Search Results (16)

Hydrofluorocarbons (HFC) are today widely used as refrigerants, solvents, or aerosols for fire protection. Due to their non-negligible environmental impact, there exists an increasing interest towards their effective separation and recovery, which still remains a major challenge. This work presents a comprehensive thermodynamic and transport modeling approach able to describe HFC sorption and transport in different amorphous polymers, including glassy, rubbery, and copolymers, as well as in supported Ionic Liquid membranes (SILMs). In particular, the literature solubility data for refrigerants such as R-32, R-125, R-134a, and R-152a is analyzed by means of the Sanchez–Lacombe Equation of State (SL-EoS), and its non-equilibrium extension (NELF), to predict gas uptake in complex polymeric materials. The Standard Transport Model (STM) is then employed to describe permeability behaviors, incorporating concentration-dependent diffusion using a mobility coefficient and thermodynamic factor. Results demonstrate that fluorinated gases exhibit strong affinity to fluorinated and high free-volume polymers, and that solubility is primarily governed by gas condensability, molecular size, and polymer structure. The combined EoS–STM approach accurately predicts both solubility and permeability across different pressures in all polymers, including SILM. The thorough study of HFC transport in polymer membranes provided both systematic insights and predictive capabilities to guide the design of next-generation materials for refrigerant recovery and low-GWP separation processes. Full article

SAYP and Bap170, subunits of the SWI/SNF remodeling complex, have the ability to support enhancer-dependent transcription when artificially recruited to the promoter on a transgene. We found that the phenomenon critically depends on two subunits of the Mediator kinase module, Med12 and Med13 but does not require the two other subunits of the module (Cdk8 and CycC) or other subunits of the core part of the complex. A cooperation of the above proteins in active transcription was also observed at endogenous loci, but the contribution of the subunits to the activity of a particular gene differed in different loci. The factors SAYP/Bap170 and Med12/Med13 did not form sufficiently stable interactions in the extract, and their cooperation was apparently local at regulatory elements, the presence of SAYP and Bap170 in a locus being necessary for stable recruitment of Med12 and Med13 to the locus. In addition to the above factors, the Nelf-A protein was found to participate in the process. The cooperation of the factors, independent of enzymatic activities of the complexes they are part of, appears to be a novel mechanism that maintains promoter activity and may be used in many loci of the genome. Extended intrinsically disordered regions of the factors were assumed to sustain the mechanism. Full article

The establishment and stable inheritance of individual patterns of gene expression in different cell types are required for the development of multicellular organisms. The important epigenetic regulators are the Polycomb group (PcG) and Trithorax group (TrxG) proteins, which control the silenced and active states of genes, respectively. In Drosophila, the PcG/TrxG group proteins are recruited to the DNA regulatory sequences termed the Polycomb response elements (PREs). The PREs are composed of the binding sites for different DNA-binding proteins, the so-called PcG recruiters. Currently, the role of the PcG recruiters in the targeting of the PcG proteins to PREs is well documented. However, there are examples where the PcG recruiters are also implicated in the active transcription and in the TrxG function. In addition, there is increasing evidence that the genome-wide PcG recruiters interact with the chromatin outside of the PREs and overlap with the proteins of differing regulatory classes. Recent studies of the interactomes of the PcG recruiters significantly expanded our understanding that they have numerous interactors besides the PcG proteins and that their functions extend beyond the regulation of the PRE repressive activity. Here, we summarize current data about the functions of the PcG recruiters. Full article

