Electrophoretic Mobility Shift Assay Research Articles

ArNAC148 induces Acer rubrum leaf senescence by activating the transcription of the ABA receptor gene ArPYR13

Acer rubrum, an ornamental tree known for its stunning autumn colors, has an elusive molecular mechanism that governs its leaf senescence. We performed the genome-wide analysis of NAC transcription factor genes and PYRABACTIN RESISTANCE1-LIKE (PYLs) and found that ArNAC148 and ArPYL13 were significantly upregulated in senescing leaves as compared to mature leaves. Subcellular localization studies confirmed the nuclear localization of ArNAC148 and the cytoplasmic localization of ArPYL13. Electrophoretic mobility shift assay and yeast one-hybrid assay demonstrated that ArNAC148 directly binds to the promoter of ArPYL13. Luciferase reporter assays further showed that ArNAC148 activates the transcription of ArPYL13. The transient expression of ArNAC148 and ArPYL13 in tobacco leaves promoted chlorophyll degradation, increased H2O2 level, MDA contents, and electrolyte leakage in response to abscisic acid (ABA). Moreover, the virus-induced gene silencing of ArNAC148 and ArPYL13 in A. rubrum produced results that were opposite to those observed in transient expression experiments. Our findings suggest that ArNAC148 induces leaf senescence by directly activating the transcription of ArPYL13, providing insights into the ABA-mediated regulatory mechanisms governing leaf senescence in A. rubrum. This study offers new perspectives for researchers to explore the roles of NAC and PYL genes in regulating leaf senescence in woody ornamental plants.

Inhibition of ATGL alleviates MASH via impaired PPARα signalling that favours hydrophilic bile acid composition in mice

Adipose triglyceride lipase (ATGL) is an attractive therapeutic target in insulin resistance and metabolic dysfunction-associated steatotic liver disease (MASLD). This study investigated the effects of pharmacological ATGL inhibition on the development of metabolic dysfunction-associated steatohepatitis (MASH) and fibrosis in mice. Streptozotocin-injected male mice were fed an HFD to induce MASH. Mice receiving the ATGL inhibitor, Atglistatin (ATGLi), were compared to controls using liver histology, lipidomics, metabolomics, 16s rRNA, and RNA sequencing. Human ileal organoids, HepG2 cells, and Caco2 cells treated with the human ATGL inhibitor NG-497, HepG2 ATGL knockdown cells, gel-shift, and luciferase assays were analysed for mechanistic insights. We validated its benefits on steatohepatitis and fibrosis in a low-methionine choline-deficient mouse model. ATGLi improved serum liver enzymes, hepatic lipid content, and histological liver injury. Mechanistically, ATGLi attenuated PPARα signalling, favouring hydrophilic bile acid (BA) synthesis with increased Cyp7a1, Cyp27a1, Cyp2c70, and reduced Cyp8b1 expression. Additionally, reduced intestinal Cd36 and Abca1, along with increased Abcg5 expression, were consistent with reduced levels of hepatic TAG-species containing PUFAs like linoleic acids as well as reduced cholesterol levels in the liver and plasma. Similar changes in gene expression associated with PPARα signaling and intestinal lipid transport were observed in ileal organoids treated with NG-497. Furthermore, HepG2 ATGL knockdown cells revealed reduced expression of PPARα target genes and upregulation of genes involved in hydrophilic BA synthesis, consistent with reduced PPARα binding and luciferase activity in the presence of the ATGL inhibitors. Inhibition of ATGL attenuates PPARα signalling, translating into hydrophilic BAs, interfering with dietary lipid absorption, and improving metabolic disturbances. The validation with NG-497 opens a new therapeutic perspective for MASLD. The global prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) is a crucial public health concern. Since adherence to behavioural interventions is limited, pharmacological strategies are necessary, as highlighted by the recent FDA approval of resmetirom. However, since our current mechanistic understanding and pathophysiology-oriented therapeutic options for MASLD are still limited, novel mechanistic insights are urgently needed. Our present work uncovers that pharmacological inhibition of ATGL, the key enzyme in lipid hydrolysis using Atglistatin (ATGLi), improves metabolic dysfunction-associated steatohepatitis (MASH), fibrosis, and associated key features of metabolic dysfunction in a mouse model of MASH and MCD-induced liver fibrosis. Mechanistically, we demonstrated that attenuation of PPARα signalling in the liver and gut favours hydrophilic bile acid composition, ultimately interfering with dietary lipid absorption. One of the drawbacks of ATGLi is its lack of efficacy against human ATGL, thus limiting its clinical applicability. Against this backdrop, we could show that ATGL inhibition using the human inhibitor NG-497 in human primary ileum-derived organoids, Caco2 cells, and HepG2 cells translated into therapeutic mechanisms similar to ATGLi. Collectively, these findings open a new avenue for MASLD treatment development by inhibiting human ATGL activity.

