Damir Đermić

Bladder cancer (BC) is a common urological malignancy that lacks the non-invasive biomarkers that would make it suitable for early diagnosis. Human alpha-satellite DNA (hASAT) is a tandemly repeated centromeric/pericentromeric DNA family associated with chromosomal stability and cancer-related genomic instability. We quantified extracellular hASAT (ec-hASAT) in plasma circulating cell-free DNA by nanoplate-based digital PCR in a pilot cohort including 29 BC-negative samples, 10 patients with non-muscle-invasive BC (NMIBC), and 7 patients with muscle-invasive BC (MIBC). Plasma ec-hASAT copy number was higher in patients with BC than in the BC-negative group (Mann–Whitney U test, p = 6.61 × 10⁻⁷). BC-negative samples ranged from 225 to 11,864 copies/µL plasma, whereas BC samples ranged from 1138 to 16,097 copies/µL plasma. ROC analysis yielded an AUC of 0.944 for discriminating BC from BC-negative samples. At an exploratory threshold of 2000 copies/µL plasma, sensitivity was 94.1% (16/17; exact 95% CI, 71.3–99.9%) and specificity was 89.7% (26/29; exact 95% CI, 72.6–97.8%). These preliminary data support plasma ec-hASAT as a candidate minimally invasive biomarker for BC detection, including NMIBC, and justify validation in larger prospective cohorts.

Satellite DNAs are highly abundant sequences that build functional centromeres and pericentromeric heterochromatin in many eukaryotes. Apart from this structural role, their involvement in gene expression modulation has been demonstrated, although a detailed understanding of the molecular mechanisms is still lacking. Here, using the major human alpha satellite as a model system, we investigate the role of satellite transcripts in gene expression regulation. We generated cell lines with forced, exogenous overexpression of alpha satellite RNA and followed the expression levels of genes containing alpha satellite repeats within introns. Our results reveal a positive correlation between exogenous alpha satellite expression and the downregulation of alpha-associated genes, strongly suggesting that alpha satellite RNA affects their transcription. Notably, the elevated levels of exogenous alpha satellite RNA did not affect histone modifications characteristic of pericentromeric heterochromatin (e.g., H3K9me3 or H3K18Ac) or euchromatin (e.g., H3K4me2) at intronic alpha satellite loci. We propose that alpha satellite RNA directly interacts with homologous DNA at dispersed intronic satellite loci by forming RNA-DNA hybrid structures, which may affect chromatin structure and transcriptional activity. The results demonstrate that alpha satellite RNA is not only involved in centromere and heterochromatin assembly but, as shown here for the first time, also plays a role in modulating the expression of alpha-associated genes.

Satellite DNAs are highly abundant sequences which build functional centromere and pericentromeric heterochromatin in many eukaryotes. Apart from this structural role, their involvement in gene expression modulation has been demonstrated, although the detailed molecular mechanism is still missing. Here, using major human alpha satellite as a model system, we investigate the role of satellite transcripts in gene expression regulation. We generated cell lines with forced, exogenous overexpression of alpha satellite RNA and followed the expression levels of genes containg alpha satellite repeats within introns. Our results reveal a positive correlation between exogenous alpha satellite expression and the downregulation of alpha-associated genes, strongly suggesting that alpha satellite RNA affects their transcription. Notably, the elevated levels of exogenous alpha satellite RNA did not affect histone modifications characteristic of pericentromeric heterochromatin (e.g. H3K9me3 or H3K18Ac) or euchromatin (e.g. H3K4me2) at intronic alpha satellite loci. We propose that alpha satellite RNA directly interacts with homologous DNA at dispersed intronic satellite loci by forming RNA-DNA hybrid structures, which may affects chromatin structure and transcriptional activity. The results demostrate that alpha satellite RNA is not only involved in centromere and heterochromatin assembly but, as shown here for the first time, also plays a role in modulating the expression of alpha-associated genes.

