Nucleic Acid Binding Research Articles

HybridRNAbind: prediction of RNA interacting residues across structure-annotated and disorder-annotated proteins.

The sequence-based predictors of RNA-binding residues (RBRs) are trained on either structure-annotated or disorder-annotated binding regions. A recent study of predictors of protein-binding residues shows that they are plagued by high levels of cross-predictions (protein binding residues are predicted as nucleic acid binding) and that structure-trained predictors perform poorly for the disorder-annotated regions and vice versa. Consequently, we analyze a representative set of the structure and disorder trained predictors of RBRs to comprehensively assess quality of their predictions. Our empirical analysis that relies on a new and low-similarity benchmark dataset reveals that the structure-trained predictors of RBRs perform well for the structure-annotated proteins while the disorder-trained predictors provide accurate results for the disorder-annotated proteins. However, these methods work only modestly well on the opposite types of annotations, motivating the need for new solutions. Using an empirical approach, we design HybridRNAbind meta-model that generates accurate predictions and low amounts of cross-predictions when tested on data that combines structure and disorder-annotated RBRs. We release this meta-model as a convenient webserver which is available at https://www.csuligroup.com/hybridRNAbind/.

Complete genome analysis of Bacillus subtilis derived from yaks and its probiotic characteristics.

Probiotics have attracted attention due to their multiple health benefits to the host. Yaks inhabiting the Tibetan plateau exhibit excellent disease resistance and tolerance, which may be associated with their inner probiotics. Currently, research on probiotics mainly focuses on their positive effects on the host, but information regarding their genome remains unclear. To reveal the potential functional genes of Bacillus subtilis isolated from yaks, we sequenced its whole genome. Results indicated that the genomic length of Bacillus subtilis was 866,044,638 bp, with 4,429 coding genes. The genome of this bacteria was composed of one chromosome and one plasmid with lengths of 4,214,774 and 54,527 bp, respectively. Moreover, Bacillus subtilis contained 86 tRNAs, 27 rRNAs (9 16S_rRNA, 9 23S_rRNA, and 9 5S_rRNA), and 114 other ncRNA. KEGG annotation indicated that most genes in Bacillus subtilis were associated with biosynthesis of amino acids, carbon metabolism, purine metabolism, pyrimidine metabolism, and ABC transporters. GO annotation demonstrated that most genes in Bacillus subtilis were related to nucleic acid binding transcription factor activity, transporter activity, antioxidant activity, and biological adhesion. EggNOG uncovered that most genes in Bacillus subtilis were related to energy production and conversion, amino acid transport and metabolism, carbohydrate transport and metabolism. CAZy annotation found glycoside hydrolases (33.65%), glycosyl transferases (22.11%), polysaccharide lyases (3.84%), carbohydrate esterases (14.42%), auxiliary activities (3.36%), and carbohydrate-binding modules (22.59%). In conclusion, this study investigated the genome and genetic properties of Bacillus subtilis derived from yaks, which contributed to understanding the potential prebiotic mechanism of probiotics from the genetic perspective.

Nonspecific Binding of Adenosine Tripolyphosphate and Tripolyphosphate Modulates the Phase Behavior of Lysozyme

Adenosine tripolyphosphate (ATP) is a small polyvalent anion that has recently been shown to interact with proteins and have a major impact on assembly processes involved in biomolecular condensate formation and protein aggregation. However, the nature of non-specific protein-ATP interactions and their effects on protein solubility are largely unknown. Here, the binding of ATP to the globular model protein is characterized in detail using X-ray crystallography and nuclear magnetic resonance (NMR). Using NMR, we identified six ATP binding sites on the lysozyme surface, with one known high-affinity nucleic acid binding site and five non-specific previously unknown sites with millimolar affinities that also bind tripolyphosphate (TPP). ATP binding occurs primarily through the polyphosphate moiety, which was confirmed by the X-ray structure of the lysozyme-ATP complex. Importantly, ATP binds preferentially to arginine over lysine in non-specific binding sites. ATP and TPP have similar effects on solution-phase protein-protein interactions. At low salt concentrations, ion binding to lysozyme causes precipitation, while at higher salt concentrations, redissolution occurs. The addition of an equimolar concentration of magnesium to ATP does not alter ATP binding affinities but prevents lysozyme precipitation. These findings have important implications for both protein crystallization and cell biology. Crystallization occurs readily in ATP solutions outside the well-established crystallization window. In the context of cell biology, the findings suggest that ATP binds non-specifically to folded proteins in physiological conditions. Based on the nature of the binding sites identified by NMR, we propose several mechanisms for how ATP binding can prevent the aggregation of natively folded proteins.

