CCNE1 amplification is synthetic lethal with PKMYT1 kinase inhibition.

Amplification of the CCNE1 locus on chromosome 19q12 is prevalent in multiple tumour types, particularly in high-grade serous ovarian cancer, uterine tumours and gastro-oesophageal cancers, where high cyclin E levels are associated with genome instability, whole-genome doubling and resistance to cytotoxic and targeted therapies1-4. To uncover therapeutic targets for tumours with CCNE1 amplification, we undertook genome-scale CRISPR-Cas9-based synthetic lethality screens in cellular models of CCNE1 amplification. Here we report that increasing CCNE1 dosage engenders a vulnerability to the inhibition of the PKMYT1 kinase, a negative regulator of CDK1. To inhibit PKMYT1, we developed RP-6306, an orally bioavailable and selective inhibitor that shows single-agent activity and durable tumour regressions when combined with gemcitabine in models of CCNE1 amplification. RP-6306 treatment causes unscheduled activation of CDK1 selectively in CCNE1-overexpressing cells, promoting early mitosis in cells undergoing DNA synthesis. CCNE1 overexpression disrupts CDK1 homeostasis at least in part through an early activation of the MMB-FOXM1 mitotic transcriptional program. We conclude that PKMYT1 inhibition is a promising therapeutic strategy for CCNE1-amplified cancers.

HELB Is a Feedback Inhibitor of DNA End Resection.

DNA double-strand break repair by homologous recombination is initiated by the formation of 3' single-stranded DNA (ssDNA) overhangs by a process termed end resection. Although much focus has been given to the decision to initiate resection, little is known of the mechanisms that regulate the ongoing formation of ssDNA tails. Here we report that DNA helicase B (HELB) underpins a feedback inhibition mechanism that curtails resection. HELB is recruited to ssDNA by interacting with RPA and uses its 5'-3' ssDNA translocase activity to inhibit EXO1 and BLM-DNA2, the nucleases catalyzing resection. HELB acts independently of 53BP1 and is exported from the nucleus as cells approach S phase, concomitant with the upregulation of resection. Consistent with its role as a resection antagonist, loss of HELB results in PARP inhibitor resistance in BRCA1-deficient tumor cells. We conclude that mammalian DNA end resection triggers its own inhibition via the recruitment of HELB.

MXene-based electrochemical devices applied for healthcare applications.

The initial part of the review provides an extensive overview about MXenes as novel and exciting 2D nanomaterials describing their basic physico-chemical features, methods of their synthesis, and possible interfacial modifications and techniques, which could be applied to the characterization of MXenes. Unique physico-chemical parameters of MXenes make them attractive for many practical applications, which are shortly discussed. Use of MXenes for healthcare applications is a hot scientific discipline which is discussed in detail. The article focuses on determination of low molecular weight analytes (metabolites), high molecular weight analytes (DNA/RNA and proteins), or even cells, exosomes, and viruses detected using electrochemical sensors and biosensors. Separate chapters are provided to show the potential of MXene-based devices for determination of cancer biomarkers and as wearable sensors and biosensors for monitoring of a wide range of human activities.

Negative Charge-Carrying Glycans Attached to Exosomes as Novel Liquid Biopsy Marker.

Prostate cancer (PCa) is the second most common cancer. In this paper, the isolation and properties of exosomes as potential novel liquid biopsy markers for early PCa liquid biopsy diagnosis are investigated using two prostate human cell lines, i.e., benign (control) cell line RWPE1 and carcinoma cell line 22Rv1. Exosomes produced by both cell lines are characterised by various methods including nanoparticle-tracking analysis, dynamic light scattering, scanning electron microscopy and atomic force microscopy. In addition, surface plasmon resonance (SPR) is used to study three different receptors on the exosomal surface (CD63, CD81 and prostate-specific membrane antigen-PMSA), implementing monoclonal antibodies and identifying the type of glycans present on the surface of exosomes using lectins (glycan-recognising proteins). Electrochemical analysis is used to understand the interfacial properties of exosomes. The results indicate that cancerous exosomes are smaller, are produced at higher concentrations, and exhibit more nega tive zeta potential than the control exosomes. The SPR experiments confirm that negatively charged α-2,3- and α-2,6-sialic acid-containing glycans are found in greater abundance on carcinoma exosomes, whereas bisecting and branched glycans are more abundant in the control exosomes. The SPR results also show that a sandwich antibody/exosomes/lectins configuration could be constructed for effective glycoprofiling of exosomes as a novel liquid biopsy marker.

