RNA Polymerase Pausing during Initial Transcription.

In bacteria, RNA polymerase (RNAP) initiates transcription by synthesizing short transcripts that are either released or extended to allow RNAP to escape from the promoter. The mechanism of initial transcription is unclear due to the presence of transient intermediates and molecular heterogeneity. Here, we studied initial transcription on a lac promoter using single-molecule fluorescence observations of DNA scrunching on immobilized transcription complexes. Our work revealed a long pause ("initiation pause," ∼20 s) after synthesis of a 6-mer RNA; such pauses can serve as regulatory checkpoints. Region sigma 3.2, which contains a loop blocking the RNA exit channel, was a major pausing determinant. We also obtained evidence for RNA backtracking during abortive initial transcription and for additional pausing prior to escape. We summarized our work in a model for initial transcription, in which pausing is controlled by a complex set of determinants that modulate the transition from a 6- to a 7-nt RNA.

DBFE: distribution-based feature extraction from structural variants in whole-genome data.

MOTIVATION: Whole-genome sequencing has revolutionized biosciences by providing tools for constructing complete DNA sequences of individuals. With entire genomes at hand, scientists can pinpoint DNA fragments responsible for oncogenesis and predict patient responses to cancer treatments. Machine learning plays a paramount role in this process. However, the sheer volume of whole-genome data makes it difficult to encode the characteristics of genomic variants as features for learning algorithms. RESULTS: In this article, we propose three feature extraction methods that facilitate classifier learning from sets of genomic variants. The core contributions of this work include: (i) strategies for determining features using variant length binning, clustering and density estimation; (ii) a programing library for automating distribution-based feature extraction in machine learning pipelines. The proposed methods have been validated on five real-world datasets using four different classification algorithms and a clustering approach. Experiments on genomes of 219 ovarian, 61 lung and 929 breast cancer patients show that the proposed approaches automatically identify genomic biomarkers associated with cancer subtypes and clinical response to oncological treatment. Finally, we show that the extracted features can be used alongside unsupervised learning methods to analyze genomic samples. AVAILABILITY AND IMPLEMENTATION: The source code of the presented algorithms and reproducible experimental scripts are available on Github at https://github.com/MNMdiagnostics/dbfe. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Gene Variants Related to Cardiovascular and Pulmonary Diseases May Correlate with Severe Outcome of COVID-19.

Background: Severe outcomes of COVID-19 account for up to 15% of all cases. The study aims to check if any gene variants related to cardiovascular (CVD) and pulmonary diseases (PD) are correlated with a severe outcome of COVID-19 in a Polish cohort of COVID-19 patients. Methods: In this study, a subset of 747 samples from unrelated individuals collected across Poland in 2020 and 2021 was used and whole-genome sequencing was performed. Results: The GWAS analysis of SNPs and short indels located in genes related to CVD identified one variant significant in COVID-19 severe outcome in the HADHA gene, while for the PD gene panel, we found two significant variants in the DRC1 gene. In this study, both potentially protective and risk variants were identified, of which variants in the HADHA gene deserve the most attention. Conclusions: This is the first study reporting the association between the HADHA and DRC1 genetic variants and COVID-19 severe outcome based on the cohort WGS analysis. Although all the identified variants are localised in introns, they may be correlated and therefore inherited along with other risk variants, potentially causative to severe outcome of COVID-19 but not discovered yet.

The Thousand Polish Genomes-A Database of Polish Variant Allele Frequencies.

