Distinguishing G-Quadruplexes Stabilizer and Chaperone for c-MYC Promoter G-Quadruplexes through Single-Molecule Manipulation.

G-quadruplex (G4) selective stabilizing ligands can regulate c-MYC gene expression, but the kinetic basis remains unclear. Determining the effects of ligands on c-MYC promoter G4s' folding/unfolding kinetics is challenging due to the polymorphic nature of G4s and the high energy barrier to unfold c-MYC promoter G4s. Here, we used single-molecule magnetic tweezers to manipulate a duplex hairpin containing a c-MYC promoter sequence to mimic the transiently denatured duplex during transcription. We measured the effects of six commonly used G4s binding ligands on the competition between quadruplex and duplex structures, as well as the folding/unfolding kinetics of G4s. Our results revealed two distinct roles for G4s selective stabilization: CX-5461 is mainly acting as c-MYC G4s stabilizer, reducing the unfolding rate (ku) of c-MYC G4s, whereas PDS and 360A also act as G4s chaperone, accelerating the folding rates (kf) of c-MYC G4s. qRT-PCR results obtained from CA46 and Raji cell lines demonstrated that G4s stabilizing ligands can downregulate c-MYC expression, while G4s stabilizer CX-5461 exhibited the strongest c-MYC gene suppression. These results shed light on the potential of manipulating G4s' folding/unfolding kinetics by ligands for precise regulation of promoter G4-associated biological activities.

Ferroptosis-related differentially expressed genes serve as new biomarkers in ischemic stroke and identification of therapeutic drugs.

Background: Iron is an essential nutrient element, and iron metabolism is related to many diseases. Ferroptosis is an iron-dependent form of regulated cell death associated with ischemic stroke (IS). Hence, this study intended to discover and validate the possible ferroptosis-related genes involved in IS. Materials and methods: GSE16561, GSE37587, and GSE58294 were retrieved from the GEO database. Using R software, we identified ferroptosis-related differentially expressed genes (DEGs) in IS. Protein-protein interactions (PPIs) and enrichment analyses were conducted. The ROC curve was plotted to explore the diagnostic significance of those identified genes. The consistent clustering method was used to classify the IS samples. The level of immune cell infiltration of different subtypes was evaluated by ssGSEA and CIBERSORT algorithm. Validation was conducted in the test sets GSE37587 and GSE58294. Results: Twenty-one ferroptosis-related DEGs were detected in IS vs. the normal controls. Enrichment analysis shows that the 21 DEGs are involved in monocarboxylic acid metabolism, iron ion response, and ferroptosis. Moreover, their expression levels were pertinent to the age and gender of IS patients. The ROC analysis demonstrated remarkable diagnostic values of LAMP2, TSC22D3, SLC38A1, and RPL8 for IS. Transcription factors and targeting miRNAs of the 21 DEGs were determined. Vandetanib, FERRIC CITRATE, etc., were confirmed as potential therapeutic drugs for IS. Using 11 hub genes, IS patients were categorized into C1 and C2 subtypes. The two subtypes significantly differed between immune cell infiltration, checkpoints, and HLA genes. The 272 DEGs were identified from two subtypes and their biological functions were explored. Verification was performed in the GSE37587 and GSE58294 datasets. Conclusion: Our findings indicate that ferroptosis plays a critical role in the diversity and complexity of the IS immune microenvironment.

Characterization of G-Quadruplexes Folding/Unfolding Dynamics and Interactions with Proteins from Single-Molecule Force Spectroscopy.

G-quadruplexes (G4s) are stable secondary nucleic acid structures that play crucial roles in many fundamental biological processes. The folding/unfolding dynamics of G4 structures are associated with the replication and transcription regulation functions of G4s. However, many DNA G4 sequences can adopt a variety of topologies and have complex folding/unfolding dynamics. Determining the dynamics of G4s and their regulation by proteins remains challenging due to the coexistence of multiple structures in a heterogeneous sample. Here, in this mini-review, we introduce the application of single-molecule force-spectroscopy methods, such as magnetic tweezers, optical tweezers, and atomic force microscopy, to characterize the polymorphism and folding/unfolding dynamics of G4s. We also briefly introduce recent studies using single-molecule force spectroscopy to study the molecular mechanisms of G4-interacting proteins.

Mechanical diversity and folding intermediates of parallel-stranded G-quadruplexes with a bulge.

A significant number of sequences in the human genome form noncanonical G-quadruplexes (G4s) with bulges or a guanine vacancy. Here, we systematically characterized the mechanical stability of parallel-stranded G4s with a one to seven nucleotides bulge at various positions. Our results show that G4-forming sequences with a bulge form multiple conformations, including fully-folded G4 with high mechanical stability (unfolding forces > 40 pN), partially-folded intermediates (unfolding forces < 40 pN). The folding probability and folded populations strongly depend on the positions and lengths of the bulge. By combining a single-molecule unfolding assay, dimethyl sulfate (DMS) footprinting, and a guanine-peptide conjugate that selectively stabilizes guanine-vacancy-bearing G-quadruplexes (GVBQs), we identified that GVBQs are the major intermediates of G4s with a bulge near the 5' or 3' ends. The existence of multiple structures may induce different regulatory functions in many biological processes. This study also demonstrates a new strategy for selectively stabilizing the intermediates of bulged G4s to modulate their functions.