There are several studies on the deregulated gene expression profiles in kidney cancer, with varying results depending on the tumor histology and other parameters. None of these, however, have identified the networks that the co-deregulated genes (co-DEGs), across different studies, create. Here, we reanalyzed 10 Gene Expression Omnibus (GEO) studies to detect and annotate co-deregulated signatures across different subtypes of kidney cancer or in single-gene perturbation experiments in kidney cancer cells and/or tissue. Using a systems biology approach, we aimed to decipher the networks they form along with their upstream regulators. Differential expression and upstream regulators, including transcription factors [MYC proto-oncogene (MYC), CCAAT enhancer binding protein delta (CEBPD), RELA proto-oncogene, NF-kB subunit (RELA), zinc finger MIZ-type containing 1 (ZMIZ1), negative elongation factor complex member E (NELFE) and Kruppel-like factor 4 (KLF4)] and protein kinases [Casein kinase 2 alpha 1 (CSNK2A1), mitogen-activated protein kinases 1 (MAPK1) and 14 (MAPK14), Sirtuin 1 (SIRT1), Cyclin dependent kinases 1 (CDK1) and 4 (CDK4), Homeodomain interacting protein kinase 2 (HIPK2) and Extracellular signal-regulated kinases 1 and 2 (ERK1/2)], were computed using the Characteristic Direction, as well as GEO2Enrichr and X2K, respectively, and further subjected to GO and KEGG pathways enrichment analyses. Furthermore, using CMap, DrugMatrix and the LINCS L1000 chemical perturbation databases, we highlight putative repurposing drugs, including Etoposide, Haloperidol, BW-B70C, Triamterene, Chlorphenesin, BRD-K79459005 and β-Estradiol 3-benzoate, among others, that may reverse the expression of the identified co-DEGs in kidney cancers. Of these, the cytotoxic effects of Etoposide, Catecholamine, Cyclosporin A, BW-B70C and Lasalocid sodium were validated in vitro. Overall, we identified critical co-DEGs across different subtypes in kidney cancer, and our results provide an innovative framework for their potential use in the future. Full article

According to previous studies, during Drosophila embryogenesis, the recruitment of RNA polymerase II precedes active gene transcription. This work is aimed at exploring whether this mechanism is used during Drosophila metamorphosis. In addition, the composition of the RNA polymerase II “paused” complexes associated with promoters at different developmental stages are described in detail. For this purpose, we performed ChIP-Seq analysis using antibodies for various modifications of RNA polymerase II (total, Pol II CTD Ser5P, and Pol II CTD Ser2P) as well as for subunits of the NELF, DSIF, and PAF complexes and Brd4/Fs(1)h that control transcription elongation. We found that during metamorphosis, similar to mid-embryogenesis, the promoters were bound by RNA polymerase II in the “paused” state, preparing for activation at later stages of development. During mid-embryogenesis, RNA polymerase II in a “pause” state was phosphorylated at Ser5 and Ser2 of Pol II CTD and bound the NELF, DSIF, and PAF complexes, but not Brd4/Fs(1)h. During metamorphosis, the “paused” RNA polymerase II complex included Brd4/Fs(1)h in addition to NELF, DSIF, and PAF. The RNA polymerase II in this complex was phosphorylated at Ser5 of Pol II CTD, but not at Ser2. These results indicate that, during mid-embryogenesis, RNA polymerase II stalls in the “post-pause” state, being phosphorylated at Ser2 of Pol II CTD (after the stage of p-TEFb action). During metamorphosis, the “pause” mechanism is closer to classical promoter-proximal pausing and is characterized by a low level of Pol II CTD Ser2P. Full article

The promoter-proximal pause induced by the binding of the DRB sensitivity-inducing factor (DSIF) and the negative elongation factor (NELF) to RNAP II is a key step in the regulation of metazoan gene expression. It helps maintain a permissive chromatin landscape and ensures a quick transcriptional response from stimulus-responsive pathways such as the innate immune response. It is also involved in the biology of several RNA viruses such as the human immunodeficiency virus (HIV), the influenza A virus (IAV) and the hepatitis delta virus (HDV). HIV uses the pause as one of its mechanisms to enter and maintain latency, leading to the creation of viral reservoirs resistant to antiretrovirals. IAV, on the other hand, uses the pause to acquire the capped primers necessary to initiate viral transcription through cap-snatching. Finally, the HDV RNA genome is transcribed directly by RNAP II and requires the small hepatitis delta antigen to displace NELF from the polymerase and overcome the transcriptional block caused by RNAP II promoter-proximal pausing. In this review, we will discuss the RNAP II promoter-proximal pause and the roles it plays in the life cycle of RNA viruses such as HIV, IAV and HDV. Full article