Although Slavic populations account for over 4.5% of world inhabitants, no centralised, open-source reference database of genetic variation of any Slavic population exists to date. Such data are crucial for clinical genetics, biomedical research, as well as archeological and historical studies. The Polish population, which is homogenous and sedentary in its nature but influenced by many migrations of the past, is unique and could serve as a genetic reference for the Slavic nations. In this study, we analysed whole genomes of 1222 Poles to identify and genotype a wide spectrum of genomic variation, such as small and structural variants, runs of homozygosity, mitochondrial haplogroups, and de novo variants. Common variant analyses showed that the Polish cohort is highly homogenous and shares ancestry with other European populations. In rare variant analyses, we identified 32 autosomal-recessive genes with significantly different frequencies of pathogenic alleles in the Polish population as compared to the non-Finish Europeans, including C2, TGM5, NUP93, C19orf12, and PROP1. The allele frequencies for small and structural variants, calculated for 1076 unrelated individuals, are released publicly as The Thousand Polish Genomes database, and will contribute to the worldwide genomic resources available to researchers and clinicians.

Single-molecule imaging of DNA gyrase activity in living Escherichia coli.

Bacterial DNA gyrase introduces negative supercoils into chromosomal DNA and relaxes positive supercoils introduced by replication and transiently by transcription. Removal of these positive supercoils is essential for replication fork progression and for the overall unlinking of the two duplex DNA strands, as well as for ongoing transcription. To address how gyrase copes with these topological challenges, we used high-speed single-molecule fluorescence imaging in live Escherichia coli cells. We demonstrate that at least 300 gyrase molecules are stably bound to the chromosome at any time, with ∼12 enzymes enriched near each replication fork. Trapping of reaction intermediates with ciprofloxacin revealed complexes undergoing catalysis. Dwell times of ∼2 s were observed for the dispersed gyrase molecules, which we propose maintain steady-state levels of negative supercoiling of the chromosome. In contrast, the dwell time of replisome-proximal molecules was ∼8 s, consistent with these catalyzing processive positive supercoil relaxation in front of the progressing replisome.

Assembly, translocation, and activation of XerCD-dif recombination by FtsK translocase analyzed in real-time by FRET and two-color tethered fluorophore motion.

The FtsK dsDNA translocase functions in bacterial chromosome unlinking by activating XerCD-dif recombination in the replication terminus region. To analyze FtsK assembly and translocation, and the subsequent activation of XerCD-dif recombination, we extended the tethered fluorophore motion technique, using two spectrally distinct fluorophores to monitor two effective lengths along the same tethered DNA molecule. We observed that FtsK assembled stepwise on DNA into a single hexamer, and began translocation rapidly (∼ 0.25 s). Without extruding DNA loops, single FtsK hexamers approached XerCD-dif and resided there for ∼ 0.5 s irrespective of whether XerCD-dif was synapsed or unsynapsed. FtsK then dissociated, rather than reversing. Infrequently, FtsK activated XerCD-dif recombination when it encountered a preformed synaptic complex, and dissociated before the completion of recombination, consistent with each FtsK-XerCD-dif encounter activating only one round of recombination.

Live-cell superresolution microscopy reveals the organization of RNA polymerase in the bacterial nucleoid.

Despite the fundamental importance of transcription, a comprehensive analysis of RNA polymerase (RNAP) behavior and its role in the nucleoid organization in vivo is lacking. Here, we used superresolution microscopy to study the localization and dynamics of the transcription machinery and DNA in live bacterial cells, at both the single-molecule and the population level. We used photoactivated single-molecule tracking to discriminate between mobile RNAPs and RNAPs specifically bound to DNA, either on promoters or transcribed genes. Mobile RNAPs can explore the whole nucleoid while searching for promoters, and spend 85% of their search time in nonspecific interactions with DNA. On the other hand, the distribution of specifically bound RNAPs shows that low levels of transcription can occur throughout the nucleoid. Further, clustering analysis and 3D structured illumination microscopy (SIM) show that dense clusters of transcribing RNAPs form almost exclusively at the nucleoid periphery. Treatment with rifampicin shows that active transcription is necessary for maintaining this spatial organization. In faster growth conditions, the fraction of transcribing RNAPs increases, as well as their clustering. Under these conditions, we observed dramatic phase separation between the densest clusters of RNAPs and the densest regions of the nucleoid. These findings show that transcription can cause spatial reorganization of the nucleoid, with movement of gene loci out of the bulk of DNA as levels of transcription increase. This work provides a global view of the organization of RNA polymerase and transcription in living cells.

