The electrochemical and statistical evaluation of isolation of mellitin and apamin from honey bee (Apis Mellifera) venom.

We present in this manuscript for the first time the electrochemical and statistical evaluation of FPLC isolation of mellitin and apamin from honey bee (Apis mellifera) venom. Venoms are extremely complex blends of diverse substances that target a myriad of receptors or ion channels. Therefore, toxins, isolated from venomous organisms can be a valuable tool with diverse biological applications. In this study we decided to optimize the purification of honey bee venom by using fast protein liquid chromatography, to obtain biologically active peptide - melittin (2846.46 Da). Due to a presence of other compounds with similar molecular weight (apamin 2027.34 Da), we optimized a differential pulse voltammetry method with adsorptive transfer technique (AdT DPV), utilizing Brdicka supporting electrolyte for measurements. Typical voltammograms - fingerprints for each substance were obtained and numerical projections of voltammograms were employed to propose an artificial neural network. Our suggested neural network can simply predict the content of each peptide in fraction with following performance: 100 % for training and 100 % for testing

Long-term treatment with rilzabrutinib in patients with immune thrombocytopenia

Immune thrombocytopenia (ITP) is an autoimmune disease associated with autoantibodymediated platelet destruction and impaired platelet production, resulting in thrombocytopenia and a predisposition to bleeding. The ongoing, global phase 1/2 study showed that rilzabrutinib, a Bruton tyrosine kinase inhibitor specifically developed to treat autoimmune disorders, could be an efficacious and well-tolerated treatment for ITP. Clinical activity, durability of response, and safety were evaluated in 16 responding patients who continued rilzabrutinib 400 mg twice daily in the long-term extension (LTE) study. At LTE entry, the median platelet count was 87 × 109/L in all patients, 68 × 109/L in those who had rilzabrutinib monotherapy (n = 5), and 156 × 109/L in patients who received concomitant ITP medication (thrombopoietin-receptor agonists and/or corticosteroids, n = 11). At a median duration of treatment of 478 days (range, 303-764), 11 of 16 patients (69%) continued to receive rilzabrutinib. A platelet count of =50 × 109/L was reported in 93% of patients for more than half of their monthly visits. The median percentage of LTE weeks with platelet counts =30 × 109/L and =50 × 109/L was 100% and 88%, respectively. Five patients discontinued concomitant ITP therapy and maintained median platelet counts of 106 × 109/L at 3 to 6 months after stopping concomitant ITP therapy. Adverse events related to treatment were grade 1 or 2 and transient, with no bleeding, thrombotic, or serious adverse events. With continued rilzabrutinib treatment in the LTE, platelet responses were durable and stable over time with no new safety signals.</p

Hormone-regulated expansins : expression, localization, and cell wall biomechanics in Arabidopsis root growth

Expansins facilitate cell expansion by mediating pH-dependent cell wall (CW) loosening. However, the role of expansins in controlling CW biomechanical properties in specific tissues and organs remains elusive. We monitored hormonal responsiveness and spatial specificity of expression and localization of expansins predicted to be the direct targets of cytokinin signaling in Arabidopsis (Arabidopsis thaliana). We found EXPANSIN1 (EXPA1) homogenously distributed throughout the CW of columella/lateral root cap, while EXPA10 and EXPA14 localized predominantly at 3-cell boundaries in the epidermis/cortex in various root zones. EXPA15 revealed cell-type-specific combination of homogenous vs. 3-cell boundaries localization. By comparing Brillouin frequency shift and AFM-measured Young’s modulus, we demonstrated Brillouin light scattering (BLS) as a tool suitable for non-invasive in vivo quantitative assessment of CW viscoelasticity. Using both BLS and AFM, we showed that EXPA1 overexpression upregulated CW stiffness in the root transition zone (TZ). The dexamethasone-controlled EXPA1 overexpression induced fast changes in the transcription of numerous CW-associated genes, including several EXPAs and XYLOGLUCAN: XYLOGLUCOSYL TRANSFERASEs (XTHs), and associated with rapid pectin methylesterification determined by in situ Fouriertransform infrared spectroscopy in the root TZ. The EXPA1-induced CW remodeling is associated with the shortening of the root apical meristem, leading to root growth arrest. Based on our results, we propose that expansins control root growth by a delicate orchestration of CW biomechanical properties, possibly regulating both CW loosening and CW remodeling.peer-reviewe