Transcription coupled DNA repair protein UVSSA binds to DNA and RNA: Mapping of nucleic acid interaction sites on human UVSSA

Transcription-coupled repair (TCR) is a dedicated pathway for the preferential repair of bulky transcription-blocking DNA lesions. These lesions stall the elongating RNA-polymerase II (RNAPII) triggering the recruitment of TCR proteins at the damaged site. UV-stimulated scaffold protein A (UVSSA) is a recently identified cofactor which is involved in stabilization of the TCR complex, recruitment of DNA-repair machinery and removal/restoration of RNAPII from the lesion site. Mutations in UVSSA render the cells TCR-deficient and have been linked to UV-sensitive syndrome. Human UVSSA is a 709-residue long protein with two short conserved domains; an N-terminal (residues 1–150) and a C-terminal (residues 495–605) domain, while the rest of the protein is predicted to be intrinsically disordered. The protein is well conserved in eukaryotes, however; none of its homologs have been characterized yet. Here, we have purified the recombinant human UVSSA and have characterized it using bioinformatics, biophysical and biochemical techniques. Using EMSA, SPR and fluorescence-based methods, we have shown that human UVSSA interacts with DNA and RNA. Furthermore, we have mapped the nucleic acid binding regions using several recombinant protein fragments containing either the N-terminal or the C-terminal domains. Our data indicate that UVSSA possesses at least two nucleic acid binding regions; the N-terminal domain and a C-terminal tail region (residues 606–662). These regions, far apart in sequence space, are predicted to be in close proximity in structure-space suggesting a coherent interaction with target DNA/RNA. The study may provide functional clues about the novel family of UVSSA proteins.

Spectrophotometric and nucleic acid-binding properties of halloysite clay nanotubes and kaolinite

Halloysite particles (HNTs) are naturally occurring aluminosilicate nanotubes of low toxicity that have shown great promise for drug and biomolecule delivery into human and animal cells. Kaolinite particles retain the same layered structure as HNT, but do not form nanotubes. In this study, the spectrophotometric and sedimentation properties of the two clays in aqueous solutions and their abilities to associate with both small and large nucleic acids have been investigated. Both clays scattered ultraviolet light strongly and this characteristic of HNT was not affected by either vacuum treatment to remove trapped gases or by sonication. Vacuum treatment increased the binding of small nucleic acids to HNT and this association was further enhanced by addition of divalent metal ions. By contrast, only small RNAs were bound efficiently by kaolinite in the presence of Mg2+ ions. Large linear double-stranded DNAs and circular plasmid DNAs bound poorly to kaolinite under all conditions, but these nucleic acids could form strong associations with HNT. Differences in binding data were largely consistent with measurements of the available surface areas of each clay. These results demonstrate that interactions with each clay are critically dependent on both the type and the conformation of each nucleic acid.

Expression analysis of transcription factors in sugarcane during cold stress.