Conformational transitions during FtsK translocase activation of individual XerCD-dif recombination complexes.

Three single-molecule techniques have been used simultaneously and in tandem to track the formation in vitro of single XerCD-dif recombination complexes. We observed the arrival of the FtsK translocase at individual preformed synaptic complexes and demonstrated the conformational change that occurs during their activation. We then followed the reaction intermediate transitions as Holliday junctions formed through catalysis by XerD, isomerized, and were converted by XerC to reaction products, which then dissociated. These observations, along with the calculated intermediate lifetimes, inform the reaction mechanism, which plays a key role in chromosome unlinking in most bacteria with circular chromosomes.

The threats for human health induced by food pests of Plodia interpunctella as reservoirs of infectious microbiota / Zagrozenia zdrowia czlowieka indukowane przez szkodniki zywnosci Plodia interpunctella jako rezerwuary infekcyjnych microbiota

INTRODUCTION: World-wide distributed pests of Plodia interpunctella occur with increasing frequency also in Poland, in areas where food is prepared and stored, in dwellings, buildings of public use, hospitals. Larvae damage various products causing economic losses. There were no data about microbiota transmission by pests. The aim of our systematic studies firstly conducted in Poland was to explain a role of pests as reservoirs of microbiota and assess health risk induced by them in human environments. MATERIAL AND METHODS: 300 adults and 200 larvae, collected in households and health facilities by traps and directly from products, were examined by light microscopy, in vitro cultivations, molecular techniques; the susceptibility /resistance of microbiota to chemicals was also assessed. RESULTS: Gram+ bacteriae of genera Enterococcus, Micrococcus, Bacillus, Gram-: Klebsiella, Escherichia, mold fungi: Aspergillus, Penicillium and yeast-like fungi were identified, including strains potentially pathogenic for humans. CONCLUSIONS: In the European Union countries, the food circulation is audited by the law; chemicals are applied to eliminate P.interpunctella pests causing economic losses. Our successive studies showed that pyralids may generate health problems as food pests and as reservoirs of microbiota. Sources of the pathogenic, drug-resistant strains revealed by us, not identified earlier, may be particularly dangerous for elder persons, with weakened immune system, persons from groups of high risk of infections. The increased awareness of the problem is necessary for more efficacy of preventive measures. A monitoring of consequences of the health risk induced by the pests may supply data useful for adequate practical approach.

[Effect of smoking on the incidence of adverse outcomes and ischemic wounds in peripheral artery disease].

Background: Smoking is a major risk factor for peripheral artery disease (PAD), which correlates with progression of the disease, the prevalence of chronic wounds, amputation incidence and mortality. On the other hand, abstinence from smoking has a beneficial effect on the bypass grafts patency after surgical revascularization. Poland fits in the steady decline in the percentage of smokers, but there is a lack of current data, whether those changes are observed also in patients with PAD and whether they lead to the improvement of health condition. Objective: This study evaluated the impact of smoking and other known risk factors for cardiovascular disease on the occurrence of PAD, the risk of adverse outcomes (death, amputation, tissue necrosis), development of chronic wounds and their healing after arterial revascularization. Materials and Methods: The study was conducted in groups of 208 patients with PAD scheduled for surgical treatment (28.4% of patients with adverse outcomes of PAD, 38.5% of patients with ischemic wounds) and 190 control subjects. The study groups were interviewed concerning smoking, characterized by known risk factors for cardiovascular disease, and in the incidence of amputation, and deaths within 30 days after surgery. In the group of 48 patients with ischemic wounds a prospective assessment of the progress of wound healing in terms of changes in the wound advancement and changes in pain severity associated with wounds was performed. Results: Over 90% of patients with PAD were smokers: 53.8% former and 36.5% past in relation to 27.4% and 14.7%, respectively, in the control group (p <0.0001). Among patients, a higher proportion of former smokers was found in those with poorer health condition: with adverse outcomes of PAD (48.0% vs 64.1%, p <0.05), and with ischemic wounds (70.7% vs 65.1%, p = 0.056). Advanced age, female sex, and the presence of diabetes were associated with both, the presence of PAD adverse outcomes, as well as the development of ischemic wounds. In the prospective study, major factors prolonging the process of wound healing were advanced age, diabetes and smoking (evaluated as peaks years of smoking). Conclusions: Smoking is still the most common risk factor for PAD, and smoking cessation is the result of the deterioration of health condition. Simultaneously this factor, in addition to diabetes, advanced age and female sex, affects both the risk of unfavorable course of PAD and decrease the progress of wound healing. Control of risk factors for cardiovascular disease should be especially careful in women in whom, despite the lower PAD incidence, further prognosis of disease progression seem worse.