Quantitative phase microscopy timelapse dataset of PNT1A, DU-145 and LNCaP cells with annotated caspase 3,7-dependent and independent cell death

Time-lapse dataset of prostatic cell lines (DU-145, PNT1A, LNCaP) exposed to cell death-inducing compounds (staurosporine, doxorubicin) and black phosphorus. The time-lapse dataset is annotated as follows: (1) cell masks and cell numbers, (2) by cell death type and timepoint of death in the attached xlsx file. This dataset is supplementary to the article: Tomas Vicar, Martina Raudenska, Jaromir Gumulec, Michal Masarik, Jan Balvan. Detection and characterization of apoptotic and necrotic cell death by time-lapse quantitative phase image analysis. bioRxiv, 589697; DOI: https://doi.org/10.1101/589697 Code is available at https://github.com/tomasvicar/CellDeathDetect Methods Cell culture and cultured cell conditions LNCaP cell line was established from a lymph node metastase of the hormone-refractory patient and contains a mutation in the AR gene. This mutation creates a promiscuous AR that can bind to different types of steroids. LNCaP cells are AR-positive, PSA-positive, PTEN-negative and harbor wild-type p53 {Skjoth, 2006 #150; Mitchell, 2000 #149}. PNT1A is immortalized non-tumorigenic epithelial cell line. PNT1A cells harbour wild-type p53. However, SV40 induced T-antigen expression inhibits the activity of p53. This cell line had lost the expression of androgen receptor (AR) and prostate-specific antigen (PSA) (Raudenska, 2019). DU-145 cell line is derived from the metastatic site in the brain and contains P223L and V274F mutations in p53. This cell line is PSA and AR-negative and androgen independent (Chappell, 2012). All cell lines used in this study were purchased from HPA Culture Collections (Salisbury, UK). and were cultured in RPMI-1640 medium with 10 % FBS. The medium was supplemented with antibiotics (penicillin 100 U/ml and streptomycin 0.1 mg/ml). Cells were maintained at 37°C in a humidified (60%) incubator with 5% CO2 (Sanyo, Japan). Correlative time-lapse quantitative phase-fluorescence imaging QPI and fluorescence imaging were performed by using multimodal holographic microscope Q-PHASE (TESCAN, Brno, Czech Republic). To determine the amount of caspase-3/7 product accumulation, cells were loaded with 2 µM CellEventTM Caspase-3/7 Green Detection Reagent (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol and visualized using FITC 488 nm filter. To detect the cells with a loss of plasma membrane integrity, cells were stained with 1 ug/ml propidium iodide (Sigma Aldrich Co., St. Louis, MO, USA) and visualized using TRITC 542 nm filter. Nuclear morphology and chromatin condensation were analyzed using Hoechst 33342 nuclear staining (ENZO, Lausen, Switzerland) and visualized using DAPI 461 nm filter. Cells were cultivated in Flow chambers μ-Slide I Lauer Family (Ibidi, Martinsried, Germany). To maintain standard cultivation conditions (37°C, humidified air (60%) with 5% CO2) during time-lapse experiments, cells were placed in the gas chamber H201 - for Mad City Labs Z100/Z500 piezo Z-stages (Okolab, Ottaviano NA, Italy). To image enough cells in one field of view, lens Nikon Plan 10/0.30 were chosen. For each cell line and each treatment, seven fields of view were observed with the frame rate 3 mins/frame for 24 or 48 h respectively. Holograms were captured by CCD camera (XIMEA MR4021 MC-VELETA), fluorescence images were captured using ANDOR Zyla 5.5 sCMOS camera. Complete quantitative phase image reconstruction and image processing were performed in Q-PHASE control software. Cell dry mass values were derived according to {Prescher, 2005 #177} and {Park, 2018 #178} from the phase (eq. (1)), where m is cell dry mass density (in pg/μm2), φ is detected phase (in rad), λ is wavelength in μm (0.65 μm in Q-PHASE), and α is specific refraction increment (≈0.18 μm3/pg). All values in the formula except the Phi are constant. Phi (Phase) is the value measured directly by the microscope. Integrated phase shift through a cell is proportional to its dry mass, which enables studying changes in cell mass distribution (Park et al., 2018). File description There are three archives included for particular cell lines: QPI_annotated_timelapse_DU145.zip for DU-145 cells QPI_annotated_timelapse_PNT1A.zip for PNT1A cells QPI_annotated_timelapse_LNCaP.zip for LNCaP cells The archive includes of following files: Tiff with time-lapse quantitative phase image (32-bit files 600x600px with values in pg/um2 with framerate 1 frame/3minutes with 1.59 px/um), named QPI_cellline_treatment_FOV.tiff Tiff file with segmentation mask for particular cells named mask_cellline_treatment_FOV.tiff xlsx table with cell death type (1 for apoptosis, 2 for necrosis, 3 for ambiguous/surviving) and time of death for representative cell number from mask, named labels_cellline_treatment_FOV.xlsx file naming has following conventions: cell names: DU145, PNT1A, LNCaP for particular cell line treatments: st, bp, do for staurosporine, black phosphorus and doxorubicin fields of view: 1 to 7 e.g. QPI_DU145_st_4.tif, mask_DU145_st_4.tif, labels_DU145_st_4.xls

