in the modulus of the g factor of the ground-state exciton. These findings indicate that strain may have major effects on the way the valley number of excitons can be used to process binary information in two-dimensional crystals.

T-psi-C: user friendly database of tRNA sequences and structures.

tRNAs have been widely studied for their role as genetic code decoders in the ribosome during translation, but have recently received new attention due to the discovery of novel roles beyond decoding, often in connection with human diseases. Yet, existing tRNA databases have not been updated for more than a decade, so they do not contain this new functional information and have not kept pace with the rate of discovery in this field. Therefore, a regularly updated database that contains information about newly discovered characteristics of tRNA molecules and can be regularly updated is strongly needed. Here, we report the creation of the T-psi-C database (http://tpsic.igcz.poznan.pl), an up-to-date collection of tRNA sequences that contains data obtained from high-throughput tRNA sequencing, e.g. all isoacceptors and isodecoders for human HEK293 cells. This database also contains 3D tRNA structures obtained from Protein Data Bank and generated using homology modeling. The T-psi-C database can be continuously updated by any member of the scientific community, and contains its own application programming interface (API), which allows users to retrieve or upload data in JSON format. Altogether, T-psi-C is user-friendly, easy to develop and an up-to-date source of knowledge about tRNAs.

Excitonic Complexes in n-Doped WS2 Monolayer.

We investigate the origin of emission lines apparent in the low-temperature photoluminescence spectra of n-doped WS2 monolayer embedded in hexagonal BN layers using external magnetic fields and first-principles calculations. Apart from the neutral A exciton line, all observed emission lines are related to the negatively charged excitons. Consequently, we identify emissions due to both the bright (singlet and triplet) and dark (spin- and momentum-forbidden) negative trions as well as the phonon replicas of the latter optically inactive complexes. The semidark trions and negative biexcitons are distinguished. On the basis of their experimentally extracted and theoretically calculated g-factors, we identify three distinct families of emissions due to exciton complexes in WS2: bright, intravalley, and intervalley dark. The g-factors of the spin-split subbands in both the conduction and valence bands are also determined.

RNAlign2D: a rapid method for combined RNA structure and sequence-based alignment using a pseudo-amino acid substitution matrix.

BACKGROUND: The functions of RNA molecules are mainly determined by their secondary structures. These functions can also be predicted using bioinformatic tools that enable the alignment of multiple RNAs to determine functional domains and/or classify RNA molecules into RNA families. However, the existing multiple RNA alignment tools, which use structural information, are slow in aligning long molecules and/or a large number of molecules. Therefore, a more rapid tool for multiple RNA alignment may improve the classification of known RNAs and help to reveal the functions of newly discovered RNAs. RESULTS: Here, we introduce an extremely fast Python-based tool called RNAlign2D. It converts RNA sequences to pseudo-amino acid sequences, which incorporate structural information, and uses a customizable scoring matrix to align these RNA molecules via the multiple protein sequence alignment tool MUSCLE. CONCLUSIONS: RNAlign2D produces accurate RNA alignments in a very short time. The pseudo-amino acid substitution matrix approach utilized in RNAlign2D is applicable for virtually all protein aligners.

Beta-defensin 1 gene polymorphisms in the pathologies of the oral cavity-Data from meta-analysis: Association only with rs1047031 not with rs1800972, rs1799946, and rs11362.

OBJECTIVES: The purpose of this meta-analysis was to reveal a potential association of the four functional polymorphisms in human Beta-defensin 1 (DEFB1) gene: rs1047031(c*5G > A) at 3'UTR and rs11362 (-20 G > A), rs1800972(-44 C > G), and rs1799946 (-52 G > A) at 5'UTR with the risk of common oral cavity pathologies that included periodontitis, caries, lichen planus, and recurrent aphthous stomatitis. METHODS: The relevant studies were obtained by the two researchers from PubMed, Scopus, and Web of Science up to April 29, 2020. The manual search of the reference lists was also performed. Studies on DEFB1 gene polymorphisms and oral cavity disorders, using the case-control genetic association analysis approach, and published as full texts in English were included. To assess the association strength, odds ratios (ORs) with their 95% confidence intervals (CIs) were extracted. RESULTS: Thirteen publications met the inclusion criteria and were incorporated in this meta-analysis. Statistically significant values of the association tests were found only for the rs1047031 polymorphism. Allele distribution in the rs1047031 polymorphism was significantly associated with susceptibility to oral cavity pathologies (adjusted P value = 0.003). The rare variant allele carriers had a significantly higher risk for oral disasters under recessive (CC vs CT + TT), and CC vs CT models. No significant correlations between rs11362, rs1800972, and rs1799946 and the risk of oral pathologies were revealed. CONCLUSIONS: Significant association between rs1047031 polymorphism and risk of oral pathologies has been found, and therefore, we suggest to include this polymorphism in future research concerning the genetic background of the oral cavity diseases.

