QN-302 demonstrates opposing effects between i-motif and G-quadruplex DNA structures in the promoter of the S100P gene

GC-rich sequences can fold into G-quadruplexes and i-motifs and are known to control gene expression in many organisms. The potent G-quadruplex experimental anticancer drug QN-302 down-regulates a number of cancer-related genes, in particular S100P. Here we show this ligand has strong opposing effects with i-motif DNA structures and is one of the most potent i-motif destabilising agents reported to date. QN-302 down-regulates the expression of numerous cancer-related genes by pan-quadruplex targeting. QN-302 exhibits exceptional combined synergistic effects compared to many other G-quadruplex and i-motif interacting compounds. This work further emphasises the importance of considering G-quadruplex and i-motif DNA structures as one dynamic system

Identification of sugar-containing natural products that interact with i-motif DNA

There are thousands of compounds shown to interact with G-quadruplex DNA, yet very few which target i-motif (iM) DNA. Previous work showed that tobramycin can interact with iM- DNA, indicating the potential for sugar-molecules to target these structures. Computational approaches indicated that the sugar-containing natural products baicalin and geniposidic acid had potential to target iM-DNA. We assessed the DNA interacting properties of these compounds using FRET-based DNA melting and a fluorescence-based displacement assay using iM-DNA structures from the human telomere and the insulin linked polymorphic region (ILPR), as well as complementary G-quadruplex and double stranded DNA. Both baicalin and geniposidic acid show promise as iM-interacting compounds with potential for use in experiments into the structure and function of i-motif forming DNA sequences and present starting points for further synthetic development of these as probes for iM-DNA

Prediction of DNA i-motifs via machine learning

i-Motifs (iMs), are secondary structures formed in cytosine-rich DNA sequences and are involved in multiple functions in the genome. Although putative iM forming sequences are widely distributed in the human genome, the folding status and strength of putative iMs vary dramatically. Much previous research on iM has focused on assessing the iM folding properties using biophysical experiments. However, there are no dedicated computational tools for predicting the folding status and strength of iM structures. Here, we introduce a machine learning pipeline, iM-Seeker, to predict both folding status and structural stability of DNA iMs. The programme iM-Seeker incorporates a Balanced Random Forest classifier trained on genome-wide iMab antibody-based CUT&Tag sequencing data to predict the folding status and an Extreme Gradient Boosting regressor to estimate the folding strength according to both literature biophysical data and our in-house biophysical experiments. iM-Seeker predicts DNA iM folding status with a classification accuracy of 81% and estimates the folding strength with coefficient of determination (R2) of 0.642 on the test set. Model interpretation confirms that the nucleotide composition of the C-rich sequence significantly affects iM stability, with a positive correlation with sequences containing cytosine and thymine and a negative correlation with guanine and adenine

The Potent G-Quadruplex-Binding Compound QN-302 Downregulates S100P Gene Expression in Cells and in an In Vivo Model of Pancreatic Cancer

The naphthalene diimide compound QN-302, designed to bind to G-quadruplex DNA sequences within the promoter regions of cancer-related genes, has high anti-proliferative activity in pancreatic cancer cell lines and anti-tumor activity in several experimental models for the disease. We show here that QN-302 also causes downregulation of the expression of the S100P gene and the S100P protein in cells and in vivo. This protein is well established as being involved in key proliferation and motility pathways in several human cancers and has been identified as a potential biomarker in pancreatic cancer. The S100P gene contains 60 putative quadruplex-forming sequences, one of which is in the promoter region, 48 nucleotides upstream from the transcription start site. We report biophysical and molecular modeling studies showing that this sequence forms a highly stable G-quadruplex in vitro, which is further stabilized by QN-302. We also report transcriptome analyses showing that S100P expression is highly upregulated in tissues from human pancreatic cancer tumors, compared to normal pancreas material. The extent of upregulation is dependent on the degree of differentiation of tumor cells, with the most poorly differentiated, from more advanced disease, having the highest level of S100P expression. The experimental drug QN-302 is currently in pre-IND development (as of Q1 2023), and its ability to downregulate S100P protein expression supports a role for this protein as a marker of therapeutic response in pancreatic cancer. These results are also consistent with the hypothesis that the S100P promoter G-quadruplex is a potential therapeutic target in pancreatic cancer at the transcriptional level for QN-302

