Targeted sequencing of cancer-related genes reveals a recurrent TOP2A variant which affects DNA binding and coincides with global transcriptional changes in glioblastoma.

High-grade gliomas are aggressive, deadly primary brain tumors. Median survival of patients with glioblastoma (GBM, WHO grade 4) is 14 months and <10% of patients survive 2 years. Despite improved surgical strategies and forceful radiotherapy and chemotherapy, the prognosis of GBM patients is poor and did not improve over decades. We performed targeted next-generation sequencing with a custom panel of 664 cancer- and epigenetics-related genes, and searched for somatic and germline variants in 180 gliomas of different WHO grades. Herein, we focus on 135 GBM IDH-wild type samples. In parallel, mRNA sequencing was accomplished to detect transcriptomic abnormalities. We present the genomic alterations in high-grade gliomas and the associated transcriptomic patterns. Computational analyses and biochemical assays showed the influence of TOP2A variants on enzyme activities. In 4/135 IDH-wild type GBMs we found a novel, recurrent mutation in the TOP2A gene encoding topoisomerase 2A (allele frequency [AF] = 0.03, 4/135 samples). Biochemical assays with recombinant, wild type (WT) and variant proteins demonstrated stronger DNA binding and relaxation activity of the variant protein. GBM patients carrying the altered TOP2A had shorter overall survival (median OS 150 vs 500 days, P = .0018). In the GBMs with the TOP2A variant we found transcriptomic alterations consistent with splicing dysregulation. luA novel, recurrent TOP2A mutation, which was found exclusively in four GBMs, results in the TOP2A E948Q variant with altered DNA binding and relaxation activities. The deleterious TOP2A mutation resulting in transcription deregulation in GBMs may contribute to disease pathology.

Exploring Novel Therapeutic Opportunities for Glioblastoma Using Patient-Derived Cell Cultures.

Glioblastomas (GBM) are the most common, primary brain tumors in adults. Despite advances in neurosurgery and radio- and chemotherapy, the median survival of GBM patients is 15 months. Recent large-scale genomic, transcriptomic and epigenetic analyses have shown the cellular and molecular heterogeneity of GBMs, which hampers the outcomes of standard therapies. We have established 13 GBM-derived cell cultures from fresh tumor specimens and characterized them molecularly using RNA-seq, immunoblotting and immunocytochemistry. Evaluation of proneural (OLIG2, IDH1R132H, TP53 and PDGFRα), classical (EGFR) and mesenchymal markers (CHI3L1/YKL40, CD44 and phospho-STAT3), and the expression of pluripotency (SOX2, OLIG2, NESTIN) and differentiation (GFAP, MAP2, ß-Tubulin III) markers revealed the striking intertumor heterogeneity of primary GBM cell cultures. Upregulated expression of VIMENTIN, N-CADHERIN and CD44 at the mRNA/protein levels suggested increased epithelial-to-mesenchymal transition (EMT) in most studied cell cultures. The effects of temozolomide (TMZ) or doxorubicin (DOX) were tested in three GBM-derived cell cultures with different methylation status of the MGMT promoter. Amongst TMZ- or DOX-treated cultures, the strongest accumulation of the apoptotic markers caspase 7 and PARP were found in WG4 cells with methylated MGMT, suggesting that its methylation status predicts vulnerability to both drugs. As many GBM-derived cells showed high EGFR levels, we tested the effects of AG1478, an EGFR inhibitor, on downstream signaling pathways. AG1478 caused decreased levels of phospho-STAT3, and thus inhibition of active STAT3 augmented antitumor effects of DOX and TMZ in cells with methylated and intermediate status of MGMT. Altogether, our findings show that GBM-derived cell cultures mimic the considerable tumor heterogeneity, and that identifying patient-specific signaling vulnerabilities can assist in overcoming therapy resistance, by providing personalized combinatorial treatment recommendations.

Inhibition of Human Respiratory Influenza A Virus and Human Betacoronavirus-1 by the Blend of Double-Standardized Extracts of Aronia melanocarpa (Michx.) Elliot and Sambucus nigra L.