Capturing reaction paths and intermediates in Cre-loxP recombination using single-molecule fluorescence.

Site-specific recombination plays key roles in microbe biology and is exploited extensively to manipulate the genomes of higher organisms. Cre is a well studied site-specific recombinase, responsible for establishment and maintenance of the P1 bacteriophage genome in bacteria. During recombination, Cre forms a synaptic complex between two 34-bp DNA sequences called loxP after which a pair of strand exchanges forms a Holliday junction (HJ) intermediate; HJ isomerization then allows a second pair of strand exchanges and thus formation of the final recombinant product. Despite extensive work on the Cre-loxP system, many of its mechanisms have remained unclear, mainly due to the transient nature of complexes formed and the ensemble averaging inherent to most biochemical work. Here, we address these limitations by introducing tethered fluorophore motion (TFM), a method that monitors large-scale DNA motions through reports of the diffusional freedom of a single fluorophore. We combine TFM with Förster resonance energy transfer (FRET) and simultaneously observe both large- and small-scale conformational changes within single DNA molecules. Using TFM-FRET, we observed individual recombination reactions in real time and analyzed their kinetics. Recombination was initiated predominantly by exchange of the "bottom-strands" of the DNA substrate. In productive complexes we used FRET distributions to infer rapid isomerization of the HJ intermediates and that a rate-limiting step occurs after this isomerization. We also observed two nonproductive synaptic complexes, one of which was structurally distinct from conformations in crystals. After recombination, the product synaptic complex was extremely stable and refractory to subsequent rounds of recombination.

Tethered fluorophore motion: studying large DNA conformational changes by single-fluorophore imaging.

We have previously introduced tethered fluorophore motion (TFM), a single-molecule fluorescence technique that monitors the effective length of a biopolymer such as DNA. TFM uses the same principles as tethered particle motion (TPM) but employs a single fluorophore in place of the bead, allowing TFM to be combined with existing fluorescence techniques on a standard fluorescence microscope. TFM has been previously been used to reveal the mechanism of two site-specific recombinase systems, Cre-loxP and XerCD-dif. In this work, we characterize TFM, focusing on the theoretical basis and potential applications of the technique. Since TFM is limited in observation time and photon count by photobleaching, we present a description of the sources of noise in TFM. Comparing this with Monte Carlo simulations and experimental data, we show that length changes of 100 bp of double-stranded DNA are readily distinguishable using TFM, making it comparable with TPM. We also show that the commonly recommended pixel size for single-molecule fluorescence approximately optimizes signal to noise for TFM experiments, thus enabling facile combination of TFM with other fluorescence techniques, such as Förster resonance energy transfer (FRET). Finally, we apply TFM to determine the polymerization rate of the Klenow fragment of DNA polymerase I, and we demonstrate its combination with FRET to observe synapsis formation by Cre using excitation by a single laser. We hope that TFM will be a useful addition to the single-molecule toolkit, providing excellent insight into protein-nucleic acid interactions.

Influence of Dispersed Phase Content on the Mechanical Properties of Electroless Nanocomposite Ni-P/Si3N4 and Hybrid Ni-P/Si3N4/Graphite Layers Deposited on the AW-7075 Alloy.