Replication-induced DNA secondary structures drive fork uncoupling and breakage

Sequences that form DNA secondary structures, such as G-quadruplexes (G4s) and intercalated-Motifs (iMs), are abundant in the human genome and play various physiological roles. However, they can also interfere with replication and threaten genome stability. Multiple lines of evidence suggest G4s inhibit replication, but the underlying mechanism remains unclear. Moreover, evidence of how iMs affect the replisome is lacking. Here, we reconstitute replication of physiologically derived structure-forming sequences to find that a single G4 or iM arrest DNA replication. Direct single-molecule structure detection within solid-state nanopores reveals structures form as a consequence of replication. Combined genetic and biophysical characterisation establishes that structure stability and probability of structure formation are key determinants of replisome arrest. Mechanistically, replication arrest is caused by impaired synthesis, resulting in helicase-polymerase uncoupling. Significantly, iMs also induce breakage of nascent DNA. Finally, stalled forks are only rescued by a specialised helicase, Pif1, but not Rrm3, Sgs1, Chl1 or Hrq1. Altogether, we provide a mechanism for quadruplex structure formation and resolution during replication and highlight G4s and iMs as endogenous sources of replication stress

The development and validation of novel biomarkers to assess the skin barrier function

Background: The uppermost skin layer, stratum corneum (SC), is a physical and biochemical barrier against environmental insults. The SC resembles a wall with terminally differentiated keratinocytes, corneocytes, as “bricks” that are embedded in lamellar layers as “mortar”. The corneocytes gain in maturity by transglutaminase enhancing rigidity and hydrophobicity. External conditions act on the skin homeostasis and thus understanding these effects on a molecular level might provide new targets for the cosmetic industry. Objectives: Tape stripping is an established approach to collect successive layers of the SC. These tapes were used to identify biomarkers for the maturation of corneocytes by measuring enzyme activities and protein expression in the photo exposed (PE) cheek and photoprotected (PP) post auricular sites. Methods: The SC integrity, cohesion and thickness was determined as a reference measurement. Immunostaining of structural proteins and enzymes was used to characterise the corneocyte from the first and ninth tape strip of both anatomical sites. The relative corneocyte envelope maturity assay was developed by determining the rigidity and hydrophobicity of corneocytes that to correlate to the SC properties. In addition, ex vivo maturation was tested at a range of relative humidities (RH) to determine the optimal RH. The corneocyte maturation and enzyme activities were investigated at low, optimal and high RH in the presence of protease inhibitors. Results: The SC of the PE cheek site is thinner with a lower integrity and higher cohesion compared to the PP post auricular site. The corneocytes from the PE cheek site are less mature than from the PP post auricular site. The corneocytes from the PE cheek site were able to increase in rigidity but not in hydrophobicity. Humidity has an impact on proteases which in turn are able to deactivate transglutaminase activity and thus influence corneocyte maturation. Conclusions: Various methods were tested and correlated with the SC integrity in human subjects. The determination of protein expression was suggested for a set of biomarkers involved in lipid processing enzymes (12R-lipoxygenase and epidermal lipoxygenase 3), structural CE proteins (involucrin and skin-specific protein 32), proteases (cathepsin D and V) and transglutaminase. The impaired ex vivo maturation of samples from the PE cheek point towards pre-mature desquamation and thus provide new pharmaceutical targets for moisturising skin care products

Interactive Videoconference Supported Teaching in Undergraduate Nursing: A Case Study for ECG