Although bladder cancer (BC) is a common urological disease, there are deficiencies in current methods used for its detection, hence the research on the identification of non- invasive biomarkers is given a high priority. Alpha satellite DNA is a major human satellite DNA (hASAT), which is important for genome stability and whose aberrant overexpression is a hallmark of many human cancers. Here we identified and quantified its release into the circulation of BC patients, demonstrating its increased copy number with respect to healthy controls. Absolute quantification of extracellular alpha satellite DNA (ecASAT) repeats was performed by nanoplate-based digital PCR using few microliters of blood plasma from cancer patients as well as from BC negative controls. We found that copy number of ecASAT repeats is significantly higher in blood plasma of BC patients than in healthy controls. The results suggest that the ec-hASAT copy number variation in human plasma could act as a reliable biomarker for early detection of BC.

Research background. Food safety is threatened by the contamination of fresh fruits and vegetables by pathogenic bacteria, among which the particularly widespread ones are coliform bacteria. Due to the continuous increase in the incidence of severe diseases caused by the consumption of fresh (tomato) fruits contaminated with Escherichia coli, antimicrobial postharvest measures are needed. The problem is that many active antimicrobial compounds have a weak and short-lasting effect and/or are not environmentally friendly. Recently, the antibacterial and antifungal activity of environmentally friendly agent phenylboronic acid (PBA), including on two tomato pathogens, has been reported. Experimental approach. The aim of this study is to determine the antibacterial effect of PBA on E. coli and three enteropathogenic Enterobacterales, and to check its ability to serve as a bacterial decontaminant of fresh tomato fruits. Results and conclusions. The minimum inhibitory concentration (MIC) of PBA against E. coli, as well as Shigella sonnei, Salmonella enteritidis and Yersinia enterocolitica was 1.0, 1.2, 1.0 and 0.8 mg/mL, respectively. In addition, we have shown that PBA has a bacteriostatic effect on E. coli at lower concentrations and a bactericidal effect at higher (>3.0 mg/mL) concentrations. Importantly, the study found that an E. coli strain resistant to seven commonly used antibiotics, as well as strains producing extended-spectrum beta-lactamases (ESBL), is as sensitive to PBA as the wild-type strain without any resistance, suggesting that the mechanism of action of PBA differs from that of all these antibiotics. Finally, we have shown that washing and incubating contaminated tomato fruits in PBA solution reduces the growth of E. coli washed from fresh tomato fruits in a concentration- (0.5–3.0 mg/mL) and time-dependent manner, while having no adverse effect on the tomato fruits. Novelty and scientific contribution. This is the first report on the antibacterial effect of PBA on medically important bacteria E. coli, S. enteritidis, S. sonnei and Y. enterocolitica. Moreover, we show that PBA kills multidrug-resistant E. coli, including those producing ESBL, making it a promising agent against such bacteria. Finally, PBA is shown to be an effective decontaminant of E. coli on fresh tomato fruits.

Single-strand DNA-binding proteins SSB/RPA are ubiquitous and essential proteins that bind ssDNA in bacteria/eukaryotes and coordinate DNA metabolic processes such as replication, repair, and recombination. SSB protects ssDNA from degradation by nucleases, while also facilitating/regulating the activity of multiple partner proteins involved in DNA processes. Using Spi⁻ assay, which detects aberrantly excised λ prophage from the E. coli chromosome as a measure of illegitimate recombination (IR) occurrence, we have shown that SSB inhibits IR in several DSB resection pathways. The conditional ssb-1 mutation produced a higher IR increase at the nonpermissive temperature than the recQ inactivation. A double ssb-1 recQ mutant had an even higher level of IR, while showing reduced homologous recombination (HR). Remarkably, the ssb gene overexpression complemented recQ deficiency in suppressing IR, indicating that the SSB function is epistatic to RecQ. Overproduced truncated SSBΔC8 protein, which binds to ssDNA, but does not interact with partner proteins, only partially complemented recQ and ssb-1 mutations, while causing an IR increase in otherwise wild-type bacteria, suggesting that ssDNA binding of SSB is required but not sufficient for effective IR inhibition, which rather entails interaction with RecQ and likely some other protein(s). Our results depict SSB as the main genome caretaker in E. coli, which facilitates HR while inhibiting IR. In enabling high-fidelity DSB repair under physiological conditions SSB is assisted by RecQ helicase, whose activity it controls. Conversely, an excess of SSB renders RecQ redundant for IR suppression.