Transcription factors (TF) are a wide class of genes in plants, and these can regulate the expression of other genes in response to various environmental stresses (biotic and abiotic). In the current study, transcription factor activity in sugarcane was examined during cold stress. Initially, RNA transcript reads of two sugarcane cultivars (ROC22 and GT08-1108) under cold stress were downloaded from SRA NCBI database. The reads were aligned into a reference genome and the differential expression analyses were performed with the R/Bioconductor edgeR package. Based on our analyses in the ROC22 cultivar, 963 TF genes were significantly upregulated under cold stress among a total of 5649 upregulated genes, while 293 TF genes were downregulated among a total of 3,289 downregulated genes. In the GT08-1108 cultivar, 974 TF genes were identified among 5,649 upregulated genes and 283 TF genes were found among 3,289 downregulated genes. Most transcription factors were annotated with GO categories related to protein binding, transcription factor binding, DNA-sequence-specific binding, transcription factor complex, transcription factor activity in RNA polymerase II, the activity of nucleic acid binding transcription factor, transcription corepressor activity, sequence-specific regulatory region, the activity of transcription factor of RNA polymerase II, transcription factor cofactor activity, transcription factor activity from plastid promoter, transcription factor activity from RNA polymerase I promoter, polymerase II and RNA polymerase III. The findings of above results will help to identify differentially expressed transcription factors during cold stress. It also provides a comprehensive analysis of the regulation of the transcription activity of many genes. Therefore, this study provides the molecular basis for improving cold tolerance in sugarcane and other economically important grasses.

Ocular protein optineurin shows reversibility from unfolded states and exhibits chaperone-like activity.

Optineurin (OPTN) is a multifunctional, ubiquitously expressed cytoplasmic protein, mutants of which are associated with primary open-angle glaucoma (POAG) and amyotrophic lateral sclerosis (ALS). The most abundant heat shock protein crystallin, known for its remarkable thermodynamic stability and chaperoning activity, allows ocular tissues to withstand stress. The presence of OPTN in ocular tissues is intriguing. Interestingly, OPTN also harbors heat shock elements in its promoter region. Sequence analysis of OPTN exhibits intrinsically disordered regions and nucleic acid binding domains. These properties hinted that OPTN might be endowed with sufficient thermodynamic stability and chaperoning activity. However, these attributes of OPTN have not yet been explored. Here, we studied these properties through thermal and chemical denaturation experiments and monitored the processes using CD, fluorimetry, differential scanning calorimetry, and dynamic light scattering. We found that upon heating, OPTN reversibly forms higher-order multimers. OPTN also displayed a chaperone-like function by reducing the thermal aggregation of bovine carbonic anhydrase. It regains its native secondary structure, RNA-binding property, and melting temperature (T m) after refolding from a thermally as well as chemically denatured state. From our data, we conclude that OPTN, with its unique ability to revert from the stress-mediated unfolded state and its unique chaperoning function, is a valuable protein of the ocular tissues.

Unwinding during stressful times: Mechanisms of helicases in meiotic recombination.

Successful meiosis I requires that homologous chromosomes be correctly linked before they are segregated. In most organisms this physical linkage is achieved through the generation of crossovers between the homologs. Meiotic recombination co-opts and modifies the canonical homologous recombination pathway to successfully generate crossovers One of the central components of this pathway are a number of conserved DNA helicases. Helicases couple nucleic acid binding to nucleotide hydrolysis and use this activity to modify DNA or protein-DNA substrates. During meiosis I it is necessary for the cell to modulate the canonical DNA repair pathways in order to facilitate the generation of interhomolog crossovers. Many of these meiotic modulations take place in pathways involving DNA helicases, or with a meiosis specific helicase. This short review explores what is currently understood about these helicases, their interaction partners, and the role of regulatory modifications during meiosis I. We focus in particular on the molecular structure and mechanisms of these helicases.

Generation of an accurate CCSD(T)/CBS data set and assessment of DFT methods for the binding strengths of group I metal-nucleic acid complexes.