This paper describes how interactive videoconference can benefit the Electrocardiography (ECG) skills of undergraduate nursing students. We have implemented a learning system that interactively transfers the visual and practical aspects of ECG from a nursing skills lab into a classroom where the theoretical part of the course is taught. The students and the instructor in the classroom observe the activities in the skill lab in real time, while communicating with the nurse in the lab via audio and video links. An experiment was performed with the participation of 13 male and 57 female (total 70) second year nursing students—36 of who were assigned to Videoconference group (experimental group-VCG) and the other 34 were assigned to Traditional Classroom groups (control group-TCG). In the experiment, ECG knowledge levels of participants were measured by repeated tests (pretest, posttest I and posttest II) and data were analyzed with repeated measures of variances and covariance, the results demonstrating that videoconferencing contributed significantly to the improvement of ECG skills of the participants. In addition, a questionnaire was given to students along with posttest II, and the result of which indicated overwhelming satisfaction with videoconference based lecture

In Vitro Evaluation of Mucoadhesive Vaginal Tablets of Antifungal Drugs Prepared with Thiolated Polymer and Development of a New Dissolution Technique for Vaginal Formulations

WOS: 000294516400006PubMed ID: 21804238The main objective of this work was to develop antifungal matrix tablet for vaginal applications using mucoadhesive thiolated polymer. Econazole nitrate (EN) and miconazole nitrate (MN) were used as antifungal drugs to prepare the vaginal tablet formulations. Thiolated poly(acrylic acid) cysteine (PAA-Cys) conjugate was synthesized by the covalent attachment of L-cysteine to PAA with the formation of amide bonds between the primary amino group of L-cysteine and the carboxylic acid group of the polymer. Vaginal mucoadhesive matrix tablets were prepared by direct compression technique. The investigation focused on the influence of modified polymer on water uptake behavior, mucoadhesive property and release rate of drug. Thiolated polymer increased the water uptake ratio and mucoadhesive property of the formulations. A new simple dissolution technique was developed to simulate the vaginal environment for the evaluation of release behavior of vaginal tablets. In this technique, daily production amount and rate of the vaginal fluid was used without any rotational movement. The drug release was found to be slower from PAA-Cys compared to that from PAA formulations. The similarity study results confirmed that the difference in particle size of EN and MN did not affect their release profile. The release process was described by plotting the fraction released drug versus time and n fitting data to the simple exponential model: M-t/M-infinity = kt(n). The release kinetics were determined as Super Case II for all the formulations prepared with PAA or PAA-Cys. According to these results the mucoadhesive vaginal tablet formulations prepared with PAA-Cys represent good example for delivery systems which prolong the residence time of drugs at the vaginal mucosal surface.The Scientific and Technological Research Council of Turkey (TUBITAK)Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [106/S/196]This work is a part of a research project (No: 106/S/196) supported by "The Scientific and Technological Research Council of Turkey (TUBITAK).

The Potent G-Quadruplex-Binding Compound QN-302 Downregulates S100P Gene Expression in Cells and in an In Vivo Model of Pancreatic Cancer

The naphthalene diimide compound QN-302, designed to bind to G-quadruplex DNA sequences within the promoter regions of cancer-related genes, has high anti-proliferative activity in pancreatic cancer cell lines and anti-tumor activity in several experimental models for the disease. We show here that QN-302 also causes downregulation of the expression of the S100P gene and the S100P protein in cells and in vivo. This protein is well established as being involved in key proliferation and motility pathways in several human cancers and has been identified as a potential biomarker in pancreatic cancer. The S100P gene contains 60 putative quadruplex-forming sequences, one of which is in the promoter region, 48 nucleotides upstream from the transcription start site. We report biophysical and molecular modeling studies showing that this sequence forms a highly stable G-quadruplex in vitro, which is further stabilized by QN-302. We also report transcriptome analyses showing that S100P expression is highly upregulated in tissues from human pancreatic cancer tumors, compared to normal pancreas material. The extent of upregulation is dependent on the degree of differentiation of tumor cells, with the most poorly differentiated, from more advanced disease, having the highest level of S100P expression. The experimental drug QN-302 is currently in pre-IND development (as of Q1 2023), and its ability to downregulate S100P protein expression supports a role for this protein as a marker of therapeutic response in pancreatic cancer. These results are also consistent with the hypothesis that the S100P promoter G-quadruplex is a potential therapeutic target in pancreatic cancer at the transcriptional level for QN-302