Telomerase RNA has long been considered to be a noncoding component of telomerase. However, the expression of the telomerase RNA gene is not always associated with telomerase activity. The existence of distinct TERC gene expression products possessing different functions were demonstrated recently. During biogenesis, hTR is processed by distinct pathways and localized in different cell compartments, depending on whether it functions as a telomerase complex component or facilitates antistress activities as a noncoding RNA, in which case it is either processed in the mitochondria or translated. In order to identify the factors responsible for the appearance and localization of the exact isoform of hTR, we investigated the roles of the factors regulating transcription DSIF (Spt5) and NELF-E; exosome-attracting factors ZCCHC7, ZCCHC8, and ZFC3H1; ARS2, which attracts processing and transport factors; and transport factor PHAX during the biogenesis of hTR. The data obtained revealed that ZFC3H1 participates in hTR biogenesis via pathways related to the polyadenylated RNA degradation mechanism. The data revealed essential differences that are important for understanding hTR biogenesis and that are interesting for further investigations of new, therapeutically significant targets. Full article

In order to identify factors involved in transcription of human snRNA genes and 3′ end processing of the transcripts, we have carried out CRISPR affinity purification in situ of regulatory elements (CAPTURE), which is deadCas9-mediated pull-down, of the tandemly repeated U2 snRNA genes in human cells. CAPTURE enriched many factors expected to be associated with these human snRNA genes including RNA polymerase II (pol II), Cyclin-Dependent Kinase 7 (CDK7), Negative Elongation Factor (NELF), Suppressor of Ty 5 (SPT5), Mediator 23 (MED23) and several subunits of the Integrator Complex. Suppressor of Ty 6 (SPT6); Cyclin K, the partner of Cyclin-Dependent Kinase 12 (CDK12) and Cyclin-Dependent Kinase 13 (CDK13); and SWI/SNF chromatin remodelling complex-associated SWI/SNF-related, Matrix-associated, Regulator of Chromatin (SMRC) factors were also enriched. Several polyadenylation factors, including Cleavage and Polyadenylation Specificity Factor 1 (CPSF1), Cleavage Stimulation Factors 1 and 2 (CSTF1,and CSTF2) were enriched by U2 gene CAPTURE. We have already shown by chromatin immunoprecipitation (ChIP) that CSTF2—and Pcf11 and Ssu72, which are also polyadenylation factors—are associated with the human U1 and U2 genes. ChIP-seq and ChIP-qPCR confirm the association of SPT6, Cyclin K, and CDK12 with the U2 genes. In addition, knockdown of SPT6 causes loss of subunit 3 of the Integrator Complex (INTS3) from the U2 genes, indicating a functional role in snRNA gene expression. CAPTURE has therefore expanded the repertoire of transcription and RNA processing factors associated with these genes and helped to identify a functional role for SPT6. Full article

Gene expression is tightly regulated during hematopoiesis. Recent studies have suggested that RNA polymerase II (Pol II) promoter proximal pausing, a temporary stalling downstream of the promoter region after initiation, plays a critical role in regulating the expression of various genes in metazoans. However, the function of proximal pausing in hematopoietic gene regulation remains largely unknown. The negative elongation factor (NELF) complex is a key factor important for this proximal pausing. Previous studies have suggested that NELF regulates granulocytic differentiation in vitro, but its in vivo function during hematopoiesis remains uncharacterized. Here, we generated the zebrafish mutant for one NELF complex subunit Nelfb using the CRISPR-Cas9 technology. We found that the loss of nelfb selectively induced excessive granulocytic development during primitive and definitive hematopoiesis. The loss of nelfb reduced hematopoietic progenitor cell formation and did not affect erythroid development. Moreover, the accelerated granulocytic differentiation and reduced progenitor cell development could be reversed by inhibiting Pol II elongation. Further experiments demonstrated that the other NELF complex subunits (Nelfa and Nelfe) played similar roles in controlling granulocytic development. Together, our studies suggested that NELF is critical in controlling the proper granulocytic development in vivo, and that promoter proximal pausing might help maintain the undifferentiated state of hematopoietic progenitor cells. Full article