Breast cancer glycan biomarkers: their link to tumour cell metabolism and their perspectives in clinical practice.

INTRODUCTION: Breast cancer (BCa) is the most common cancer type diagnosed in women and 5th most common cause of deaths among all cancer deaths despite the fact that screening program is at place. This is why novel diagnostics approaches are needed in order to decrease number of BCa cases and disease mortality. AREAS COVERED: In this review paper, we aim to cover some basic aspects regarding cellular metabolism and signalling in BCa behind altered glycosylation. We also discuss novel exciting discoveries regarding glycan-based analysis, which can provide useful information for better understanding of the disease. The final part deals with clinical usefulness of glycan-based biomarkers and the clinical performance of such biomarkers is compared to already approved BCa biomarkers and diagnostic tools based on imaging. EXPERT OPINION: Recent discoveries suggest that glycan-based biomarkers offer high accuracy for possible BCa diagnostics in blood, but also for better monitoring and management of BCa patients. The review article was written using Web of Science search engine to include articles published between 2019 and 2021.

Detection of N,N-diacetyllactosamine (LacdiNAc) containing free prostate-specific antigen for early stage prostate cancer diagnostics and for identification of castration-resistant prostate cancer patients.

Prostate cancer (PCa) is one of the most common cancer types among men and also acommon cause of death globally. With an increasing incidence, there is aneed for low-cost, reliable biomarkers present in samples, which could be provided non-invasively (without a need to perform prostate biopsy). Glycosylation changes of free-PSA (fPSA) are considered cancer-specific, while the level of different PSA forms can increase under other than cancerous conditions. In the present study, we investigated the role ofN,N-diacetyllactosamine (LacdiNAc) epitope of fPSA (i.e. glycoprofile of fPSA or gPSA) in combination with total-PSA (tPSA), prostate volume, and tPSA density (tPSA level divided by prostate volume i.e. PSAd) as biomarkers for monitoring of PCa development and progression in 105 men. Furthermore, we applied an genetic (evolutionary) algorithm to identify any suspicious individuals in abenign cohort having benign prostatic hyperplasia (BPH). We identified 3 suspicious men originally diagnosed with BPH using gPSA analysis. In thefollow-up we found out that two men should not be considered as BPH patients since multiparametric magnetic resonance imaging (mpMRI) identified one man with clinically significant PCa via Prostate Imaging - Reporting and Data System (PI RADS v2 = 4) and the second man was with High-gradeprostatic intraepithelial neoplasia (HG PIN), commonly described as apre-cancerous stage. Moreover, in the study we described for the first time that changed LacdiNAc on PSA can be applied to identify prostatitis patients and most importantly this is the first study suggesting that changed glycosylation on PSA can be applied to identify castration-resistant prostate cancer (CRPCa) patients.

A cell cycle-dependent regulatory circuit composed of 53BP1-RIF1 and BRCA1-CtIP controls DNA repair pathway choice.

DNA double-strand break (DSB) repair pathway choice is governed by the opposing activities of 53BP1 and BRCA1. 53BP1 stimulates nonhomologous end joining (NHEJ), whereas BRCA1 promotes end resection and homologous recombination (HR). Here we show that 53BP1 is an inhibitor of BRCA1 accumulation at DSB sites, specifically in the G1 phase of the cell cycle. ATM-dependent phosphorylation of 53BP1 physically recruits RIF1 to DSB sites, and we identify RIF1 as the critical effector of 53BP1 during DSB repair. Remarkably, RIF1 accumulation at DSB sites is strongly antagonized by BRCA1 and its interacting partner CtIP. Lastly, we show that depletion of RIF1 is able to restore end resection and RAD51 loading in BRCA1-depleted cells. This work therefore identifies a cell cycle-regulated circuit, underpinned by RIF1 and BRCA1, that governs DSB repair pathway choice to ensure that NHEJ dominates in G1 and HR is favored from S phase onward.

Analysis of serum glycome by lectin microarrays for prostate cancer patients - a search for aberrant glycoforms.