Accurate information about interactions between group I metals and nucleic acids is required to understand the roles these metals play in basic cellular functions, disease progression, and pharmaceuticals, as well as to aid the design of new energy storage materials and nucleic acid sensors that target metal contaminants, among other applications. From this perspective, this work generates a complete CCSD(T)/CBS data set of the binding energies for 64 complexes involving each group I metal (Li+, Na+, K+, Rb+, or Cs+) directly coordinated to various sites in each nucleic acid component (A, C, G, T, U, or dimethylphosphate). This data have otherwise been challenging to determine experimentally, with highly accurate information missing for many group I metal-nucleic acid combinations and no data available for the (charged) phosphate moiety. Subsequently, the performance of 61 DFT methods in combination with def2-TZVPP is tested against the newly generated CCSD(T)/CBS reference values. Detailed analysis of the results reveals that functional performance is dependent on the identity of the metal (with increased errors as group I is descended) and nucleic acid binding site (with larger errors for select purine coordination sites). Over all complexes considered, the best methods include the mPW2-PLYP double-hybrid and ωB97M-V RSH functionals (≤1.6% MPE; <1.0kcal/mol MUE). If more computationally efficient approaches are required, the TPSS and revTPSS local meta-GGA functionals are reasonable alternatives (≤2.0% MPE; <1.0kcal/mol MUE). Inclusion of counterpoise corrections to account for basis set superposition error only marginally improves the computed binding energies, suggesting that these corrections can be neglected with little loss in accuracy when using larger models that are necessary for describing biosystems and biomaterials. Overall, the most accurate functionals identified in this study will permit future works geared towards uncovering the impact of group I metals on the environment and human biology, designing new ways to selectively sense harmful metals, engineering modern biomaterials, and developing improved computational methods to more broadly study group I metal-nucleic acid interactions.

Abstract 2145: Structural and functional studies of nucleic acid binding domain from rabbit SAMD9

Abstract 2145: Structural and functional studies of nucleic acid binding domain from rabbit SAMD9

METTL3 Promotes the Growth and Invasion of Melanoma Cells by Regulating the lncRNA SNHG3/miR-330-5p Axis

Accumulating evidence indicates that m6A methyltransferase 3 (METTL3) plays a pivotal role in different malignancies including melanoma. However, the function and underlying mechanisms by which METTL3 contributes to the tumorigenesis of melanoma remain undocumented. The association of METTL3 and long noncoding RNA (lncRNA) small nucleolar RNA host gene 3 (SNHG3) with clinicopathological characteristics and prognosis in patients with melanoma was analyzed by real-time quantitative polymerase chain reaction (RT-qPCR), Western blotting, and The Cancer Genome Atlas data sets. The role of METTL3 in melanoma cells was assessed by in vitro and in vivo experiments. The m6A dot blot, methylated RNA immunoprecipitation (MeRIP), and RT-qPCR were used to verify METTL3-mediated m6A modification of lncRNA SNHG3. The effect of METTL3 on lncRNA SNHG3 was determined by luciferase gene reporter assay, RT-qPCR, and Western blotting. We found that METTL3 was upregulated in melanoma tissue samples and associated with poor survival in patients with melanoma. Knockdown of METTL3 suppressed the growth and invasion of melanoma cells in vitro and in vivo, whereas restored expression of METTL3 promoted these effects. Mechanistic investigations showed that knockdown of METTL3 reduced SNHG3 m6A levels and its messenger ribonucleic acid (mRNA) expression levels. SNHG3 could act as a sponge of microRNA (miR)-330-5p to upregulate the expression of CCHC-type zinc finger nucleic acid binding protein (CNBP). SNHG3 overexpression reversed METTL3-knockdown-caused antitumor effects, miR-330-5p upregulation and CNBP downregulation. SNHG3 had a positive correlation with METTL3 expression but a negative correlation with miR-330-5p expression in melanoma tissue samples. In conclusion, our findings demonstrated that METTL3-mediated m6A modification of lncRNA SNHG3 promoted the growth and invasion of melanoma cells by regulating the miR-330-5p/CNBP axis.

Theoretical Computational Analysis Predicts Interaction Changes Due to Differences of a Single Molecule in DNA

Theoretical methods, such as molecular mechanics and molecular dynamics, are very useful in understanding differences in interactions at the single molecule level. In the life sciences, small conformational changes, including substituent modifications, often have a significant impact on function in vivo. Changes in binding interactions between nucleic acid molecules and binding proteins are a prime example. In this study, we propose a strategy to predict the complex structure of DNA-binding proteins with arbitrary DNA and analyze the differences in their interactions. We tested the utility of our strategy using the anticancer drug trifluoro-thymidine (FTD), which exerts its pharmacological effect by incorporation into DNA, and confirmed that the binding affinity of the BCL-2-associated X sequence to the p53 tetramer is increased by FTD incorporation. On the contrary, in p53-binding sequences extracted from FTD-resistant cells, the binding affinity of DNA containing FTD was found to be greatly reduced compared to normal DNA. This suggests that thymidine randomly substituted for FTD in resistant cells may acquire resistance by entering a position that inhibits binding to DNA-binding proteins. We believe that this is a versatile procedure that can also take energetics into account and will increase the importance of computational science in the life sciences.