Viral and bacterial diseases are among the greatest concerns of humankind since ancient times. Despite tremendous pharmacological progress, there is still a need to search for new drugs that could treat or support the healing processes. A rich source of bioactive compounds with antiviral potency include plants such as black chokeberry and elderberry. The aim of this study was to assess the in vitro antiviral ability of an originally designed double-standardized blend of extracts from Aronia melanocarpa (Michx.) Elliot and Sambucus nigra L. (EAM-ESN) or separated extracts of A. melanocarpa (EAM) or S. nigra (ESN) against four human respiratory tract viruses: influenza A virus (A/H1N1), betacoronavirus-1 (HCoV-OC43) belonging to the same ß-coronaviruses as the current pandemic SARS-CoV-2, human herpesvirus type 1 (HHV-1), and human adenovirus type 5 (HAdV-5). Antiviral assays (AVAs) were used to evaluate the antiviral activity of the plant extracts in a cell-present environment with extracts tested before, simultaneously, or after viral infection. The virus replication was assessed using the CPE scale or luminescent assay. The EAM-ESN blend strongly inhibited A/H1N1 replication as well as HCoV-OC43, while having a limited effect against HHV-1 and HAdV-5. This activity likely depends mostly on the presence of the extract of S. nigra. However, the EAM-ESN blend possesses more effective inhibitory activity toward virus replication than its constituent extracts. A post-infection mechanism of action of the EAM-ESN make this blend the most relevant for potential drugs and supportive treatments; thus, the EAM-ESN blend might be considered as a natural remedy in mild, seasonal respiratory viral infections.

Stent-Assisted Coiling of Unruptured MCA Aneurysms Using the LVIS Jr. Device: A Multicenter Registry.

PURPOSE: The low-profile visualized intraluminal support junior (LVIS Jr.) is a new generation low-profile braided stent. Our aim was to evaluate the safety and efficacy of the LVIS Jr. in the stent-assisted coiling of unruptured middle cerebral artery (MCA) aneurysms. This was a multicenter retrospective study. Patient demographics, aneurysm characteristics, procedural details, complications, and the results of clinical and imaging follow-up were analyzed. Four centers participated in the study. A total of 162 consecutive patients with 162 unruptured MCA aneurysms were included for the analysis. The mean aneurysm size was 7.6 mm (range 2 to 37 mm) and 97.5% were wide-necked. Immediate postprocedural angiograms showed Raymond-Roy class 1 in 118 (72.8%), class 2 in 23 (14.2%), and class 3 in 21 patients (13%). Periprocedural complications occurred in 14 patients (8.6%). There were no procedure-related deaths. Follow-up imaging at 12-18 months post-procedure showed Raymond-Roy class 1 in 132 (81.5%), class 2 in 17 (10.5%), and class 3 in 13 patients (8%). There were 3 cases of in-stent stenosis (1.9%). All 162 patients had good clinical outcome (mRS score 0-2) at 90 days post-procedure. Stent-assisted coiling of unruptured MCA aneurysms with the LVIS Jr. stent is safe and effective, with high immediate and long-term total occlusion rates.

Aberrantly Expressed RECQL4 Helicase Supports Proliferation and Drug Resistance of Human Glioma Cells and Glioma Stem Cells.

Anti-tumour therapies eliminate proliferating tumour cells by induction of DNA damage, but genomic aberrations or transcriptional deregulation may limit responses to therapy. Glioblastoma (GBM) is a malignant brain tumour, which recurs inevitably due to chemo- and radio-resistance. Human RecQ helicases participate in DNA repair, responses to DNA damage and replication stress. We explored if a helicase RECQL4 contributes to gliomagenesis and responses to chemotherapy. We found upregulated RECQL4 expression in GBMs associated with poor survival of GBM patients. Increased levels of nuclear and cytosolic RECQL4 proteins were detected in GBMs on tissue arrays and in six glioma cell lines. RECQL4 was detected both in cytoplasm and mitochondria by Western blotting and immunofluorescence. RECQL4 depletion in glioma cells with siRNAs and CRISPR/Cas9 did not affect basal cell viability, slightly impaired DNA replication, but induced profound transcriptomic changes and increased chemosensitivity of glioma cells. Sphere cultures originated from RECQL4-depleted cells had reduced sphere forming capacity, stronger responded to temozolomide upregulating cell cycle inhibitors and pro-apoptotic proteins. RECQL4 deficiency affected mitochondrial network and reduced mitochondrial membrane polarization in LN18 glioblastoma cells. We demonstrate that targeting RECQL4 overexpressed in glioblastoma could be a new strategy to sensitize glioma cells to chemotherapeutics.