Flow chart showing the number of citations retrieved by database searching.

<p>Flow chart showing the number of citations retrieved by database searching.</p

Forrest plot showing associations between metallothionein staining and tumors (tumors versus healthy controls).

<p>The result of meta-analysis for particular tumor types displayed instead of individual studies. For more detailed results see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085346#pone-0085346-t001" target="_blank">Table 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085346#pone-0085346-t002" target="_blank">2</a>. Sorted alphabetically by tumor types. Forrest plot displayed as odds ratio and 95% confidence intervals. Red dashed line indicates the result for all tumor types together. OR, odds ratio; CI, confidence interval.</p

Association of MT staining and clinicopatological factors. Tumor type not taken into account.

<p>Heterogeneity of studies analyzed using Cochran's Q-test (p-value displayed) and using I². Egger's two-tailed test used for publication bias analysis (p-value displayed). * effect measure is odds ratio, OR, except for survival analysis using hazard ratio, HR. CI, confidence interval</p

Forrest plot of studies reporting the association of metallothionein staining and nodal and distant metastases.

<p>Random effects model used for both outcomes, Relative weight of individual studies displayed in %. For more detailed results see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085346#pone-0085346-t001" target="_blank">Table 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085346#pone-0085346-t002" target="_blank">2</a>. Sorted alphabetically by tumor types. Forrest plot displayed as odds ratio and 95% confidence intervals. Red dashed line indicates the result for all studies together. OR, odds ratio; CI, confidence interval.</p

Forrest plot of studies reporting the association of metallothionein staining and tumor grading.

<p>Random effects model used. Relative weight of individual studies displayed in %. For more detailed results see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085346#pone-0085346-t001" target="_blank">Table 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085346#pone-0085346-t002" target="_blank">2</a>. * indicates studies using Gleason grading. Sorted alphabetically by tumor types. Forrest plot displayed as odds ratio and 95% confidence intervals. Red dashed line indicates the result for all studies together. OR, odds ratio; CI, confidence interval.</p

ELECTROCHEMICAL SCIENCE Femtogram Electroanalytical Detection of Prostatic Specific Antigen by Brdicka Reaction

Prostatic-specific antigen is considered as the best marker for prostate cancer. Due to the importance of PSA for diagnostic purposes it is not surprising that there are tested and optimized various methods for its determination. In spite of such intensive research in the field of electrochemical detection of some by-products connected with concentration of PSA, electrochemical behaviour of PSA has not been studied yet. The aim of this study was to investigate electrochemical catalytic signals of PSA using differential pulse voltammetry Brdicka reaction. The catalytic signals were studied using adsorptive transfer stripping technique as well as directly in the electrochemical cell. Nevertheless, we primarily tested detection of PSA by standard immunoanalysis and by gel and capillary chip electrophoresis to investigate behaviour of this protein in electric field. Both electrophoretic methods showed that the most intensive band of PSA was determined at 37 kDa under reducing conditions and at 26 kDa under non-reducing. Band at 37 kDa corresponds to a reduced, and at 26 kDa to non-reduced PSA. Studying of basic electrochemical behaviour of PSA was primarily carried out using standard electrochemical cell and HMDE as a working electrode. Co(NH 3