Computational Modeling of IN-CTD/TAR Complex to Elucidate Additional Strategies to Inhibit HIV-1 Replication.

HIV-1 integrase (IN) is a key enzyme that is essential for mediating the insertion of retroviral DNA into the host chromosome. IN also exhibits additional functions which are not fully elucidated, including its ability to bind to viral genomic RNA. Lack of binding of IN to RNA within the virions has been shown to be associated with production of morphologically defective virus particles. However, the exact structure of HIV-1 IN bound to RNA is not known. Based on the studies that C-terminal domain (CTD) of IN binds to TAR RNA region and based on the observation that TAR and the host factor INI1 binding to IN-CTD are identical, we computationally modelled the IN-CTD/TAR complex structure. Computational modeling of nucleic acid binding to proteins is a valuable method to understand the macromolecular interaction when experimental methods of solving the complex structures are not feasible. The current model of the IN-CTD/TAR complex may facilitate further understanding of this interaction and may lead to therapeutic targeting of IN-CTD/RNA interactions to inhibit HIV-1 replication.

Molecular interactions underlying the phase separation of HP1α: role of phosphorylation, ligandand nucleic acid binding.

Heterochromatin protein 1α (HP1α) is a crucial element of chromatin organization. It has been proposed that HP1α functions through liquid-liquid phase separation (LLPS), which allows it to compact chromatin into transcriptionally repressed heterochromatin regions. In vitro, HP1α can undergo phase separation upon phosphorylation of its N-terminus extension (NTE) and/or through interactions with DNA and chromatin. Here, we combine computational and experimental approaches to elucidate the molecular interactions that drive these processes. In phosphorylation-driven LLPS, HP1α can exchange intradimer hinge-NTE interactions with interdimer contacts, which also leads to a structural change from a compacted to an extended HP1α dimer conformation. This process can be enhanced by the presence of positively charged HP1α peptide ligands and disrupted by the addition of negatively charged or neutral peptides. In DNA-driven LLPS, both positively and negatively charged peptide ligands can perturb phase separation. Our findings demonstrate the importance of electrostatic interactions in HP1α LLPS where binding partners can modulate the overall charge of the droplets and screen or enhance hinge region interactions through specific and non-specific effects. Our study illuminates the complex molecular framework that can fine-tune the properties of HP1α and that can contribute to heterochromatin regulation and function.

Zinc Finger Protein BCL11A Contributes to the Abortive Infection of Hirame novirhabdovirus (HIRRV) in B Lymphocytes of Flounder (Paralichthys olivaceus).

Hirame novirhabdovirus (HIRRV) infection is characterized by a pronounced viremia, and the high viral load is typically detected in immune-related organs and the circulatory system. In the present study, we demonstrated that HIRRV has the capacity to invade part of flounder membrane-bound IgM (mIgM+) B lymphocyte. Eight quantitative real-time PCR (qRT-PCR) standard curves involving HIRRV genomic RNA (gRNA), cRNA, and six mRNAs were established based on the strand-specific reverse transcription performed with tagged primers. It was revealed that viral RNA synthesis, especially the replication of gRNA, was inhibited in B cells, and the intracellular HIRRV even failed to produce infectious viral particles. Moreover, a range of genes with nucleic acid binding activity or related to viral infection were screened out based on the transcriptome analysis of HIRRV-infected B cells, and five molecules were further selected because of their different expression patterns in HIRRV-infected B cells and hirame natural embryo (HINAE) cells. The overexpression of these genes followed by HIRRV infection and RNA binding protein immunoprecipitation (RIP) assay revealed that the flounder B cell lymphoma/leukemia 11A (BCL11A), a highly conserved zinc finger transcription factor, is able to inhibit the proliferation of HIRRV by binding with full-length viral RNA mainly via its zinc finger domains at the C terminus. In conclusion, these data indicated that the high transcriptional activity of BCL11A in flounder mIgM+ B lymphocytes is a crucial factor for the abortive infection of HIRRV, and our findings provide new insights into the interaction between HIRRV and teleost B cells. IMPORTANCE HIRRV is a fish rhabdovirus that is considered as an important pathogen threatening the fish farming industry represented by flounder because of its high infectivity and fatality rate. To date, research toward understanding the complex pathogenic mechanism of HIRRV is still in its infancy and faces many challenges. Exploration of the relationship between HIRRV and its target cells is interesting and necessary. Here, we revealed that flounder mIgM+ B cells are capable of suppressing viral RNA synthesis and result in an unproductive infection of HIRRV. In addition, our results demonstrated that zinc finger protein BCL11A, a transcription factor in B cells, is able to suppress the replication of HIRRV. These findings increased our understanding of the underlying characteristics of HIRRV infection and revealed a novel antiviral mechanism against HIRRV based on the host restriction factor in teleost B cells, which sheds new light on the research into HIRRV control.