How much, if anything, do we know about sperm chromosomes of Robertsonian translocation carriers?

In men with oligozoospermia, Robertsonian translocations (RobTs) are the most common type of autosomal aberrations. The most commonly occurring types are rob(13;14) and rob(14;21), and other types of RobTs are described as 'rare' cases. Based on molecular research, all RobTs can be broadly classified into Class 1 and Class 2. Class 1 translocations produce the same breakpoints within their RobT type, but Class 2 translocations are predicted to form during meiosis or mitosis through a variety of mechanisms, resulting in variation in the breakpoint locations. This review seeks to analyse the available data addressing the question of whether the molecular classification of RobTs into Classes 1 and 2 and/or the type of DD/GG/DG symmetry of the involved chromosomes is reflected in the efficiency of spermatogenesis. The lowest frequency value calculated for the rate of alternate segregants was found for rob(13;15) carriers (Class 2, symmetry DD) and the highest for rob(13;21) carriers (Class 2, DG symmetry). The aneuploidy values for the rare RobT (Class 2) and common rob(14;21) (Class 1) groups together exhibited similarities while differing from those for the common rob(13;14) (Class 1) group. Considering the division of RobT carriers into those with normozoospermia and those with oligoasthenozoospermia, it was found that the number of carriers with elevated levels of aneuploidy was unexpectedly quite similar and high (approx. 70%) in the two subgroups. The reason(s) that the same RobT does not always show a similar destructive effect on fertility was also pointed out.

Luminescent Emission of Excited Rydberg Excitons from Monolayer WSe2.

We report the experimental observation of radiative recombination from Rydberg excitons in a two-dimensional semiconductor, monolayer WSe2, encapsulated in hexagonal boron nitride. Excitonic emission up to the 4 s excited state is directly observed in photoluminescence spectroscopy in an out-of-plane magnetic field up to 31 T. We confirm the progressively larger exciton size for higher energy excited states through diamagnetic shift measurements. This also enables us to estimate the 1 s exciton binding energy to be about 170 meV, which is significantly smaller than most previous reports. The Zeeman shift of the 1 s to 3 s states, from both luminescence and absorption measurements, exhibits a monotonic increase of the g-factor, reflecting nontrivial magnetic-dipole-moment differences between ground and excited exciton states. This systematic evolution of magnetic dipole moments is theoretically explained from the spreading of the Rydberg states in momentum space.

Seminal Plasma Analysis of Oxidative Stress in Different Genitourinary Topographical Regions Involved in Reproductive Tract Disorders Associated with Genital Heat Stress.

The pathophysiological mechanisms responsible for male subfertility/infertility caused by or complicated by genital heat stress remains unclear in many respects. Because seminal plasma creates the environment for the proper functioning of spermatozoa, in this study, we verified the associations among standard spermiograms, seminal biochemical parameters (neutral alpha-glucosidase, fructose, and citric acid) and oxidative stress markers (total antioxidant capacity, catalase activity, superoxide dismutase activity, and malondialdehyde concentration) in distinct entities associated with male infertility with and without long-time exposure to local hyperthermia. We demonstrated that men exposed to prolonged environmental or clinically recognized local heat stress in adulthood may suffer from dysregulation of seminal antioxidant components, which can be directly associated with epididymal and prostate function. The comparative analysis of the studied parameters showed numerous correlations among all biochemical parameters (particularly neutral alpha-glucosidase) with low standard semen quality in almost all the investigated infertile groups. In light of the data obtained in this originally designed study, we conclude that more attention should be paid to the epididymis and accessory gland function in subfertile and infertile men exposed to genital heat stress, especially in the context of novel treatment algorithms (targeted therapies).