Direct DNA binding by BRCA1 on β-hCG promoter and its clinical implications

ObjectiveThe role of β-hCG in breast cancer is largely unknown, this study aims to analyse the gene expression and clinical implications of β-hCG and its isoforms in various cancers focussing particularly in Breast Invasive Carcinoma (BRCA). A mechanistic approach deciphering the transcriptional regulation of β-hCG by BRCA1 was also explored. MethodsData from various comprehensive gene expression platforms like UALCAN, GEPIA2, GENT2, TIMER2, LinkedOmics, and STRING were used to analyse the expression of β-hCG and its clinical implications; Immunohistochemistry and ELISA for β-hCG expression analysis from human breast cancer patients; Electrophoretic mobility shift assay (EMSA) to analyse the direct binding of BRCA1 on β-hCG; Immunoblotting and Luciferase assay to understand the regulation of β-hCG by p53 were performed. ResultsResults from UALCAN and GENT2 gene expression cancer database revealed that TNBC subtypes and high-grade metaplastic carcinoma shows elevated expression of β-hCG and infiltration of various immune cells were also identified in BRCA by TIMER2. It was observed that most of the isoforms of β-hCG (CGB) are upregulated in breast cancers irrespective of hormonal status when BRCA1 gene is mutated according to TIMER2. Similar results were observed with Lymphoid neoplasm diffuse large B-cell lymphoma (LGG) and DLBC (Brain lower grade glioma) when BRCA1 is mutated. These results correlate with our earlier reports indicating expression of β-hCG in BRCA1 defective condition. We have also identified direct binding of BRCA1 on β-hCG promoter. ConclusionAll these findings demonstrate the importance of β-hCG as a potential target in BRCA1-deficient carcinomas.

Involvement of PG1037 in the repair of 8-oxo-7,8-dihydroguanine caused by oxidative stress in Porphyromonas gingivalis.

The PG1037 gene is part of the uvrA-PG1037-pcrA operon in Porphyromonas gingivalis. It encodes for a protein of unknown function upregulated under hydrogen peroxide (H2O2)-induced oxidative stress. Bioinformatic analysis shows that PG1037 has a zinc-finger motif, two peroxidase motifs, and one cytidylate kinase domain. The aim of this study is to characterize further the role of the PG1037 recombinant protein in the unique 8-oxoG repair system in P. gingivalis. PG1037 recombinant proteins with deletions in the zinc-finger or peroxidase motifs were created. Electrophoretic mobility shift assays were used to evaluate the ability of the recombinant proteins to bind 8-oxoG-containing oligonucleotides. Zinc binding, peroxidase, and Fenton reaction assays were used to assess the functional roles of the rPG1037 protein. A bacterial adenylate cyclase two-bride assay was used to identify the partner protein of PG1037 in the repair of 8-oxoG. The recombinant PG1037 (rPG1037) protein carrying an N-terminal His-tag demonstrated an ability to recognize and bind 8-oxoG-containing oligonucleotide. In contrast to the wild-type rPG1037 protein, the zinc-finger motif deletion resulted in the loss of zinc and 8-oxoG binding activities. A deletion of the peroxidase motif-1 showed a decrease in peroxidase activity. Using a bacterial adenylate cyclase two-hybrid system, there was no observed protein-protein interaction of PG1037 with UvrA (PG1036), PcrA (PG1038), or mismatch repair system proteins. Taken together, the results show that PG1037 is an important member of a novel mechanism that recognizes and repairs oxidative stress-induced DNA damage in P. gingivalis.

A cold-inducible MYB like transcription factor, CsHHO2, positively regulates chilling tolerance of cucumber fruit by enhancing CsGR-RBP3 expression