Evaluation of Antioxidant and Anti-Inflammatory Activity of Anthocyanin-Rich Water-Soluble Aronia Dry Extracts.

Aronia fruits contain many valuable components that are beneficial to human health. However, fruits are characterized by significant variations in chemical composition dependent on the growing conditions and harvesting period. Therefore, there is a need to formulate the extracts with a precisely defined content of health-promoting substances. Aronia dry extracts (ADE) were prepared from frozen pomace applying water extraction, followed by purification and spray-drying. Subsequently, the content of anthocyanins, phenolic acids, and polyphenols was determined. The high-quality chokeberry pomace enabled obtaining extracts with anthocyanin content much higher than the typical market standards. Moreover, it was found that the antioxidant capacity of aronia extracts exceeded those found in other fruit preparations. Antioxidant and free-radical scavenging properties were evaluated using a 2,2'-diphenyl-1-picrylhydrazyl using Electron Paramagnetic Resonance (EPR) spectroscopy (DPPH-EPR) test and Oxygen Radical Absorbance Capacity (ORAC) assay. The inhibition of lipid peroxidation and the level of inflammatory markers have been also investigated using lipopolysaccharide (LPS)-stimulated RAW 264 cells. It was revealed that ADE standardized to 25% of anthocyanins depresses the level of markers of inflammation and lipid peroxidation (Interleukin 1 beta (IL-1ß), tumor necrosis factor alpha (TNF-α), and malondialdehyde (MDA)) in in vitro conditions. Additionally, it was confirmed that ADE at all analyzed concentrations did not show any cytotoxic effect as demonstrated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

Analysis of changes in cervical spinal curvature and intervertebral disk space height following ACDF surgery in a group of 100 patients followed up for 12 months.

We analysed 100 patients following anterior cervical discectomy and fusion with interbody stabilisation with PEEK cages. Radiographs obtained preoperatively and during the 12-month follow-up were compared to track changes in overall and local cervical lordosis and disk space height. Subsidence was defined as cage migration ≥ 3 mm into the adjacent endplates. Mean change in operated disk space height was 1.13 ±â€¯1.33 mm. Subsidence was detected in 10.23% of the operated spaces. Mean change in overall cervical lordosis was 1.31 ±â€¯5.71 degrees, and mean change in local lordosis was 0.19 ±â€¯4.71 degrees. Change in overall cervical lordosis correlated with change in local lordosis (r = 0.61, p < 0.01). The greatest changes in lordosis and disk space height were noted immediately post-surgery. Baseline values were approximated gradually over time, but the post-operative values at 12 months were still higher than baseline. Disk space height change did not correlate with changes in patient-reported pain intensity at baseline (VAS 0) vs. at 12 months post-operatively (VAS 12) (r = 0.12, p < 0.05) or changes in the Neck Disability Index (NDI) at baseline (NDI 0) vs. at 12 months post-operatively (NDI 12) (r = -0.02, p = 0.05). Changes in overall cervical lordosis did not directly influence treatment outcomes assessed by comparing VAS 0 vs. VAS 12 (r = 0.13, p = 0.24) or NDI 0 vs. NDI 12 (r = -0.0005, p = 0.96). Surgical outcomes depend primarily on adequate decompression of the spinal cord and nerve roots. Post-operative radiological changes did not directly influence patients' pain level or quality of life.

Percutaneous Vertebroplasty for Pathological Vertebral Compression Fractures Secondary to Multiple Myeloma--Medium-Term and Long-Term Assessment of Pain Relief and Quality of Life.