This is the first work focused on glycoprofiling of whole N- and O- glycome using lectins in an array format applied for analysis of serum samples from healthy individuals, benign prostate hyperplasia (BPH) patients, and prostate cancer (PCa) patients. Lectin microarray was prepared using traditional lectins with the incorporation of 2 recombinant bacterial lectins and 3 human lectins (17 lectins in total). Clinical validation of glycans as biomarkers was done in two studies: discrimination of healthy individuals with BPH patients vs. PCa patients (C vs. PCa) and discrimination of healthy individuals vs. BPH and PCa patients (H vs. PCond). Single lectins (17 lectins) and a combination of two lectins (136 binary lectin combinations) were applied in the clinical validation of glycan biomarkers providing 153 AUC values from ROC curves for both studies (C vs. PCa and H vs. PCond). Potential N- and O-glycans as biomarkers were identified and possible carriers of these glycans are shortly discussed.

Electrochemical Nanobiosensors for Detection of Breast Cancer Biomarkers.

This comprehensive review paper describes recent advances made in the field of electrochemical nanobiosensors for the detection of breast cancer (BC) biomarkers such as specific genes, microRNA, proteins, circulating tumor cells, BC cell lines, and exosomes or exosome-derived biomarkers. Besides the description of key functional characteristics of electrochemical nanobiosensors, the reader can find basic statistic information about BC incidence and mortality, breast pathology, and current clinically used BC biomarkers. The final part of the review is focused on challenges that need to be addressed in order to apply electrochemical nanobiosensors in a clinical practice.

Glycomics of prostate cancer: updates.

Introduction: Prostate cancer (PCa) is a life-threatening disease affecting millions of men. The current best PCa biomarker (level of prostate-specific antigen in serum) lacks specificity for PCa diagnostics and this is why novel PCa biomarkers in addition to the conventional ones based on biomolecules such as DNA, RNA and proteins need to be identified. Areas covered: This review details the potential of glycans-based biomarkers to become diagnostic, prognostic, predictive and therapeutic PCa biomarkers with a brief description of the innovative approaches applied to glycan analysis to date. Finally, the review covers the possibility to use exosomes as a rich source of glycans for future innovative and advanced diagnostics of PCa. The review covers updates in the field since 2016. Expert commentary: The summary provided in this review paper suggests that glycan-based biomarkers can offer high-assay accuracy not only for diagnostic purposes but also for monitoring/surveillance of the PCa disease.

Sulfobetaines Meet Carboxybetaines: Modulation of Thermo- and Ion-Responsivity, Water Structure, Mechanical Properties, and Cell Adhesion.

A procedure for the preparation of copolymers bearing sulfobetaine and carboxybetaine methacrylic-based monomers by free-radical polymerization is described and discussed. A combination of monomers affects the upper critical solution temperature (UCST) in water and in the presence of a simple NaCl electrolyte while retaining the zwitterionic character. In addition, hydrogel samples were prepared and showed tunable water structure and mechanical properties. The total nonfreezable water content decreases with the amount of carboxybetaine segment in the hydrogel feed and the compression moduli were in a range of 0.7-1.6 MPa. Responses to external conditions such as temperature and ion strength were investigated and a potential application such as modulated thermal detection is proposed. The presence of the carboxylate group in the carboxybetaine segment enables a small fluorescence probe and peptide bearing RDG motif to be attached to polymer and hydrogel samples, respectively. The hydrogel samples functionalized with the RGD motif exhibit controlled cell adhesion. Such synthetic strategy based on combination of different zwitterionic segments offers a simple pathway for the development of zwitterionic materials with programmable properties.

Polyzwitterionic Hydrogels in Engines Based on the Antipolyelectrolyte Effect and Driven by the Salinity Gradient.

In this paper, we propose and investigate an original approach to energy conversion based on polyzwitterionic hydrogels, which exhibit an antipolyelectrolyte effect that enables them to swell in salt water and shrink in water of a different (i.e., desalinated water) salinity. The swelling and shrinking processes run cyclically and can move a piston up or down reversibly, thus transforming the antipolyelectrolyte effect into a mechanical force based on the salinity gradient. This phenomenon makes polyzwitterionic hydrogels suitable for use in a smart, polymeric engine. We apply this approach to investigate energy recovery from a polysulfobetaine-based hydrogel. The cross-linking density, external load, particle size, and repeatability of energy recoverability of hydrogels are examined. The maximum energy recovery from 0.4 g of hydrogel in feed (calculated based on dry form) of 102 mJ/kg was obtained by a hydrogel with a 3% cross-linking density, a 200-300 µm particle size, and 100 g external load. Excellent reproducibility of engine cycles was achieved over 10 cycles. This concept is complementary to the osmotic engine concept based on a polyelectrolyte hydrogel. In addition, polyzwitterionic materials have become a benchmark material for preventing biofouling, and the swelling properties of such materials can be further modulated and tuned.