Homologous recombination repairs potentially lethal DNA lesions such as double-strand DNA breaks (DSBs) and single-strand DNA gaps (SSGs). In Escherichia coli, DSB repair is initiated by the RecBCD enzyme that resects double-strand DNA ends and loads RecA recombinase to the emerging single-strand (ss) DNA tails. SSG repair is mediated by the RecFOR protein complex that loads RecA onto the ssDNA segment of gaped duplex. In both repair pathways, RecA catalyses reactions of homologous DNA pairing and strand exchange, while RuvABC complex and RecG helicase process recombination intermediates. In this work, we have characterised cytological changes in various recombination mutants of E. coli after three different DNA-damaging treatments: (i) expression of I-SceI endonuclease, (ii) γ-irradiation, and (iii) UV-irradiation. All three treatments caused severe chromosome segregation defects and DNA-less cell formation in the ruvABC, recG, and ruvABC recG mutants. After I-SceI expression and γ-irradiation, this phenotype was efficiently suppressed by the recB mutation, indicating that cytological defects result mostly from incomplete DSB repair. In UV-irradiated cells, the recB mutation abolished cytological defects of recG mutants and also partially suppressed the cytological defects of ruvABC recG mutants. However, neither recB nor recO mutation alone could suppress the cytological defects of UV-irradiated ruvABC mutants. The suppression was achieved only by simultaneous inactivation of the recB and recO genes. Cell survival and microscopic analysis suggest that chromosome segregation defects in UV-irradiated ruvABC mutants largely result from defective processing of stalled replication forks. The results of this study show that chromosome morphology is a valuable marker in genetic analyses of recombinational repair in E. coli.

Bacterial SSB proteins, as well as their eukaryotic RPA analogues, are essential and ubiquitous. They avidly bind single-stranded DNA and regulate/coordinate its metabolism, hence enabling essential DNA processes such as replication, transcription, and repair. The prototypic Escherichia coli SSB protein is encoded by an ssb gene. Although the ssb gene promoters harbor an SOS box, multiple studies over several decades failed to elucidate whether ssb gene expression is inducible and SOS dependent. The SOS regulon is comprised of about 50 genes, whose transcription is coordinately induced under stress conditions. Using quantitative real-time PCR, we determined the ssb gene expression kinetics in UV- and γ-irradiated E. coli and revealed that ssb gene expression is elevated in irradiated cells in an SOS-dependent manner. Additionally, the expression of the sulA gene was determined to indicate the extent of SOS induction. In a mutant with a constitutively induced SOS regulon, the ssb gene was overexpressed in the absence of DNA damage. Furthermore, we measured ssb gene expression by droplet digital PCR during unaffected bacterial growth and revealed that ssb gene expression was equal in wild-type and SOS⁻ bacteria, whereas sulA expression was higher in the former. This study thus reveals a complex pattern of ssb gene expression, which under stress conditions depends on the SOS regulon, whereas during normal bacterial growth it is unlinked to SOS induction. The E. coli ssb gene is SOS regulated in such a way that its basal expression is relatively high and can be increased only through stronger SOS induction. The remarkable SOS induction observed in undisturbed wild-type cells may challenge our notion of the physiological role of the SOS response in bacteria.

Finding a suitable alternative to the small pool of existing antifungal agents is a vital task in contemporary agriculture. Therefore, intensive research has been conducted globally to uncover environmentally friendly and efficient agents that can suppress pathogens resistant to the currently used antimycotics. Here, we tested the activity of boric acid (BA) and its derivative phenylboronic acid (PBA) in controlling the early blight symptoms in tomato plants infected with pathogenic fungus Alternaria alternata. By following the appearance and intensity of the lesions on leaves of the tested plants, as well as by measuring four selected physiological factors that reflect plant health, we have shown that both BA and PBA act prophylactically on fungal infection. They did it by reducing the amount and severity of early blight symptoms, as well as by preventing deterioration of the physiological traits, occurring upon fungal inoculation. Phenylboronic acid was more efficient in suppressing the impact of A. alternata infection. Therefore, we conclude that BA, and even more so PBA, may be used as agents for controlling early blight on tomato plants, as they are both quite effective and environmentally friendly.