High Mechanical Stability and Slow Unfolding Rates Are Prevalent in Parallel-Stranded DNA G-Quadruplexes.

Guanine-rich repeat sequences are known to adopt diverse G-quadruplex (G4) topologies. Determining the unfolding rates of individual G4 species is challenging due to the coexistence of multiple G4 conformations in a solution. Here, using single-molecule magnetic tweezers, we systematically measured the unfolding force distributions of 4 oncogene promoter G4s, 12 model sequences with two 1-nucleotide (nt) thymine loops that predominantly adopt parallel-stranded G4 structures, and 6 sequences forming multiple G4 structures. All parallel-stranded G4s reveal an unfolding force peak at 40-60 pN, which is associated with extremely slow unfolding rates on the order of 10-5-10-7 s-1. In contrast, nonparallel G4s and partially folded intermediate states reveal an unfolding force peak <40 pN. These results suggest a strong correlation between the parallel-stranded G4s folding topology and the slow unfolding rates and provide important insights into the mechanism that govern the stability and the transition kinetics of G4s.

Single-Molecule Mechanical Unfolding of AT-Rich Chromosomal Fragile Site DNA Hairpins: Resolving the Thermodynamic and Kinetic Effects of a Single G-T Mismatch.

Chromosomal fragile sites (CFSs) contain AT-rich sequences that tend to form hairpins on lagging strands in DNA replication, making them hotspots for chromosomal rearrangements in cancers. Here, we investigate the structural stability of the AT-rich CFS DNA hairpins with a single non-AT base pair using magnetic tweezers. Strikingly, a single G-T mismatched base pair in the short CFS DNA hairpin gives a 38.7% reduction of the unfolding Gibbs free energy and a 100-fold increase of the transition kinetics compared to a single G-C matched base pair, which are deviated from the theoretical simulations. Our study reveals the unique features of CFSs to provide profound insights into chromosomal instability and structure-specific genome targeting therapeutics for genetic disorder-related diseases.

Typing and evaluating heat resistance of Bacillus cereus sensu stricto isolated from the processing environment of powdered infant formula.

Bacillus cereus sensu lato is one of the most harmful bacterial groups affecting the quality and safety of powdered infant formula (PIF). In this study, samples were collected from the raw materials and processing environments of PIF. A total of 84 isolates were identified as Bacillus cereus sensu stricto (B. cereus s. s.) by 16S rRNA analysis, molecular typing technology, and physiological and biochemical tests. The 84 B. cereus s. s. strains were assigned to panC group II, group III, and group IV. Then, the 7 housekeeping genes glpF, gmk, ilvD, pta, pur, pycA, and tpi were selected for multilocus sequence typing. Results showed that the 84 isolates were clustered into 24 sequence types (ST), and 14 novel ST were detected. Among the 24 ST, ST999 (19/84, 22.62%) and ST1343 (13/84, 15.48%) predominated. The correlation between processing areas and ST showed that the processing environments of the production and packing areas were the most susceptible to contamination by B. cereus s. s. Spores of these ST showed different heat resistance phenotypes evaluated by the analysis of DT (time in minutes of spore decimal reduction at each temperature) and Z values (temperature increase required to reduce the DT value to one-tenth of the original). Spores from group III according to panC gene analysis were the most heat resistant. These findings will help us to better understand B. cereus s. s. contamination and control in PIF processing environments.

Transcriptomic responses of Caco-2 cells to Lactobacillus rhamnosus GG and Lactobacillus plantarum J26 against oxidative stress.

Oxidative stress is the basic reason for aging and age-related diseases. In this study, we investigated the protective effect of 2 strains of lactic acid bacteria (LAB), Lactobacillus rhamnosus GG and L. plantarum J26, against oxidative stress in Caco-2 cells, and gave an overview of the mechanisms of lactic acid bacteria antioxidant activity using digital gene expression profiling. The 2 LAB strains provided significant protection against hydrogen peroxide (H2O2)-induced reduction in superoxide dismutase activity and increase in glutathione peroxidase activity in Caco-2 cells. However, inactive bacteria had little effect on alleviating oxidation stress in Caco-2 cells. Eight genes related to oxidative stress-FOSB, TNF, PPP1R15A, NUAK2, ATF3, TNFAIP3, EGR2, and FBN2-were significantly upregulated in H2O2-induced Caco-2 cells compared with untreated Caco-2 cells. After incubation of the H2O2-induced Caco-2 cells with L. rhamnosus GG and L. plantarum J26, 5 genes (TNF, EGR2, NUAK2, FBN2, and TNFAIP3) and 2 genes (NUAK2 and FBN2) were downregulated, respectively. In addition, the Kyoto Encyclopedia of Genes and Genomes indicated that some signaling pathways associated with inflammation, immune response, and apoptosis, such as Janus kinase/signal transducers and activators of transcription (Jak-STAT), mitogen-activated protein kinase (MAPK), nuclear factor-κB, and tumor necrosis factor, were all negatively modulated by the 2 strains, especially L. rhamnosus GG. In this paper, we reveal the mechanism of LAB in relieving oxidative stress and provide a theoretical basis for the rapid screening and evaluation of new LAB resources.