Computational Fluid Dynamics as an Engineering Tool for the Reconstruction of Hemodynamics after Carotid Artery Stenosis Operation: A Case Study.

Background and objectives: Brain ischemic stroke is caused by impaired or absolutely blocked blood flow into the brain regions. Despite the large number of possible origins, there is no general strategy for preventive treatment. In this paper, we aimed to predict the hemodynamics in a patient who experienced a critical stenosis operation in the carotid artery. This is a unique study where we used medical data together with the computational fluid (CFD) technique not to plan the surgery, but to predict its outcome. Materials and Methods: AngioCT data and blood perfusion of brain tissue (CT-perfusion) together with CFD technique were applied for stroke formation reconstruction in different clinical conditions. With the use of self-made semiautomatic algorithm for image processing and 3DDoctror software, 3D-vascular geometries before and after surgical intervention were reconstructed. As the paper is focused on the analysis of stroke appearance, apparent stroke was simulated as higher and lower pressure values in the cranial part due to different outcomes of the surgical intervention. This allowed to investigate the influence of spatial configuration and pressure values on blood perfusion in the analyzed circulatory system. Results: Application of CFD simulations for blood flow reconstruction for clinical conditions in the circulatory system accomplished on average 98.5% and 98.7% accuracy for CFD results compared to US-Doppler before and after surgical intervention, respectively. Meanwhile, CFD results compared to CT-perfusion indicated an average 89.7% and 92.8% accuracy before and after surgical intervention, respectively. Thus, the CFD is a reliable approach for predicting the patient hemodynamics, as it was confirmed by postoperative data. Conclusions: Our study indicated that the application of CFD simulations for blood flow reconstruction for clinical conditions in circulatory system reached 98% and 90% accuracy for US-Doppler and CT-perfusion, respectively. Therefore, the proposed method might be used as a tool for reconstruction of specific patients' hemodynamics after operation of critical stenosis in the carotid artery. However, further studies are necessary to confirm its usefulness in clinical practice.

[Lublin - the capital of polish speech and language therapy. Half a century of slt education in UMCS].

Lublin is the capital of Polish speech and language therapy (SLT) and this fact is justified by both historical as well as support in evaluating the potential of science - research and teaching, particularly in connection with the activities of the Department of Logopedics/SLT and Applied Linguistics of University of Maria Curie-Sklodowska and Polish Logopedic Society. The article discusses the history of the formation of SLT in Poland, strongly associated with Lublin, and also presents Lublin SLT educational traditions and the current teaching and research activities of the Department of Logopedics/SLT and Applied Linguistics of UMCS.

Education of speech and language therapists/logopedists in selected Central and Southeastern European countries: challenges and new horizons.

OBJECTIVE: To provide an overview of student training in speech and language therapy/logopedics (SLT) in selected Central and Southeastern European countries (Poland, Slovenia, Bulgaria, Czech Republic, Serbia, Bosnia and Herzegovina and Turkey). METHOD: Data were collected using a special questionnaire developed by Söderpalm and supplemented by Georgieva. Results from 23 SLT programs in the seven countries were collected and organized. RESULTS: In all these countries, SLT has roots in special education or health and is centralized in the university environment. The training programs have positive accreditation provided by the national agencies of accreditation and evaluation. Results were examined specifically for evidence of the new paradigm of evidence-based practice (EBP) according to the revised International Association of Logopedics and Phoniatrics (IALP) guidelines and the application of research-based teaching in SLT. The professional bodies that govern clinical practice in public health and/or educational fields are in the process of EBP implementation. Most speech and language therapists/logopedists in the selected countries work in an educational setting, clinical organization and/or hospital as well as in social day care centers. Except in Turkey, private practices are not regulated by the law. CONCLUSIONS: In the seven countries examined in this survey, SLT is progressing as a professional discipline but must be supported by government funding of SLT education and services to relevant populations.

Resonant Raman scattering of few layers CrBr3.