BACKGROUND: In patients with multiple myeloma (MM) there is a high risk of compression fractures of the spine. In the majority of cases, the method of treatment is percutaneous vertebroplasty (PV) or kyphoplasty (PK). The number of studies verifying their efficacy in MM is still relatively small. OBJECTIVES: The aim of this study has been to assess medium- and long-term pain relief as well as improvement in the quality of life (QL) after PV in MM cases. MATERIAL AND METHODS: There was a prospective group of 34 MM cases in which a total of 131 vertebral bodies were augmented by means of PV. It was possible to follow up 22 patients who agreed to take part in the assessment. Their level of daily activity and the level of pain were assessed using the Oswestry Back Pain scale and a visual analogue scale (VAS) before PV and at a later date (medium-term follow up was a mean of 10 months after the last operation). Five out of eight cases in which 4.5-5 years had elapsed since the first PV were tested again (long-term follow-up). RESULTS: Relief of pain and improvement of QL, assessed a mean of 10 months after PV, proved to be statistically significant. On the average, pain decreased by 4.7 points as measured on the VAS scale and the average improvement in the QL measured on the Oswestry scale was 27.7%. There were no neurological or general complications. After 4.5-5 years, there has not been any significant change in the level of pain relief or the improvement in the QL in the 5 cases in which long-term assessment was possible. CONCLUSIONS: In MM cases, PV is a simple, effective and safe method for the treatment of vertebral infiltration and compression fractures, giving permanent long-term pain relief and concomitant improvement in the QL.

Reliability and validity of the Polish version of the Core Outcome Measures Index for the neck.

PURPOSE: Patient reported outcome measures play an increasingly important role in the outcomes research. The Core Outcome Measures Index (COMI) is a short, multidimensional instrument initially developed for the use by patients with low back pain. This study is an evaluation of a Polish version of COMI adapted for neck pain. METHODS: One hundred twenty-three patients complaining of neck pain were enrolled. All of them completed a questionnaire booklet containing COMI-neck, Neck Disability Index and Likert-type questions regarding the frequency of use of pain medications and pain frequency. Ninety-eight patients returned the retest questionnaire. Data quality was also assessed. Assessment of psychometric properties included examination of data quality, construct validity, test-retest reliability and factor analysis. RESULTS: The quality of data was good with no missing answers and a little floor effect. Exploratory factor analysis revealed a single-factor structure. Reliability expressed as intraclass correlation coefficient was 0.88 (95% CI 0.84-0.92) for the overall COMI score and was generally good for most of individual core items. The minimum detectable change (MDC95%) was 1.97. CONCLUSION: This version of the COMI-neck is a valid and reliable instrument, with good psychometric properties. It can be recommended for Polish-speaking patients.

Isolation and bioinformatic analysis of seven genes encoding potato apyrase. Bacterial overexpresssion, refolding and initial kinetic studies on some recombinant potato apyrases.

Here we have isolated seven apyrase encoding cDNA sequences (StAPY4-StAPY10) from the potato variety Saturna tuber cDNA library by affecting necessary modifications in the screening protocol. The cDNA sequences were identified with a pair of primers complementary to the most conserved sequences identified in potato variety Desiree apyrase genes. Our data strongly suggest the multigenic nature of potato apyrase. All deduced amino acid sequences contain a putative signal sequence, one transmembrane region at the amino terminus and five apyrase conserved regions (ACRs) (except StAPY6). Phylogenetic analysis revealed that encoded proteins shared high level of DNA sequence identity among themselves, representing a family of proteins markedly distinct from other eukaryotic as well as prokaryotic apyrases. Two cDNA sequences (StAPY4 and StAPY6) were overexpressed in bacteria and recombinant proteins were found accumulated in inclusion bodies, even thought they were fused with thioredoxin-tag. Additionally, we present the first successful in vitro attempt at reactivation and purification of recombinant potato apyrase StAPY6. The ratio of ATPase/ADPase hydrolysis of recombinant StAPY6 was determined as 1.5:1. Unlike other apyrases the enzyme lacked ACR5 and was endowed with lower molecular weight, high specificity for purine nucleotides and very low specificity for pyrimidine, suggesting that StAPY6 is a potato apyrase, not described so far.