Plant HRS1 (hypersensitive to low phosphate (Pi)-elicited primary root shortening 1) and its homologs have been primarily characterized in Pi and nitrogen responses. However, their regulatory roles in cold responses remain largely unexplored. Here, we characterized the functional roles of HRS1 homolog 2 (CsHHO2) in regulating cold resistance in harvested cucumber fruit and examined the dependency of the role on glycine-rich RNA-binding protein 3 (CsGR-RBP3) gene expression. Cold treatments strongly induced CsHHO2 expression in cucumber fruit. GUS staining and expression analysis revealed that cold treatment stimulated CsHHO2 expression, at least partially, by enhancing the promoter activity. Subcellular localization results indicated that the green fluorescent protein (GFP)-tagged CsHHO2 proteins were localized to both the nucleus and the cytoplasm (potentially the plasma membrane), with confirmed nuclear localization when co-overexpressed with mCherry. Reducing CsHHO2 expression in cucumber fruit through VIGS significantly compromised the chilling tolerance, while its overexpression in cucumber fruit and Arabidopsis plants enhanced cold tolerance. The results of electrophoretic mobility shift assay (EMSA), yeast one-hybrid (Y1H) assay and dual-luciferase reporter (DLR) assay indicated that CsHHO2 specifically bound to the promoter of CsGR-RBP3, and enhanced its transcription. Transcriptomic analysis indicated that most differentially expressed genes (DEGs) modulated by CsHHO2 and CsGR-RBP3 proteins were identical, and the common DEGs were mainly enriched in several stress defense pathways, such as plant-pathogen interaction, phenylpropanoid biosynthesis, and plant hormone signal transduction. Functional complementation analysis of CsHHO2 and CsGR-RBP3 in cucumber fruit revealed that CsGR-RBP3 expression were indispensable for CsHHO2-modulated cold resistance. These results collectively suggest that CsHHO2, activated by cold signals, positively regulates the chilling tolerance of cucumber fruit by enhancing CsGR-RBP3 expression. Overall, this work suggests CsHHO2 as a key regulator of cold tolerance in cucumber fruit, provides key genetic resources for breeding cold-resistant cucumbers, and sheds novel insight into the roles of HRS1 family members in regulating plant development under environmental stresses.

Programmable Aggregation of Self-Assembled DNA Constructs.

Biomolecular aggregates ensure the optimum concentration and proximity required for biochemical processes to take place. Synthetic aggregating systems are becoming increasingly essential to study/mimic dynamic condensates in nature. Herein the ratiometric DNA aggregation of self-assembled DNA constructs using lanthanide salts is reported. In addition, the aggregation is shown to be reversed by the addition of specific lanthanide-binding ligands. The aggregate formation is confirmed by dynamic light scatteringexperiment, electrophoretic mobility shift assay, and field emission scanning electron microscope. This programmed DNA aggregation and its reversion are applied to evaluating the lanthanide-DNA and lanthanide-ligand binding constants, respectively. To achieve this, Forster resonance energy transfer (FRET)pair dyes at the 3' or 5' end of the DNA strands are strategically placed that generate unique fluorescence patterns upon interaction with the DNA constructs and different triggers such as lanthanides/ligands/monovalent cations, thus enabling the tracking of various states of binding. It also demonstrates a "fast method" to form and stabilize G-quadruplex (GQ) using lanthanides which complements the existing slow formation of GQs with Na+/K+ ions. The formation of GQ by lanthanides is corroborated by FRET, circular dichroism (CD), and enzyme linked immunosorbent assay (ELISA) experiments. These DNA constructs, formed by lanthanides, have shown resistance to cleavage by DNase I, and distinctive binding to Protoporphyrin dyes and Thioflavin T.

The yellowhorn MYB transcription factor MYB30 is required for wax accumulation and drought tolerance.

Yellowhorn (Xanthoceras sorbifolium Bunge) is an economically important tree species in northern China, mainly distributed in arid and semi-arid areas where water resources are scarce. Drought affects its yield and the expansion of its suitable growth area. It was found that the wax content in yellowhorn leaves varied significantly among different germplasms, which had a strong correlation with the drought resistance of yellowhorn. In this study, XsMYB30 was isolated from 'Zhongshi 4' of yellowhorn, a new highly waxy variety. DAP-Seq technology revealed that the pathways associated with fatty acids were significantly enriched in the target genes of XsMYB30. Moreover, the results of electrophoretic mobility shift assay, yeast one hybrid assay and dual-luciferase assay demonstrated that XsMYB30 could directly and specifically bind with the promoters of genes involved in wax biosynthesis (XsFAR4, XsCER1 and XsKCS1), lipid transfer (XsLTPG1 and XsLTP1) and fatty acid synthesis (XsKASIII), thus enhancing their expression. In addition, the overexpression of XsMYB30 in poplar promoted the expression levels of these target genes and increased the wax deposition on poplar leaves leading to a notable improvement in the plant's ability to withstand drought. These findings indicate that XsMYB30 is an important regulatory factor in cuticular wax biosynthesis and the drought resistance of yellowhorn.

Betaine delays age-related muscle loss by mitigating Mss51-induced impairment in mitochondrial respiration via Yin Yang1.