NONO enhances mRNA processing of super‐enhancer‐associated GATA2 and HAND2 genes in neuroblastoma

High‐risk neuroblastoma patients have poor survival rates and require better therapeutic options. High expression of a multifunctional DNA and RNA‐binding protein, NONO, in neuroblastoma is associated with poor patient outcome; however, there is little understanding of the mechanism of NONO‐dependent oncogenic gene regulatory activity in neuroblastoma. Here, we used cell imaging, biochemical and genome‐wide molecular analysis to reveal complex NONO‐dependent regulation of gene expression. NONO forms RNA‐ and DNA‐tethered condensates throughout the nucleus and undergoes phase separation in vitro, modulated by nucleic acid binding. CLIP analyses show that NONO mainly binds to the 5′ end of pre‐mRNAs and modulates pre‐mRNA processing, dependent on its RNA‐binding activity. NONO regulates super‐enhancer‐associated genes, including HAND2 and GATA2. Abrogating NONO RNA binding, or phase separation activity, results in decreased expression of HAND2 and GATA2. Thus, future development of agents that target RNA‐binding activity of NONO may have therapeutic potential in this cancer context.

The regulatory function of tumor-infiltrating Th9 cells to anti-tumor activity of CD8(+) T cells in patients with gastric cancer

Objective: To investigate the levels of Th9 cells and interleukin-9 (IL-9), and to assess the regulatory activity of Th9/IL-9 to anti-tumor immune response in patients with gastric cancer. Methods: Thirty-four patients with gastric cancer who received operation in the First Affiliated Hospital of Xinxiang Medical University between October 2018 and August 2019 were included. Twenty individuals who received physical examination in the same period were also enrolled. Peripheral blood was collected, and then plasma and peripheral blood mononuclear cells (PBMCs) were isolated. Tumor-infiltrating lymphocytes (TILs) and autologous gastric cancer cells were isolated from resected gastric cancer tissues. CD4(+) T cells, CD8(+) T cells, and CD4(+) CCR4(-)CCR6(-)CXCR3(-) cells were purified from PBMCs and TILs. Plasma IL-9 level was measured by enzyme linked immunosorbent assay (ELISA). The percentage of CD3(+) CD4(+) IL-9(+) Th9 cells in PBMCs and TILSs was assessed by flow cytometry. The mRNA levels of IL-9 and transcriptional factors purine-rich nucleic acid binding protein 1 (PU.1) were semi-quantified by real-time quantitative polymerase chain reaction (RT-qPCR). PBMCs and TILs from gastric cancer patients were stimulated with recombinant human IL-9. Cellular proliferation was measured by cell counting kit-8. The phosphorylation levels of signal transducer and activator of transcription 3 (STAT3) and STAT6 were investigated by western blot. Cytokine production was measured by ELISA. Purified CD8(+) T cells from TILs of gastric cancer patients were stimulated with recombinant human IL-9. CD8(+) T cells and autologous gastric cancer cells were cocultured in direct contact and indirect contact manner. The percentage of target cell death was calculated by measuring the lactate dehydrogenase (LDH) level. These cretion of γ-Interferon (γ-IFN) and tumor necrosis factor-α (TNF-α) was measured by ELISA. CD4(+) CCR4(-)CCR6(-)CXCR3(-)cells, CD8(+) T cells, and autologous gastric cancer cells were directly cocultured, and