Validation and cross-cultural adaptation of the Polish version of the Oswestry Disability Index.

STUDY DESIGN: Validation of a translated, culturally adapted questionnaire. OBJECTIVE: To translate and culturally adapt a Polish version of the Oswestry Disability Index (ODI) and to validate its use in Polish patients. SUMMARY OF BACKGROUND DATA: The ODI is among the most popular questionnaires used to evaluate back pain-related disability. To our knowledge no validated Polish version of the index was available at the time our study was initiated. METHODS: The questionnaire was translated and culturally adapted by 2 independent translators and approved by expert committee. Final version was included in the booklet consisting in addition of a previously validated Roland-Morris disability questionnaire, VAS for low back and leg and 3 Likert scale questions (pain medications, pain frequency, disability). It was tested on 169 patients with chronic low back pain, 164 (97%) of them were enrolled, and 84 of 164 (53%) returned the completed retest booklet within 2 to 14 days after the baseline test. There were no differences between the 2 groups in demographic and clinical parameters. Test-retest reliability, internal consistency, and construct validity were investigated. RESULTS: The mean ODI (standard deviation [SD]) was 48.45 (18.94); minimum 2, maximum 94. The Cronbach α for baseline questionnaires (n = 164) was 0.90. Concurrent validity, measured by comparing ODI responses with the results of the Roland-Morris disability questionnaire score was very good (r = 0.607, P < 0.001). The correlation with VAS back was fair (r = 0.37, P < 0.001) and with VAS leg was good (r = 0.56, P < 0.001). The tested ODI had excellent test-retest reliability, the intraclass correlation coefficient was 0.97 and standard error of measurements was 3.54, the resulting minimal detectable changes at the 95% confidence level was 10. CONCLUSION: The results of this study indicate that the Polish version of the ODI is a reliable and valid instrument for the measurement of disability in Polish-speaking patients with lower back pain.

Cross-cultural adaptation and validation of the Polish version of the core outcome measures index for low back pain.

PURPOSE: The core outcome measures index (COMI) is a short, multidimensional outcome scale validated for the use by patients with spinal disorders. It is a recommended instrument in the Spine Society of Europe Spine Tango Registry. The purpose of this study was to produce a cross-culturally adapted and validated Polish COMI. METHODS: The cross-cultural adaptation was carried out using the established guidelines. One-hundred and sixty-nine patients with chronic low back pain were enrolled, 89 took part in the reproducibility part of the study. Data quality, construct validity and reproducibility were assessed. RESULTS: The quality of data was very good with very few missing answers and modest floor effect. Reliability expressed as intraclass correlation coefficient (ICC) was 0.90 (95 % CI 0.85-0.93) for the overall COMI score and for most of the individual core items. The minimum detectable change (MDC95%) was 1.79. CONCLUSIONS: The Polish version of COMI showed a favorable reproducibility similar to that of previously tested language versions. The COMI scores correlated sufficiently with existing measures. This version of the COMI is a valuable instrument for the use by Polish-speaking patients with spinal disorders.

Modeling studies of potato nucleoside triphosphate diphosphohydrolase NTPDase1: an insight into the catalytic mechanism.

Nucleoside triphosphate diphosphohydrolase--NTPDase1 (apyrase, EC 3.6.1.5) was modeled based on sequence homology. The single polypeptide chain of apyrase is folded into two domains. The putative catalytic site with the apyrase conserved regions (ACR 1-5) is located between these two domains. Modeling confirmed that apyrase belongs to the actin superfamily of proteins. The amino acids interacting with the nucleoside triphosphate substrate and probably involved in the catalyzed hydrolysis were identified. The proposed two-step catalytic mechanism of hydrolysis involves Thr127 and Thr55 as potential nucleophilic factors responsible for the cleavage of the Pgamma and Pbeta anhydride bonds, respectively. Their action seems to be assisted by Glu170 and Glu78 residues, respectively. The presence of two nucleophiles in the active site of apyrase explains the differences in the hydrolytic activity between apyrases and other enzymes belonging to the NTPDase family.