Remarkable differences in the voltammetric response towards hydrogen peroxide, oxygen and Ru(NH3)63+ of electrode interfaces modified with HF or LiF-HCl etched Ti3C2Tx MXene.

An electrochemical study was performed on the behavior of Ti3C2Tx MXenes prepared by using either HF (MXene1) or LiF/HCl as etchants (MXene2). The use of two redox probes indicates the presence of a higher negative charge density on MXene2 in comparison to MXene1. The characterization of two nanomaterials shows that titanium and fluoride are present higher by one order of magnitude at the interface of MXene2, compared to MXene1. The high Ti and F content is accompanied by a 82-fold larger (249 µA·cm-2 vs. 5.64 µA·cm-2) anodic peak at the peak potential near 0.4 V (vs. Ag/AgCl). Similarly, the peak current on MXene2 is 317-fold higher for the oxygen reduction at pH 7.0 (at a voltage of -0.84 V) and 215-fold higher for the reduction of H2O2 at -0.89 V, when compared to MXene1. Graphical abstractDifference in electrochemical behavior of MXene prepared by HF (MXene1) and LiF/HCl (MXene2) as etchants.

A Graphene-Based Glycan Biosensor for Electrochemical Label-Free Detection of a Tumor-Associated Antibody.

The study describes development of a glycan biosensor for detection of a tumor-associated antibody. The glycan biosensor is built on an electrochemically activated/oxidized graphene screen-printed electrode (GSPE). Oxygen functionalities were subsequently applied for covalent immobilization of human serum albumin (HSA) as a natural nanoscaffold for covalent immobilization of Thomsen-nouvelle (Tn) antigen (GalNAc-O-Ser/Thr) to be fully available for affinity interaction with its analyte-a tumor-associated antibody. The step by step building process of glycan biosensor development was comprehensively characterized using a battery of techniques (scanning electron microscopy, atomic force microscopy, contact angle measurements, secondary ion mass spectrometry, surface plasmon resonance, Raman and energy-dispersive X-ray spectroscopy). Results suggest that electrochemical oxidation of graphene SPE preferentially oxidizes only the surface of graphene flakes within the graphene SPE. Optimization studies revealed the following optimal parameters: activation potential of +1.5 V vs. Ag/AgCl/3 M KCl, activation time of 60 s and concentration of HSA of 0.1 g L-1. Finally, the glycan biosensor was built up able to selectively and sensitively detect its analyte down to low aM concentration. The binding preference of the glycan biosensor was in an agreement with independent surface plasmon resonance analysis.

Nanotechnology in Glycomics: Applications in Diagnostics, Therapy, Imaging, and Separation Processes.

This review comprehensively covers the most recent achievements (from 2013) in the successful integration of nanomaterials in the field of glycomics. The first part of the paper addresses the beneficial properties of nanomaterials for the construction of biosensors, bioanalytical devices, and protocols for the detection of various analytes, including viruses and whole cells, together with their key characteristics. The second part of the review focuses on the application of nanomaterials integrated with glycans for various biomedical applications, that is, vaccines against viral and bacterial infections and cancer cells, as therapeutic agents, for in vivo imaging and nuclear magnetic resonance imaging, and for selective drug delivery. The final part of the review describes various ways in which glycan enrichment can be effectively done using nanomaterials, molecularly imprinted polymers with polymer thickness controlled at the nanoscale, with a subsequent analysis of glycans by mass spectrometry. A short section describing an active glycoprofiling by microengines (microrockets) is covered as well.

Optimization of the Small Glycan Presentation for Binding a Tumor-Associated Antibody: Application to the Construction of an Ultrasensitive Glycan Biosensor.