anti-IL-9 neutralizing antibody was added. The target cell death was measured. Results: The percentages of CD3(+) CD4(+) IL-9(+) Th9 cells in PBMCs of control group and PBMCs of gastric cancer group were (1.21±0.25)% and (1.14±0.19)%, respectively. The difference was not statistically significant (P=0.280). The percentage of CD3(+) CD4(+) IL-9(+) Th9 cells in TILs of gastric cancer group was (2.30±0.55)%, which was higher than those in PBMCs of control group and PBMCs of gastric cancer group (P<0.001). The plasma IL-9 level in control group and gastric cancer group were (5.04±1.51) and (4.93±1.25) ng/ml. The difference was not statistically significant (P=0.787). The relative levels of IL-9 mRNA in PBMCs of control group and PBMCs of gastric cancer group were 1.33±0.39 and 1.36±0.27. The difference was not statistically significant (P=0.691). The relative level of IL-9 mRNA in TILs of gastric cancer group was 2.90±0.75, which was higher than those in PBMCs of control group (P<0.001) and PBMCs of gastric cancer group (P<0.001). The relative levels of PU.1 mRNA in PBMCs of control group and PBMCs of gastric cancer group were 1.21±0.12 and 1.20±0.11. The difference was not statistically significant (t=0.21, P=0.833). PU.1 mRNA relative level in TILs of gastric cancer group was 2.81±0.65, which was higher than those in PBMCs of control group (P<0.001) and PBMCs of gastric cancer group (P<0.001). Recombinant human IL-9 stimulation did not affect the proliferation of PBMCs and TILs of gastric cancer patients (P>0.05), but elevated the phosphorylation level of STAT6 and induced the secretions of γ-IFN, IL-17, and IL-22 by TILs (P<0.05). In direct contact culture system, IL-9 stimulation promoted tumor-infiltrating CD8(+) T cells-induced autologous gastric cancer cell death [(20.62±2.27)% vs. (16.08±2.61)%, P<0.01)]. In indirect contact culture system, IL-9 stimulation did not increase CD8(+) T cell-induced autologous gastric cancer cell death [(5.21±0.70)% vs. (5.31±1.22)%, P=0.998)]. However, the secretion levels of γ-IFN were elevated in response to IL-9 stimulation in both culture systems [direct contact culture system: (100.40±12.05) pg/ml vs. (76.45±8.56) pg/ml; indirect contact culture system: (78.00±9.98) pg/ml vs. (42.09±10.71) pg/ml; P<0.01]. The TNF-α secretion level did not significantly changed (P>0.05). In direct contact culture system, the percentage of target cells was (22.01±3.05) % and γ-IFN secretion level was (104.5±12.84) pg/ml in CD4(+) CCR4(-)CCR6(-)CXCR3(-) cells+ CD8(+) T cells+ gastric cancer cells group, which was higher than (16.08±2.61)% and (76.45±8.56) pg/ml in CD8(+) T cells+ gastric cancer cells group (P<0.01). However, the percentage of target cells was (14.47±3.14)% and γ-IFN secretion level was (70.45±19.43) pg/ml in CD4(+) CCR4(-)CCR6(-)CXCR3(-) cells+ CD8(+) T cells+ gastric cancer cells+ anti-IL-9 neutralizing antibody group, which were lower than those in CD4(+) CCR4(-)CCR6(-)CXCR3(-) cells+ CD8(+) T cells+ gastric cancer cells group (P<0.01). Conclusion: Tumor-infiltrating Th9 cells and the secreting IL-9 promote the activity of CD8(+) T cells in gastric cancer patients, and enhance anti-tumor immune response.

Thermostable Proteins from HaCaT Keratinocytes Identify a Wide Breadth of Intrinsically Disordered Proteins and Candidates for Liquid-Liquid Phase Separation.