Mitochondrial dysfunction is one of the hallmarks of aging and a leading contributor to sarcopenia. Nutrients are essential for improving mitochondrial function and skeletal muscle health during the aging process. Betaine is a nutrient with potential muscle-preserving properties. However, whether and how betaine could regulate the mitochondria function in aging muscle are poorly understood. We aimed to explore the molecular target and underlying mechanism of betaine in attenuating the age-related mitochondrial dysfunction in skeletal muscle. Young mice (YOU, 2months), old mice (OLD, 15months), and old mice with betaine treatment (BET, 15months) were fed for 12weeks. The effects of betaine on muscle mass, strength, function, and subcellular structure of muscle fibres were assessed. RNA sequencing (RNA-seq) was conducted to identify the molecular target of betaine. The impacts of betaine on mitochondrial-related molecules, superoxide accumulation, and oxidative respiration were examined using western blotting (WB), immunofluorescence (IF) and seahorse assay. The underlying mechanism of betaine regulation on the molecular target to maintain mitochondrial function was investigated by luciferase reporter assay, chromatin immunoprecipitation and electrophoretic mobility shift assay. Adenoassociated virus transfection, succinate dehydrogenase staining (SDH), and energy expenditure assessment were performed on 20-month-old mice for validating the mechanism in vivo. Betaine intervention demonstrated anti-aging effects on the muscle mass (P=0.017), strength (P=0.010), and running distance (P=0.013). Mitochondrial-related markers (ATP5a, Sdha, and Uqcrc2) were 1.1- to 1.5-fold higher in BET than OLD (all P≤0.036) with less wasted mitochondrial vacuoles accumulating in sarcomere. Bioinformatic analysis from RNA-seq displayed pathways related to mitochondrial respiration activity was higher enriched in BET group (NES=-0.87, FDR=0.10). The quantitative real time PCR (qRT-PCR) revealed betaine significantly reduced the expression of a novel mitochondrial regulator, Mss51 (-24.9%, P=0.002). In C2C12 cells, betaine restored the Mss51-mediated suppression in mitochondrial respiration proteins (all P≤0.041), attenuated oxygen consumption impairment, and superoxide accumulation (by 20.7%, P=0.001). Mechanically, betaine attenuated aging-induced repression in Yy1 mRNA expression (BET vs. OLD: 2.06 vs. 1.02, P=0.009). Yy1 transcriptionally suppressed Mss51 mRNA expression both in vitro and in vivo. This contributed to the preservation of mitochondrial respiration, improvement for energy expenditure (P=0.008), and delay of muscle loss during aging process. Altogether, betaine transcriptionally represses Mss51 via Yy1, improving age-related mitochondrial respiration in skeletal muscle. These findings suggest betaine holds promise as a dietary supplement to delay skeletal muscle degeneration and improve age-related mitochondrial diseases.

In vitro and In silico studies of terpenes extracted from Cymbopogon flexuous leaves against human myeloid leukemia as an inhibitor of NF-κB activation signaling pathway

Despite recent progress in treating myeloid leukemia (ML) challenges such as the emergence of drug resistance and the persistence of minimal residual disease pose significant obstacles in treating these patients. This highlights the importance of devising novel and innovative strategies to enhance therapeutic outcomes. Citral, myrcene and geranic acid are the major bio-actives of Cymbopogon flexuosus volatile oil (CFVO) that possess strong anticancer potential. This study explored the cytotoxic potential, chemical profiling, and the inhibitory effect of CFVO on nuclear factor-κB (NF-κB) activation pathways. The 3-(4–5-dimethylthiazol-2- yl) 2–5-diphenyl-tetrazolium (MTT) protocol was employed to assess the cell viability. Flow cytometry analysis was performed for assessing the cell cycle and conducting apoptosis assays. Western blot analysis showed that CFVO triggered apoptosis in cancer cells via activating caspases which led to proteolysis of poly ADP ribose polymerase (PARP). Furthermore, electrophoretic mobility shift assay (EMSA) showed that CFVO was capable of suppressing the activation of NF-kB triggered by TNF-α and also resulted in decrease in NF-κB-dependent proteins such as antiapoptotic(cFLIP, Bcl-xL and XIAP) proliferative (Cyclin E1, Cyclin D, C-myc) and invasion (ICAM-1) proteins. Our in sililco study also revealed the binding of anticancer phytochemicals with the P50 protein of NF-kB that favors the downregulation of this protein. Inclusively CFVO inhibits the proliferation and metastasis of tumor cells and we can suggest that it will be an effective therapy in fighting against cancer.