We investigate the vibrational and magnetic properties of thin layers of chromium tribromide (CrBr3) with a thickness ranging from three to twenty layers (3-20 L) revealed by the Raman scattering (RS) technique. Systematic dependence of the RS process efficiency on the energy of the laser excitation is explored for four different excitation energies: 1.96 eV, 2.21 eV, 2.41 eV, and 3.06 eV. Our characterization demonstrates that for 12 L CrBr3, 3.06 eV excitation could be considered resonant with interband electronic transitions due to the enhanced intensity of the Raman-active scattering resonances and the qualitative change in the Raman spectra. Polarization-resolved RS measurements for 12 L CrBr3 and first-principles calculations allow us to identify five observable phonon modes characterized by distinct symmetries, classified as the A g and E g modes. The evolution of phonon modes with temperature for a 16 L CrBr3 encapsulated in hexagonal boron nitride flakes demonstrates alterations of phonon energies and/or linewidths of resonances indicative of a transition between the paramagnetic and ferromagnetic state at Curie temperature ( T C ≈ 50  K). The exploration of the effects of thickness on the phonon energies demonstrated small variations pronounces exclusively for the thinnest layers in the vicinity of 3-5 L. We propose that this observation can be due to the strong localization in the real space of interband electronic excitations, limiting the effects of confinement for resonantly excited Raman modes to atomically thin layers.

Public attitudes toward stuttering in Poland.

BACKGROUND: People who stutter often experience negative judgments and reactions to their stuttering from the nonstuttering majority. Many are stigmatized because of their stuttering and threatened with social exclusion, placing them at risk for compromised quality of life. AIMS: The purpose of this investigation was to measure public attitudes toward stuttering in Poland. METHODS & PROCEDURES: A sample of 268 respondents (mean age = 29 years; range = 15-60 years) from numerous different geographic and urban-rural settings in Poland filled out a Polish translation of the Public Opinion Survey of Human Attributes-Stuttering (POSHA-S). OUTCOMES & RESULTS: Polish respondents displayed attitudes toward stuttering and people who stutter that were generally similar or "average" in comparison with other samples around the world from the POSHA-S database. CONCLUSIONS & IMPLICATIONS: Although generally typical of other Western societies studied, attitudes of adolescents and adults from Poland were notably different in some ways, such as in the beliefs that emotional trauma or viruses and disease can cause stuttering as well as in the self reaction that they would feel uncomfortable speaking with a stuttering person. Overall, social exclusion and stigma are as likely among Poles who stutter as among most other populations studied.

The effect of temperature and excitation energy on Raman scattering in bulkHfS2.

Raman scattering (RS) in bulk hafnium disulfide (HfS2) is investigated as a function of temperature (5 K - 350 K) with polarization resolution and excitation of several laser energies. An unexpected temperature dependence of the energies of the main Raman-active (A1gand Eg) modes with the temperature-induced blueshift in the low-temperature limit is observed. The low-temperature quenching of a modeω1(134 cm-1) and the emergence of a new mode at approx. 184 cm-1, labeledZ, is reported. The optical anisotropy of the RS inHfS2is also reported, which is highly susceptible to the excitation energy. The apparent quenching of the A1gmode atT = 5 K and of the Egmode atT= 300 K in the RS spectrum excited with 3.06 eV excitation is also observed. We discuss the results in the context of possible resonant character of light-phonon interactions. Analyzed is also a possible effect of the iodine molecules intercalated in the van der Waals gaps between neighboringHfS2layers, which inevitably result from the growth procedure.

CRISPR/Cas9 screen for genome-wide interrogation of essential MYC-bound E-boxes in cancer cells.

The transcription factor MYC is a proto-oncogene with a well-documented essential role in the pathogenesis and maintenance of several types of cancer. MYC binds to specific E-box sequences in the genome to regulate gene expression in a cell-type- and developmental-stage-specific manner. To date, a combined analysis of essential MYC-bound E-boxes and their downstream target genes important for growth of different types of cancer is missing. In this study, we designed a CRISPR/Cas9 library to destroy E-box sequences in a genome-wide fashion. In parallel, we used the Brunello library to knock out protein-coding genes. We performed high-throughput screens with these libraries in four MYC-dependent cancer cell lines-K562, ST486, HepG2, and MCF7-which revealed several essential E-boxes and genes. Among them, we pinpointed crucial common and cell-type-specific MYC-regulated genes involved in pathways associated with cancer development. Extensive validation of our approach confirmed that E-box disruption affects MYC binding, target-gene expression, and cell proliferation in vitro as well as tumor growth in vivo. Our unique, well-validated tool opens new possibilities to gain novel insights into MYC-dependent vulnerabilities in cancer cells.