Folding/unfolding kinetics of G-quadruplexes upstream of the P1 promoter of the human BCL-2 oncogene.

The G-rich Pu39 region of the P1 promoter of the oncogene BCL-2, an apoptosis regulator, can fold into multiple G-quadruplex (G4) structures. Bcl2-2345 and Bcl2-1245 are two major G4 species forming with high thermal stability and distinct topologies in the Pu39 region, but their folding/unfolding kinetics have not yet been investigated. Here, we used magnetic tweezers to measure the mechanical stability and the folding/unfolding kinetics of the Bcl2-2345 and Bcl2-1245 G4 structures. We report that the hybrid-stranded Bcl2-2345 G4 had a lower mechanical stability than the parallel-stranded Bcl2-1245 G4. We observed that the Bcl2-2345 G4 is a kinetically favored structure, whereas the Bcl2-1245 G4, with a slow unfolding rate, may function as a kinetic barrier for transcription. We also determined that in addition to the Bcl2-2345 and Bcl2-1245 G4s, other stable DNA secondary structures, such as a hybrid-stranded Bcl2-1234 G4, can also form in the Pu39 sequence. The characterization of the folding/unfolding kinetics of specific G4s reported here sheds light on the participation of G4s during gene transcription and provides information for designing G4-targeting small molecules that could modulate BCL-2 gene expression.

Silver nanoparticles: A novel antibacterial agent for control of Cronobacter sakazakii.

Silver nanoparticles (AgNP) have been widely applied because of their broad spectrum of antimicrobial activities against bacteria, fungi, and viruses. However, little research has been done to evaluate their effects on Cronobacter sakazakii, an opportunistic pathogen usually infecting infants and having a high fatality rate. The aims of this work were to investigate the antibacterial property of novel, synthesized, positively charged silver nanoparticles against C. sakazakii and to discuss the potential antibacterial mechanisms involved. In this study, the spherical and face-centered cubic silver nanoparticles had a mean particle size of 31.2 nm and were synthesized by reducing Ag+ using citrate and dispersed by glycerol and polyvinylpyrrolidone (PVP) under alkaline conditions. Minimum inhibitory concentrations (MIC) and inhibition zone tests showed that the AgNP exhibited strong antibacterial activity against 4 tested C. sakazakii strains with mean MIC of 62.5 to 125 mg/L and average inhibition zone diameters of 13.8 to 16.3 mm. Silver nanoparticles caused cell membrane injury accompanied by adsorption of AgNP onto the cell surface, as shown by changes in cell morphology, cell membrane hyperpolarization, and accelerated leakage of intracellular reducing sugars and proteins outward from the cytoplasm. In addition, dysfunction of the respiratory chain was induced after treatment with AgNP, which was supported by a decrease in intracellular ATP and inhibition of related dehydrogenases. This research indicates that AgNP could be a novel and efficient antibacterial agent to control C. sakazakii contamination in environments producing powdered infant formulas from milk.

Determination of the transfer of lead and chromium from feed to raw milk in Holstein cows.

In this study, we aimed to determinate the transfer of lead (Pb) and chromium (Cr) from feed to raw milk in Holstein cows. Solutions of lead acetate and chromium (III) nicotinate were added together at different levels to individual portions of feed on a daily basis. Lead administration for the low-, middle- and high-dosage groups was controlled as 0.9, 1.8 and 3.6 g/day for 30, 30 and 25 days, while chromium was feed at 0.47, 1.5 and 4.7 g/d for 40, 40, and 40 days, respectively. A sensitive graphite furnace atomic absorption spectrometry (GFAAS) method was developed and optimised with a respective limit of detection of lead and chromium found to be 1 and 5 µg kg-1 raw milk. The optical wavelengths for detection of lead and chromium were 283.31 nm and 357.87 nm respectively. The results showed that the highest concentrations of lead in raw milk for the low-, middle- and high-dosage groups were 0.083 ± 0.021, 0.215 ± 0.064 and 0.232 ± 0.035 mg kg-1, which displayed a positive correlation with dosage levels. In addition, a high dosage level accompanied a greater rate of loss of lead contents after dosing withdrawal. However, chromium concentrations maintained a relatively stable range around 0.1 mg kg-1 raw milk for all dosing groups and showed little transfer from feed to raw milk through the whole experiment.