Background: SARS-CoV-2 enters the body through inhalation or self-inoculation to mucosal surfaces. The kinetics of the ocular and nasal mucosal-specific-immunoglobulin A(IgA) responses remain under-studied. Methods: Conjunctival fluid (CF, n = 140) and nasal epithelial lining fluid (NELF, n = 424) obtained by paper strips and plasma (n = 153) were collected longitudinally from SARS-CoV-2 paediatric (n = 34) and adult (n = 47) patients. The SARS-CoV-2 spike protein 1(S1)-specific mucosal antibody levels in COVID-19 patients, from hospital admission to six months post-diagnosis, were assessed. Results: The mucosal antibody was IgA-predominant. In the NELF of asymptomatic paediatric patients, S1-specific IgA was induced as early as the first four days post-diagnosis. Their plasma S1-specific IgG levels were higher than in symptomatic patients in the second week after diagnosis. The IgA and IgG levels correlated positively with the surrogate neutralization readout. The detectable NELF “receptor-blocking” S1-specific IgA in the first week after diagnosis correlated with a rapid decline in viral load. Conclusions: Early and intense nasal S1-specific IgA levels link to a rapid decrease in viral load. Our results provide insights into the role of mucosal immunity in SARS-CoV-2 exposure and protection. There may be a role of NELF IgA in the screening and diagnosis of SARS-CoV-2 infection. Full article

Cyclin Dependent Kinase 9 (CDK9) is one of the most important transcription regulatory members of the CDK family. In conjunction with its main cyclin partner—Cyclin T1, it forms the Positive Transcription Elongation Factor b (P-TEFb) whose primary function in eukaryotic cells is to mediate the positive transcription elongation of nascent mRNA strands, by phosphorylating the S2 residues of the YSPTSPS tandem repeats at the C-terminus domain (CTD) of RNA Polymerase II (RNAP II). To aid in this process, P-TEFb also simultaneously phosphorylates and inactivates a number of negative transcription regulators like 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) Sensitivity-Inducing Factor (DSIF) and Negative Elongation Factor (NELF). Significantly enhanced activity of CDK9 is observed in multiple cancer types, which is universally associated with significantly shortened Overall Survival (OS) of the patients. In these cancer types, CDK9 regulates a plethora of cellular functions including proliferation, survival, cell cycle regulation, DNA damage repair and metastasis. Due to the extremely critical role of CDK9 in cancer cells, inhibiting its functions has been the subject of intense research, resulting the development of multiple, increasingly specific small-molecule inhibitors, some of which are presently in clinical trials. The search for newer generation CDK9 inhibitors with higher specificity and lower potential toxicities and suitable combination therapies continues. In fact, the Phase I clinical trials of the latest, highly specific CDK9 inhibitor BAY1251152, against different solid tumors have shown good anti-tumor and on-target activities and pharmacokinetics, combined with manageable safety profile while the phase I and II clinical trials of another inhibitor AT-7519 have been undertaken or are undergoing. To enhance the effectiveness and target diversity and reduce potential drug-resistance, the future of CDK9 inhibition would likely involve combining CDK9 inhibitors with inhibitors like those against BRD4, SEC, MYC, MCL-1 and HSP90. Full article

Sarcoidosis is characterized by multiorgan involvement and granulomatous inflammation. Its origin is unknown and the potential role of autoimmunity has not been sufficiently determined. We investigated the presence of autoantibodies in sarcoidosis using protein array technology. The derivation cohort consisted of patients with sarcoidosis (n = 25) and controls including autoimmune disease and blood donors (n = 246). In addition, we tested a validation cohort including pulmonary sarcoidosis patients (n = 58) and healthy controls (n = 13). Initially, sera of three patients with sarcoidosis were screened using a protein array with 28.000 proteins against controls. Thereby we identified the Negative Elongation Factor E (NELF-E) as an autoantigen. With confirmatory Enzyme-linked Immunosorbent Assay (ELISA)testing, 29/82 patients (35%) with sarcoidosis had antibodies against NELF-E of the Immunoglobulin (Ig) G type, whereas 18/253 (7%) sera of the controls were positive for NELF-E. Clinically, there was an association of the frequency of NELF-E antibody detection with lung parenchymal involvement and corresponding x-ray types. NELF-E autoantibodies are associated with sarcoidosis and should be further investigated. Full article