Intrinsically disordered proteins (IDPs) move through an ensemble of conformations which allows multitudinous roles within a cell. Keratinocytes, the predominant cell type in mammalian epidermis, have had only a few individual proteins assessed for intrinsic disorder and its possible contribution to liquid-liquid phase separation (LLPS), especially in regard to what functions or structures these proteins provide. We took a holistic approach to keratinocyte IDPs starting with enrichment via the isolation of thermostable proteins. The keratinocyte protein involucrin, known for its resistance to heat denaturation, served as a marker. It and other thermostable proteins were identified by liquid chromatography tandem mass spectrometry and subjected to extensive bioinformatic analysis covering gene ontology, intrinsic disorder, and potential for LLPS. Numerous proteins unique to keratinocytes and other proteins with shared expression in multiple cell types were identified to have IDP traits (e.g., compositional bias, nucleic acid binding, and repeat motifs). Among keratinocyte-specific proteins, many that co-assemble with involucrin into the cell-specific structure known as the cornified envelope scored highly for intrinsic disorder and potential for LLPS. This suggests intrinsic disorder and LLPS are previously unrecognized traits for assembly of the cornified envelope, echoing the contribution of intrinsic disorder and LLPS to more widely encountered features such as stress granules and PML bodies.

Transcriptome Analysis of Wheat-Tilletia indica Interaction Provides Defense and Pathogenesis-Related Genes.

Karnal bunt (Tilletia indica Mitra) is an internationally quarantined disease of wheat. Until now, very little information has been available on the molecular basis of resistance and pathogenicity of T.&amp;nbsp;indica. To investigate the molecular basis of host-pathogen interaction, the transcriptome of T. indica inoculated resistant (HD29) and susceptible (WH542) genotypes of wheat were analyzed. Approximately 58 million reads were generated using RNA sequencing by the Illumina NextSeq500 platform. These sequence reads were aligned to a reference genome of wheat to compare the expression level of genes in resistant and susceptible genotypes. The high-quality reads were deposited in the NCBI SRA database (SRP159223). More than 80,000 genes were expressed in both the resistant and susceptible wheat genotypes. Of these, 76,088 were commonly expressed genes, including 3184 significantly upregulated and 1778 downregulated genes. Four thousand one hundred thirteen and 5604 genes were exclusively expressed in susceptible and resistant genotypes, respectively. Based on the significance, 503 genes were upregulated and 387 genes were downregulated. Using gene ontology, the majority of coding sequences were associated with response to stimuli, stress, carbohydrate metabolism, developmental process, and catalytic activity. Highly differentially expressed genes (integral component of membrane, exonuclease activity, nucleic acid binding, DNA binding, metal ion binding) were validated in resistant and susceptible genotypes using qPCR analysis and similar expression levels were found in RNA-Seq. Apart from the wheat, the mapping of T. indica was 7.07% and 7.63% of resistant and susceptible hosts, respectively, upon infection, which revealed significant pathogenesis-related genes. This first study provided in-depth information and new insights into wheat-T. indica interaction for managing Karnal bunt disease of wheat.

SILAC-based quantitative proteomics and microscopy analysis of cancer cells treated with the N-glycolyl GM3-specific anti-tumor antibody 14F7.

Cancer immunotherapy represents a promising approach to specifically target and treat cancer. The most common mechanisms by which monoclonal antibodies kill cells include antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity and apoptosis, but also other mechanisms have been described. 14F7 is an antibody raised against the tumor-associated antigen NeuGc GM3, which was previously reported to kill cancer cells without inducing apoptotic pathways. The antibody was reported to induce giant membrane lesions in tumor cells, with apparent changes in the cytoskeleton. Here, we investigated the effect of humanized 14F7 on HeLa cells using stable isotope labeling with amino acids in cell culture (SILAC) in combination with LC-MS and live cell imaging. 14F7 did not kill the HeLa cells, however, it caused altered protein expression (MS data are available via ProteomeXchange with identifier PXD024320). Several cytoskeletal and nucleic-acid binding proteins were found to be strongly down-regulated in response to antibody treatment, suggesting how 14F7 may induce membrane lesions in cells that contain higher amounts of NeuGc GM3. The altered expression profile identified in this study thus contributes to an improved understanding of the unusual killing mechanism of 14F7.