Exploring the manganese-dependent interaction between a transcription factor and its corresponding DNA: insights from gas-phase electrophoresis on a nES GEMMA instrument

Manganese ion homeostasis is vital for bacteria and is achieved via manganese-dependent transcription factors. Manganese mediation of transcription factor attachment to the corresponding oligonucleotide sequences can be investigated, e.g. via electrophoretic mobility shift assays (EMSA). Formation of specific biocomplexes leads to differences in the migration pattern upon gel electrophoresis. Focusing on electrophoresis in the gas-phase, applying a nano electrospray gas-phase electrophoretic mobility molecular analyzer (nES GEMMA) also known as nES differential mobility analyzer (nES DMA), and on transcription factors (MntR proteins) from Bacillus subtilis and Mycobacterium tuberculosis, we took interest in the gas-phase electrophoresis of the corresponding biospecific complexes. We compared nES GEMMA, separating analytes in the nanometer regime (a few to several hundred nm in diameter) in the gas-phase in their native state according to particle size, to EMSA data. Indeed we were able to demonstrate manganese-mediated attachment of MntR to target genomic sequences with both analytical techniques. Despite some inherent pitfalls of the nES GEMMA method like analyte/instrument surface interactions, we were able to detect the target complexes. Moreover, we were able to calculate the molecular weight (MW) of the obtained species by application of a correlation function based on nES GEMMA obtained data. As gas-phase electrophoresis also offers the possibility of offline hyphenation to orthogonal analysis techniques, we are confident that nES GEMMA measurements are not just complementary to EMSA, but will offer the possibility of further in-depth characterization of biocomplexes in the future.Graphical

Box-Behnken design assisted approach in optimizing lipid composition for cationic liposome formulation as gene carrier

BackgroundCationic liposomes represent a promising non-viral carrier platform for gene delivery. The successful intracellular delivery of genes to the target cell is highly influenced by lipid compositions in the liposomal formulation. In the present study, a Box-Behnken design was applied to investigate the optimal lipid composition for the liposome-based transfection agent. MethodsThe concentrations of DOTAP, DSPE-PEG, and cholesterol were set as independent factors. A total of 15 lipid compositions were generated and tested for specific responses, including particle size, encapsulation efficiency, cell viability, and cell transfection. The data were then analyzed to predict the optimal composition using response surface methodology (RSM). ResultsThe results for particle size, encapsulation efficiency, cell viability and fluorescence intensity ranged from 158.7 to 2064 nm, 48.19–95.72%, 81.50–122.67%, and 0.0–9.08, respectively. Compositions of liposome-based transfection agent without DOTAP, those without cholesterol, and those containing DSPE-PEG2000 with a molar ratio equal to or greater than that of cholesterol tended to exhibit low encapsulation efficiency. The ability of the liposome to complex DNA, as determined through electrophoresis gel retardation assay, showed that the composition without DOTAP produced DNA bands, indicating that the prepared liposomes had a less ability to complex DNA. The cytotoxicity test results indicated that all lipid compositions were considered non-toxic, as they exhibited >80% cell viability. The cell transfection assay demonstrated that the lipid composition containing a combination of DOTAP and cholesterol was able to transfect DNA into cells. According to response analysis, RSM predicted that the optimal lipid composition consisted of 2.75 μmol DOTAP and 0.91 μmol cholesterol, with a desirability value of 0.85. ConclusionsAlthough the equation model is still acceptable for predicting the optimal lipid composition, further study is needed to obtain a model with higher desirability, such as by using more lipid compositions, increased replications, and different variable responses.

T3SS protein EsrC binds to the lacI-like operator of type 1 fimbrial operon to suppress adhesion of Edwardsiella piscicida.