At the central region of the mammalian major histocompatibility complex (MHC) is a complement gene cluster that codes for constituents of complement C3 convertases (C2, factor B and C4). Complement activation drives the humoral effector functions for immune response. Sandwiched between the genes for serine proteinase factor B and anchor protein C4 are four less known but critically important genes coding for essential functions related to metabolism and surveillance of RNA during the transcriptional and translational processes of gene expression. These four genes are NELF-E (RD), SKIV2L (SKI2W), DXO (DOM3Z) and STK19 (RP1 or G11) and dubbed as NSDK. NELF-E is the subunit E of negative elongation factor responsible for promoter proximal pause of transcription. SKIV2L is the RNA helicase for cytoplasmic exosomes responsible for degradation of de-polyadenylated mRNA and viral RNA. DXO is a powerful enzyme with pyro-phosphohydrolase activity towards 5′ triphosphorylated RNA, decapping and exoribonuclease activities of faulty nuclear RNA molecules. STK19 is a nuclear kinase that phosphorylates RNA-binding proteins during transcription. STK19 is also involved in DNA repair during active transcription and in nuclear signal transduction. The genetic, biochemical and functional properties for NSDK in the MHC largely stay as a secret for many immunologists. Here we briefly review the roles of (a) NELF-E on transcriptional pausing; (b) SKIV2L on turnover of deadenylated or expired RNA 3′→5′ through the Ski-exosome complex, and modulation of inflammatory response initiated by retinoic acid-inducible gene 1-like receptor (RLR) sensing of viral infections; (c) DXO on quality control of RNA integrity through recognition of 5′ caps and destruction of faulty adducts in 5′→3′ fashion; and (d) STK19 on nuclear protein phosphorylations. There is compelling evidence that a dysregulation or a deficiency of a NSDK gene would cause a malignant, immunologic or digestive disease. Full article

The transport of gases in glassy polymeric membranes has been analyzed by means of a fundamental approach based on the nonequilibrium thermodynamic model for glassy polymers (NET-GP) that considers the penetrant chemical potential gradient as the actual driving force of the diffusional process. The diffusivity of a penetrant is thus described as the product of a purely kinetic quantity, the penetrant mobility, and a thermodynamic factor, accounting for the chemical potential dependence on its concentration in the polymer. The NET-GP approach, and the nonequilibrium lattice fluid (NELF) model in particular, describes the thermodynamic behavior of penetrant/polymer mixtures in the glassy state, at each pressure or composition. Moreover, the mobility is considered to follow a simple exponential dependence on penetrant concentration, as typically observed experimentally, using only two adjustable parameters, the infinite dilution penetrant mobility L₁₀ and the plasticization factor β, both determined from the analysis of the dependence of steady state permeability on upstream pressure. The available literature data of diffusional time lag as a function of penetrant upstream pressure has been reviewed and compared with model predictions, obtained after the values of the two model parameters (L₁₀ and β), have been conveniently determined from steady state permeability data. The model is shown to be able to describe very accurately the experimental time lag behaviors for all penetrant/polymer pairs inspected, including those presenting an increasing permeability with increasing upstream pressure. The model is thus more appropriate than the one based on Dual Mode Sorption, which usually provides an unsatisfactory description of time lag and required an ad hoc modification. Full article

Solubility and permeability of gases in glassy polymers have been considered with the aim of illustrating the applicability of thermodynamically-based models for their description and prediction. The solubility isotherms are described by using the nonequilibrium lattice fluid (NELF) (model, already known to be appropriate for nonequilibrium glassy polymers, while the permeability isotherms are described through a general transport model in which diffusivity is the product of a purely kinetic factor, the mobility coefficient, and a thermodynamic factor. The latter is calculated from the NELF model and mobility is considered concentration-dependent through an exponential relationship containing two parameters only. The models are tested explicitly considering solubility and permeability data of various penetrants in three glassy polymers, PSf, PPh and 6FDA-6FpDA, selected as the reference for different behaviors. It is shown that the models are able to calculate the different behaviors observed, and in particular the permeability dependence on upstream pressure, both when it is decreasing as well as when it is increasing, with no need to invoke the onset of additional plasticization phenomena. The correlations found between polymer and penetrant properties with the two parameters of the mobility coefficient also lead to the predictive ability of the transport model. Full article