The article presents the results of mechanical testing of Ni-P/Si3N4 nanocomposite and hybrid Ni-P/Si3N4/graphite coatings deposited on AW-7075 aluminum alloy using the chemical reduction method. In terms of mechanical testing, microhardness was measured, and surface roughness and adhesion of the coatings to the aluminum substrate were determined using the "scratch test" method. The surface morphology of the deposited layers was also analyzed using light microscopy and scanning electron microscopy. Samples made of AW-7075 aluminum alloy with electroless deposited Ni-P/Si3N4 nanocomposite, Ni-P/graphite composite and hybrid Ni-P/Si3N4/graphite coatings with different content of dispersed phases were tested, and also, for comparison purposes, the Ni-P layer that constituted the matrix of the tested materials. Reinforcing phases in the form of silicon nitride nanoparticles and graphite particles were used in the layers. The purpose of the research was a thorough characterization of the coating materials used on aluminum alloys in terms of mechanical properties. Graphite is considered in this paper as it enables the reduction of the coefficient of friction through its lubricating properties. Unfortunately, graphite is difficult to use in selected layers as the only dispersion phase, because it has much lower hardness than the Ni-P coating. For this reason, a layer with a single dispersion phase in the form of graphite will be characterized by worse mechanical properties. It is necessary to add particles or nanoparticles with hardness higher than the base Ni-P coating, e.g., Si3N4, which improve the mechanical properties of the coating. The presented analyses of the results of the conducted research complement the previous studies on selected properties of nanocomposite layers with an amorphous structure and supplement the knowledge regarding their suitability for application to aluminum machine parts.

The Influence of Osteon Orientation on Surface Topography Parameters after Machining of Cortical Bone Tissue.

Mechanical processing of cortical bone tissue is one of the most common surgical procedures. A critical issue accompanying this processing is the condition of the surface layer, which can stimulate tissue growth and serve as a drug carrier. A comparison of the surface condition before and after orthogonal and abrasive processing was conducted to validate the influence of bone tissue's processing mechanism and orthotropic properties on the surface topography. A cutting tool with a defined geometry and a custom-made abrasive tool was used. The bone samples were cut in three directions, depending on the orientation of the osteons. The cutting forces, acoustic emission, and surface topography were measured. The level of isotropy and the topography of the grooves showed statistical differences relative to the anisotropy directions. After orthogonal processing, the surface topography parameter Ra was determined from 1.38 ± 0.17 µm to 2.82 ± 0.32. In the case of abrasive processing, no correlation was found between the orientation of osteons and topographical properties. The average groove density for abrasive machining was below 1004 ± 0.7, and for orthogonal, it was above 1156 ± 58. Due to the positive properties of the developed bone surface, it is advisable to cut in the transverse direction and parallel to the axis of the osteons.

State of the Art in Temporomandibular Joint Arthrocentesis-A Systematic Review.

Temporomandibular joint disorders are a heterogenic group of clinical conditions, which impair physiological functioning of the masticatory system. Arthrocentesis of the temporomandibular joint has become a widely approved method for non-invasive treatment, bridging the gap between conservative and surgical approaches. Regardless of technique, treatment is based upon joint lavage and lysis of the inflammatory fibrous tissue adhesions, which, in turn, improves joint mobility and reduces pain and closed lock. Recently, approaches for intra-articular injections have been proposed as adjuvant or replacement therapy. The aim of this study was to assess the most efficient technique of arthrocentesis. A systematic search based on PRISMA guidelines, including a computer search with specific keywords, a reference list search and a manual search, was performed. Relevant articles were selected after three search rounds for final review. The studies pulled for the analysis presented information about the relevant predictors, including the technique of arthrocentesis (single- or two-needle method), fluid used for lavage (Ringer lactate or saline), volume of the fluid, application of the injectable, number of interventions, pain (VAS) and mouth opening scores (MMO) and follow-up. All cohorts showed improvement in mouth opening, but significant pain reduction was observed only in cohorts treated either by arthrocentesis alone or arthrocentesis followed by intra-articular injectables. Intra-articular injectables used alone failed to reduce pain post-operatively when compared to other cohorts. We concluded that both double-needle and single-puncture arthrocentesis techniques are equally efficient. Application of the adjuvant injectable did not improve the outcomes of arthrocentesis performed alone. The volume of the fluid used for joint lavage and its chemical composition were not significant in clinical outcomes. However, due to the lack of homogeneity in the study settings, a meta-analysis could not be applied and a systematic review was conducted. We still, however, state that there is a knowledge gap in the current literature regarding the use of injectables alone, as well as a longitudinal follow-up, which provides information about treatment efficiency. More high-quality and randomized controlled trials are required to shed light on this subject.