Type 1 fimbria, the short hair-like appendage assembled on the bacterial surface, plays a pivotal role in adhesion and invasion in Edwardsiella piscicida. The type III secretion system (T3SS), another bacterial surface appendage, facilitates E. piscicida's replication in vivo by delivering effectors into host cells. Our previous research demonstrated that E. piscicida T3SS protein EseJ inhibits adhesion and invasion of E. piscicida by suppressing type 1 fimbria. However, how EseJ suppresses type 1 fimbria remains elusive. In this study, a lacI-like operator (nt -245 to -1 of fimA) upstream of type 1 fimbrial operon in E. piscicida was identified, and EseJ inhibits type 1 fimbria through the lacI-like operator. Moreover, through DNA pull-down and electrophoretic mobility shift assay, an AraC-type T3SS regulator, EsrC, was screened and verified to bind to nt -145 to -126 and nt -50 to -1 of fimA, suppressing type 1 fimbria. EseJ is almost abolished upon the depletion of EsrC. EsrC and EseJ impede type 1 fimbria expression. Intriguingly, nutrition and microbiota-derived indole activate type 1 fimbria through downregulating T3SS, alleviating EsrC or EseJ's inhibitory effect on lacI-like operator of type 1 fimbrial operon. By this study, it is revealed that upon entering the gastrointestinal tract, rich nutrients and indole downregulate T3SS and thereof upregulate type 1 fimbria, stimulating efficient adhesion and invasion; upon being internalized into epithelium, the limit in indole and nutrition switches on T3SS and thereof switches off type 1 fimbria, facilitating effector delivery to guarantee E. piscicida's survival/replication in vivo.IMPORTANCEIn this work, we identified the lacI-like operator of type 1 fimbrial operon in E. piscicida, which was suppressed by the repressors-T3SS protein EseJ and EsrC. We unveiled that E. piscicida upregulates type 1 fimbria upon sensing rich nutrition and the microbiota-derived indole, thereof promoting the adhesion of E. piscicida. The increase of indole and nutrition promotes type 1 fimbria by downregulating T3SS. The decrease in EseJ and EsrC alleviates their suppression on type 1 fimbria, and vice versa.

RNA interacts with topoisomerase I to adjust DNA topology

Topoisomerase I (TOP1) is an essential enzyme that relaxes DNA to prevent and dissipate torsional stress during transcription. However, the mechanisms underlying the regulation of TOP1 activity remain elusive. Using enhanced cross-linking and immunoprecipitation (eCLIP) and ultraviolet-cross-linked RNA immunoprecipitation followed by total RNA sequencing (UV-RIP-seq) in human colon cancer cells along with RNA electrophoretic mobility shift assays (EMSAs), biolayer interferometry (BLI), and invitro RNA-binding assays, we identify TOP1 as an RNA-binding protein (RBP). We show that TOP1 directly binds RNA invitro and in cells and that most RNAs bound by TOP1 are mRNAs. Using a TOP1 RNA-binding mutant and topoisomerase cleavage complex sequencing (TOP1cc-seq) to map TOP1 catalytic activity, we reveal that RNA opposes TOP1 activity as RNA polymerase II (RNAPII) commences transcription of active genes. We further demonstrate the inhibitory role of RNA in regulating TOP1 activity by employing DNA supercoiling assays and magnetic tweezers. These findings provide insight into the coordinated actions of RNA and TOP1 in regulating DNA topological stress intrinsic to RNAPII-dependent transcription.

Tetrazine Amino Acid Encoding for Rapid and Complete Protein Bioconjugation.

Generating protein conjugates using the bioorthogonal ligation between tetrazines and trans-cyclooctene groups avoids the need to manipulate cysteine amino acids; this ligation is rapid, site-specific, and stoichiometric and allows for labeling of proteins in complex biological environments. Here, we provide a protocol for the expression of conjugation-ready proteins at high yields in Escherichia coli with greater than 95% encoding and labeling fidelity. This protocol focuses on installing the Tet2 tetrazine amino acid using an optimized genetic code expansion (GCE) machinery system, Tet2 pAJE-E7, to direct Tet2 encoding at TAG stop codons in BL21 E. coli strains, enabling reproducible expression of Tet2-proteins that quantitatively react with trans-cyclooctene (TCO) groups within 5 min at room temperature and physiological pH. The use of the BL21 derivative B95(DE3) minimizes premature truncation byproducts caused by incomplete suppression of TAG stop codons, which makes it possible to use more diverse protein construct designs. Here, using a superfolder green fluorescent protein construct as an example protein, we describe in detail a four-day process for encoding Tet2 with yields of ~200 mg per liter of culture. Additionally, a simple and fast diagnostic gel electrophoretic mobility shift assay is described to confirm Tet2-Et encoding and reactivity. Finally, strategies are discussed to adapt the protocol to alternative proteins of interest and optimize expression yields and reactivity for that protein. Key features • Protocol describes site-specific encoding of the tetrazine amino acid Tet2-Et into proteins for bioorthogonal, quantitative, and rapid attachment of trans-cyclooctene-containing labels. • Protocol uses auto-induction methods for the production Tet2-Et protein in E. coli. • This protocol focuses on Tet-protein expressions in BL21(DE3) and B95(DE3) strains, which take approximately 4 days to complete. • SDS-PAGE mobility shift assay using a strained TCO-PEG5000 (sTCO-PEG5000) reagent provides a simple, generalizable method for testing Tet-protein reactivity.