The main aim of the study was to optimize the interfacial presentation of a small antigen-a Tn antigen (N-acetylgalactosamine)-for binding to its analyte anti-Tn antibody. Three different methods for the interfacial display of a small glycan are compared here, including two methods based on the immobilization of the Tn antigen on a mixed self-assembled monolayer (SAM) (2D biosensor) and the third one utilizing a layer of a human serum albumin (HSA) for the immobilization of a glycan forming a 3D interface. Results showed that the 3D interface with the immobilized Tn antigen is the most effective bioreceptive surface for binding its analyte. The 3D impedimetric glycan biosensor exhibited a limit of detection of 1.4 aM, a wide linear range (6 orders of magnitude), and high assay reproducibility with an average relative standard deviation of 4%. The buildup of an interface was optimized using various techniques with the visualization of the glycans on the biosensor surface by atomic force microscopy. The study showed that the 3D biosensor is not only the most sensitive compared to other two biosensor platforms but that the Tn antigen on the 3D biosensor surface is more accessible for antibody binding with better kinetics of binding (t50% = 137 s, t50% = the time needed to attain 50% of a steady-state signal) compared to the 2D biosensor configuration with t50% = 354 s. The 3D glycan biosensor was finally applied for the analysis of a human serum sample spiked with an analyte.

pH-Switchable Interaction of a Carboxybetaine Ester-Based SAM with DNA and Gold Nanoparticles.

We describe a self-assembled monolayer (SAM) on a gold surface with a carboxybetaine ester functionality to control the interaction between DNA and gold nanoparticles via pH. The negatively charged phosphate backbone of DNA interacts with and adsorbs to the positively charged carboxybetaine esters on the SAM. DNA release can be achieved by the hydrolysis of carboxybetaine ester (CBE) to a zwitterionic carboxybetaine state. Furthermore, the adsorption of negatively charged citrate-capped gold nanoparticles to a SAM-modified plain gold surface can be controlled by the pH. The SAM based on carboxybetaine ester allows for the homogeneous adsorption of particles, whereas the SAM after hydrolysis at high pH repels AuNP adsorption. The antifouling surface properties of the surface modified with carboxybetaine were investigated with protein samples.

Full-length antibodies versus single-chain antibody fragments for a selective impedimetric lectin-based glycoprofiling of prostate specific antigen.

The main aim of the research was to design a functional impedimetric biosensor able to glycoprofile prostate specific antigen (PSA), a biomarker for prostate cancer (PCa), with high specificity using lectins as glycan recognising proteins. Traditionally, full-length antibody is immobilised on the biosensor interface for specific capture of PSA with subsequent glycoprofiling of PSA by addition of lectins. Since full-length antibodies contain glycans in the Fc domain, particular attention has to be paid to suppress direct binding of lectins to immobilised full-length antibodies, which would compromise accurate glycoprofiling. This issue is addressed here using a recombinant single-chain antibody fragments (scAb), which do not contain any carbohydrate moiety. Surface plasmon resonance was applied to prove negligible interaction of lectins with immobilised scAb fragments, while substantial binding of lectins to full length antibodies was observed. Eight different biosensor designs were tested for their ability to detect PSA. The biosensor device based on scAb fragments covalently immobilised on the gold electrode surface, patterned by a mixed SAM using standard amine coupling chemistry, proved to be the most sensitive. The scAb fragment-based biosensor exhibited sensitivity of 15.9 ± 0.8% decade-1 (R2 = 0.991 with an average RSD of 4.9%), while the full antibody-based biosensor offered sensitivity towards PSA of 4.2 ± 0.1% decade-1 (R2 = 0.999 with an average RSD of 4.8%). Moreover, the selectivity of the scAb-based biosensor was tested using a kallikrein 2 protein, a protein structurally similar to PSA, and the results indicated high selectivity for PSA detection.

Electrochemical performance of Ti3C2Tx MXene in aqueous media: towards ultrasensitive H2O2 sensing.

An extensive characterization of pristine and oxidized Ti3C2Tx (T: =O, -OH, -F) MXene showed that exposure of MXene to an anodic potential in the aqueous solution oxidizes the nanomaterial forming TiO2 layer or TiO2 domains with subsequent TiO2 dissolution by F- ions, making the resulting nanomaterial less electrochemically active compared to the pristine Ti3C2Tx. The Ti3C2Tx could be thus applied for electrochemical reactions in a cathodic potential window i.e. for ultrasensitive detection of H2O2 down to nM level with a response time of approx. 10 s. The manuscript also shows electrochemical behavior of Ti3C2Tx modified electrode towards oxidation of NADH and towards oxygen reduction reactions.