Effect of screw angulation and multiple insertions on load-to-failure of polyaxial locking system.

PURPOSE: Polyaxial locking plates rely on the alignment between the thread-to-thread connections of the screw head and the plate hole. These implants have provided substantial support for surgeons. In particular, extended screw positioning have proven to be beneficial in the fixation of challenging fractures. This study aimed to investigate the mechanical properties of ChM 5.0 ChLP polyaxial screws inserted in off-axis trajectories, including multiple insertions and to correlate these parameters with the screw head and the plate hole thread-to-thread engagement. METHODS: Polyaxial locking screws were inserted into the plates at various angles (0°,10°,15°, -15° off-axis). Multiple time inserted screws were placed firstly at 15°, then 0° and finally -15° off-axis in the same plate hole. A microCT scan of the plate-hole and screw-head interface was conducted before destructive tests. Representative screws from each group were also examined by Scanning Electron Microscope. RESULTS: The standard insertion at 0° sustained the greatest maximum bending strength without relocation in the screw hole. Screws inserted at 10° and 15° (one time) showed a significant reduction in load-to-failure of up to 36% and 55%, (p = 0.001) (p = 0.001) respectively. Screws inserted at -15° after a maximum of three multiple insertions with angle shift, showed a total reduction in force of up to 70% (p = 0.001). A microCT analysis of thread engagement showed significant correlations. However, the results obtained for multiple insertions were highly variable. CONCLUSIONS: ChM 5.0 ChLP polyaxial locking system has valuable properties that foster fracture fixation, providing various surgical options. Nevertheless, the freedom of off-axis placement and multiple insertions of the screws comes at the price of reduced force. When possible surgeons should minimize the angles of insertions.

Effect of screw angulation on the bending performance of polyaxial locking interfaces: a micro-CT evaluation.

Polyaxial locking plates rely on a specific thread-to-thread interface of the screw head and the plate hole. The objective of this study was to evaluate the mechanical performance of single screw interfaces when inserted off-axis and to establish correlations between those parameters and the engagement of the screw head and the plate hole thread. Three polyaxial locking screw systems were inserted into the corresponding plates at various angles (0°, 5°, 10°, and 15° off-axis). The screws were tested until failure. A micro-CT was performed to examine the interface between the plate hole and the screw head. The standard insertion at 0° sustained the greatest maximum bending strength without relocation in the screw hole. Screws inserted at 15° showed a significant reduction in force of up to 44%, 55% and 57%, respectively. Micro-CT analysis of the interface showed a significant loss of thread engagement for off-axis insertion. Polyaxial plates offer additional advantages for off-axis placement of screws. However, this flexibility is related to a significant decrease in both thread engagement and bending strength compared to monoaxial insertion. Regardless the insertion angle, the loss of stability is comparable when screws are placed off-axis. Surgeons are advised to consider off-axis insertion as a salvage option, providing access to better bone stock.

Biomechanical evaluation of variable angle locking systems. A micro-CT analysis.