Strigolactones positively regulate HY5-dependent autophagy and the degradation of ubiquitinated proteins in response to cold stress in tomato.

Autophagy, involved in protein degradation and amino acid recycling, plays a key role in plant development and stress responses. However, the relationship between autophagy and phytohormones remains unclear. We used diverse methods, including CRISPR/Cas9, ultra-performance liquid chromatography coupled with tandem mass spectrometry, chromatin immunoprecipitation, electrophoretic mobility shift assays, and dual-luciferase assays to explore the molecular mechanism of strigolactones in regulating autophagy and the degradation of ubiquitinated proteins under cold stress in tomato (Solanum lycopersicum). We show that cold stress induced the accumulation of ubiquitinated proteins. Mutants deficient in strigolactone biosynthesis were more sensitive to cold stress with increased accumulation of ubiquitinated proteins. Conversely, treatment with the synthetic strigolactone analog GR245DS enhanced cold tolerance in tomato, with elevated levels of accumulation of autophagosomes and transcripts of autophagy-related genes (ATGs), and reduced accumulation of ubiquitinated proteins. Meanwhile, cold stress induced the accumulation of ELONGATED HYPOCOTYL 5 (HY5), which was triggered by strigolactones. HY5 further trans-activated ATG18a transcription, resulting in autophagy formation. Mutation of ATG18a compromised strigolactone-induced cold tolerance, leading to decreased formation of autophagosomes and increased accumulation of ubiquitinated proteins. These findings reveal that strigolactones positively regulate autophagy in an HY5-dependent manner and facilitate the degradation of ubiquitinated proteins under cold conditions in tomato.

PH modification of gel mobility shift improves polyplex selection In Vivo

Cationic polymers that bind with the plasmids to form polyplexes protect the DNA from enzymatic degradation and improve cellular and tissue uptake. Complete or near complete gel retardation of the polyplex is an important assay to determine the optimal polymer: plasmid ratio for in vitro and in vivo studies. Nevertheless, despite minimal to moderate gel retardation of histidine-lysine (HK) polyplexes formed with low peptide: plasmid DNA ratios (1:2 and 1:4; w:w), the polyplexes effectively targeted the tumor in vivo. To understand the lack of predictability of the initial gel mobility shift assays, we revisited the retardation and stability of polyplexes with these electrophoresis assays. Because the histidine component with a pKa of about 6.0 will have a greater positive charge and may bind plasmids with a higher affinity at lower pHs, we compared the retardation of the two HK polyplexes when the pH of the running buffer of the gel mobility shift assay was altered. Both HK polyplexes were retarded significantly more when the running buffer had a pH of 7.3 instead of the standard pH of 8.3. Indeed, the HK polyplexes formed at the 1:2 ratio showed complete retardation at pH 7.3. Consequently, while both HK polyplexes formed at these low ratios targeted the tumor, the polyplex formed with the 1:2 ratio had reduced tumor gene expression variability and lower lung and liver values. Thus, the selection of the optimal ratios for the linear HK and plasmid for transfection studies in vivo was improved with a running buffer pH of 7.3.

HSP70 promotes pancreatic cancer cell epithelial-mesenchymal transformation and growth via the NF-κB signaling pathway.

To study the effects of HSP70 on proliferation, migration, invasion, and epithelial-mesenchymal transformation (EMT) of pancreatic cancer cells and explore its underlying mechanisms. Pancreatic cancer cell models with either reduced HSP70 or increased HSP70 expression were established. RT-qPCR and Western blot assays were used to determine mRNA and protein levels of HSP70, IKK/IκBa/NF-κB signaling pathway-related genes, and EMT markers. The CCK-8 and cell cloning assays were used to evaluate cell proliferation and cloning abilities. Transwell and wound healing assays were used to assess the invasive and migratory properties of the cells. Effects of NF-κB signaling modulation were explored using an IKK inhibitor (BAY11-7082) and an IKK overexpression vector (pCMV-IKK). Electrophoretic mobility shift assay (EMSA) and luciferase reporter assays were conducted to analyze NF-κB's promoter binding and transcriptional activities. HSP70 knockdown inhibited p-p65 nuclear translocation and reduced the expression of p-p65, p-IKKα/β, p-IκBα, N-cadherin, Vimentin, and Twist. It also decreased NF-κB's promoter binding and transcriptional activities, increased E-cadherin levels, and suppressed pancreatic cancer cell proliferation, cloning, migration, and invasion. In contrast, HSP70 overexpression led to increased expression of p-p65, p-IKKα/β, p-IκBα, N-cadherin, Vimentin, and Twist, decreased E-cadherin levels, and enhanced cell proliferation, cloning, migration, and invasion capabilities. NF-κB signaling pathway modulation reversed EMT changes induced by altered HSP70 expression levels. rhHSP70 also increased p-IKKα/β and p-IκBα protein levels. HSP70 promotes the EMT and enhances pancreatic cancer cell proliferation, migration, and invasion by activating the NF-κB pathway.