PURPOSE: Formerly poor bone stock and periprosthetic fractures used to jeopardize monaxial constructs. Polyaxial locking screws have substantially supported those particular fixation constructs. However, those systems rely on complex alignment between the screw head and the plate hole. This study aimed to investigate the mechanical properties of several polyaxial systems and to correlate these parameters with the screw head and the plate hole engagement. METHODS: Polyaxial locking systems were tested with screws inserted into the corresponding plates at various angles (0°, 5°, 10°, 15°). A micro-CT scan of the plate-hole and screw-head interface with the quantification of average thread engagement was performed before destructive tests. The screw-plate interface of each system was tested in a cantilever bending setup. Representative screws and plates were also examined by SEM. RESULTS: The standard insertion at 0° sustained the greatest maximum bending strength in all analyzed systems. Point-loading thread-in and cut-in screws inserted off-axis showed a significant reduction in bending strength (p<0.001) (p=0.041) (p<0.001). In contrast, locking cap screws maintained similar bending strength with disregard to the angle of insertion (p<0.4849). A micro-CT analysis confirmed that the average thread engagement of point-loading thread-in, cut-in and locking-cap screws was significantly reduced when placed off-axis (p=0.005) (p<0.001) (p=0.002) (p<0.001). The locking-cap mechanisms maintained the highest average thread engagement among all analyzed systems. CONCLUSIONS: The mechanical performance of polyaxial locking plates usually comes at the price of reduced bending strength. Surgeons should limit polyaxial insertions depending on the particular system's characteristics.

Successive Acanthamoeba Corneal Isolates Identified in Poland Monitored in Terms of In Vitro Dynamics.

BACKGROUND: Amoebae of the genus Acanthamoeba cause a sight-threatening infection called Acanthamoeba keratitis. It is considered a rare disease in humans but poses an increasing threat to public health worldwide, including in Poland. We present successive isolates from serious keratitis preliminary examined in terms of the identification and monitoring of, among others, the in vitro dynamics of the detected strains. METHODS: Clinical and combined laboratory methods were applied; causative agents of the keratitis were identified at the cellular and molecular levels; isolates were cultivated in an axenic liquid medium and regularly monitored. RESULTS: In a phase-contrast microscope, Acanthamoeba sp. cysts and live trophozoites from corneal samples and in vitro cultures were assessed on the cellular level. Some isolates that were tested at the molecular level were found to correspond to A. mauritanensis, A. culbertsoni, A. castellanii, genotype T4. There was variability in the amoebic strain dynamics; high viability was expressed as trofozoites' long duration ability to intense multiply. CONCLUSIONS: Some strains from keratitis under diagnosis verification and dynamics assessment showed enough adaptive capability to grow in an axenic medium, allowing them to exhibit significant thermal tolerance. In vitro monitoring that was suitable for verifying in vivo examinations, in particular, was useful to detect the strong viability and pathogenic potential of successive Acanthamoeba strains with a long duration of high dynamics.

The cancer-risk variant frequency among Polish population reported by the first national whole-genome sequencing study.

Introduction: Population-based cancer screening has raised many controversies in recent years, not only regarding the costs but also regarding the ethical nature and issues related to variant interpretation. Nowadays, genetic cancer screening standards are different in every country and usually encompass only individuals with a personal or family history of relevant cancer. Methods: Here we performed a broad genetic screening for cancer-related rare germline variants on population data from the Thousand Polish Genomes database based on 1076 Polish unrelated individuals that underwent whole genome sequencing (WGS). Results: We identified 19 551 rare variants in 806 genes related to oncological diseases, among them 89% have been located in non-coding regions. The combined BRCA1/BRCA2 pathogenic/likely pathogenic according to ClinVar allele frequency in the unselected population of 1076 Poles was 0.42%, corresponding to nine carriers. Discussion: Altogether, on the population level, we found especially problematic the assessment of the pathogenicity of variants and the relation of ACMG guidelines to the population frequency. Some of the variants may be overinterpreted as disease-causing due to their rarity or lack of annotation in the databases. On the other hand, some relevant variants may have been overseen given that there is little pooled population whole genome data on oncology. Before population WGS screening will become a standard, further studies are needed to assess the frequency of the variants suspected to be pathogenic on the population level and with reporting of likely benign variants.