Host plant-derived benzoic acid interferes with 4-hydroxybenzoic acid degradation in the phytopathogen Xanthomonas campestris by competitively binding to PobR

Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot in Brassica vegetables, which can induce the host plant to produce salicylic acid and 4-hydroxybenzoic acid (4-HBA) during infection. Xcc was previously shown to sense and degrade host plant-derived 4-HBA via the sensor PobR and a PobA-dependent pathway. The degradation of 4-HBA is associated with Xcc virulence in cabbage. The present study generated a reporter strain XC1::PpobA-gusA to monitor pobA transcription. 4-HBA-like compounds were screened for their ability to interfere with pobA transcription. Benzoic acid (BA) was found to efficiently decrease pobA transcription in a dose-dependent manner. Xcc neither produced nor degraded BA; however, the exogenous addition of BA to the 4-HBA-containing Xcc culture significantly decreased the 4-HBA degradation rate. Furthermore, addition of BA into the Xcc culture did not significantly affect the transcription of pobA or pobR; however, addition of BA into the 4-HBA-containing culture significantly decreased the transcription of both genes. Isothermal titration calorimetry and an electrophoretic mobility shift assay revealed that BA binds to PobR with a moderate affinity, which interfered with the binding of 4-HBA/PobR complex to the pobA promoter and thereby inhibiting pobA transcription and 4-HBA degradation. The endogenous BA level of the infected cabbage leaves increased in response to Xcc infection. In the presence of BA, the virulence of Xcc on cabbage decreased significantly. Taken together, these results suggest that cabbage utilizes BA to interfere with 4-HBA degradation, thereby reducing Xcc virulence. Thus, BA has the potential to be developed as a bactericide against Xcc infection.

The MYC transcription factor PbrMYC8 negatively regulates PbrMSL5 expression to promote pollen germination in Pyrus

The successful germination of pollen is essential for double fertilization in flowering plants. Mechanosensitive channels of small conductance (MscS-like, MSL) inhibit pollen germination and maintains cellular integrity of pollen during this process. Therefore, it is vital to carefully regulate the expression of MSL to promote successful pollen germination. Despite its importance, the molecular mechanisms governing MSL expression in plants remain poorly understood. Here, we had identified 15 MSL genes in the pear, among which PbrMSL5 was expressed in pollen development. Subcellular localization experiments revealed that PbrMSL5 was located in both plasma membrane and cytoplasm. Functional investigations, including complementation experiments using the atmsl8 mutant background, demonstrated the involvement of PbrMSL5 in preserving pollen cell integrity and inhibiting germination. Antisense oligonucleotide experiments further confirmed that PbrMSL5 suppressed pear pollen germination by reducing osmotic pressure and Cl− content. Yeast one-hybrid, electrophoretic mobility shift assays, and dual luciferase reporter assay elucidated that PbrMYC8 interacts directly with the N-box element, leading to the suppression of PbrMSL5 expression and promoted pollen germination. These results represented a significant advancement in unraveling the molecular mechanisms controlling plant MSL expression. This study showed valuable contribution to advancing our comprehension of the mechanism underlying pollen germination.

High-throughput identification of functional regulatory SNPs in systemic lupus erythematosus

Genome-wide association studies implicate multiple loci in risk for systemic lupus erythematosus (SLE), but few contain exonic variants, rendering systematic identification of non-coding variants essential to decoding SLE genetics. We utilized SNP-seq and bioinformatic enrichment to interrogate 2180 single-nucleotide polymorphisms (SNPs) from 87 SLE risk loci for potential binding of transcription factors and related proteins from B cells. 52 SNPs that passed initial screening were tested by electrophoretic mobility shift and luciferase reporter assays. To validate the approach, we studied rs2297550 in detail, finding that the risk allele enhanced binding to the transcription factor Ikaros (encoded by IKZF1), thereby modulating expression of IKBKE. Correspondingly, primary cells from genotyped healthy donors bearing the risk allele expressed higher levels of the interferon / NF-κB regulator IKKε. Together, these findings define a set of likely functional non-coding lupus risk variants and identify a regulatory pathway involving rs2297550, Ikaros, and IKKε implicated by human genetics in risk for SLE.