The growth performance of pond-reared common carp (Cyprinus carpio) larvae propagated using cryopreserved sperm.

The aim of our study was to determine the efficacy of utilizing cryopreserved common carp sperm (in comparison to fresh sperm) for propagation at a Hungarian aquaculture facility. The sperm was frozen in 5 mL straws using an extender method that was previously tested in common carp. Sperm motility was monitored using a computer-assisted sperm analysis system. The hatching and malformation rates among the specimens were recorded before the stocking of larvae in both groups. The growth (body weight, total length) and survival rates of the fish were measured during the pre-nursing (from May to June: between 1 and 26 days post hatching) and grow-out periods (from June to October: between 26 and 105 days post hatching) of the same year. The fresh sperm, which was collected and pooled prior to fertilization, showed high MOT (97%), pMOT (92%), VCL (106 µm s-1), LIN (75%), and ALH (1.84 µm). Prior to the fertilization trial of the cryopreserved sperm, low MOT (34%), pMOT (14%), and VCL (61 µm s-1) values were observed in frozen-thawed sperm. A significantly higher hatching rate was measured in the fresh sperm group (87%) when compared to the cryopreserved sperm group (42%). No significant difference in the overall malformation rate was observed in larvae originating from either the fresh or frozen sperm. A significant difference between the two test groups was observed in the incidence of deformed tails (fresh: 20%, cryopreserved: 55%). Except for one sampling period, no significant difference in the body weight and total length of the fish larvae was found between the two groups throughout the pre-nursing and grow-out periods. A significantly higher larvae survival rate was noted in the fresh sperm (72%) as compared to the cryopreserved group (43%) by the end of the pre-nursing stage. However, no significant difference in survival rate was observed for the cryopreserved sperm (96%) in comparison to the fresh sperm (95%) by the end of the grow-out stage. The results of this study showed, for the first time in large-scale pond culturing, an equal growth and viability in larvae propagated from cryopreserved sperm when compared to fresh sperm (despite the limited available rearing ponds provided by the commercial company).

Black Soldier Fly (Hermetia illucens) Meal as Direct Replacement of Complex Fish Feed for Rainbow Trout (Oncorhynchus mykiss) and African Catfish (Clarias gariepinus).

In the experiments, defatted black soldier fly meal reared on vegetable byproducts was used in the fry rearing of two economically important fish species, African catfish and rainbow trout. Both fish species were reared in a recirculation system and 0-33-66-100% of the complex fry feed was replaced by a defatted prepupae meal of black soldier flies during a 28-day feeding experiment. African catfish was reared at 25 ± 1 °C while rainbow trout was reared at 12 ± 1 °C. The results showed that the growth of African catfish was not significantly reduced when 66% of the feed was replaced by soldier fly meal (mean weight in the control fish group at the end of the experiment was 0.4632 ± 0.2469 g, while the 66% group resulted mean weights of 0.4150 ± 0.1886 g) and the survival did not show any statistically different results (mean survival in control group was 57.48 ± 13.76% while it was 56.6 ± 7.763% in the 66% group). In the case of rainbow trout, replacing the feed entirely with insect meal did not cause a decrease in weight gain (final mean weight in the control group was measured at 1.9640 ± 0.4154 g, while in the group consuming only insect meal, it was 1.9410 ± 0.4248 g) or in survival (in the control group 98.5%, while in the group consuming only insect meal 99.5%). All these preliminary results indicate that black soldier fly meal can be used directly as a nursery feed in fish farming as a partial or total replacement of complete feeds. The results showed that black soldier fly meal could replace 66% of the complex brood feed of African catfish and up to 100% of rainbow trout feed without deterioration of production results. Our experiments have therefore opened the way for further experiments on insect meal in larval rearing.

Biochemical Marker Assessment of Chronic Carbamazepine Exposure at Environmentally Relevant Concentrations in Juvenile Common Carp (Cyprinus carpio).

Worldwide, the anticonvulsant drug carbamazepine (CBZ) is the most frequently identified pharmaceutical residue detected in rivers. Reported chronic effects of CBZ in non-target freshwater organisms, particularly fish, include oxidative stress and damage to liver tissues. Studies on CBZ effects in fish are mostly limited to zebrafish and rainbow trout studies. Furthermore, there are only a few chronic CBZ studies using near environmental concentrations. In this study, we provide data on subacute effects of CBZ exposure (28 days) to common carp (Cyprinus carpio), employing a set of biochemical markers of damage and exposure. CBZ was found to induce a significant change in the hepatic antioxidant status of fish subjected to 5 µg/L. Moreover, with increasing concentrations, enzymatic and non-enzymatic biomarkers of oxidative defence (catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR), DNA strand breaks)), toxicant biotransformation (ethoxyresorufin-o-demethylase (EROD), glutathione-S-transferase (GST)), and organ and tissue damage (lactate dehydrogenase (LDH), cetylcholinesterase (AChE)) were altered. The AChE, LDH, and lipid peroxidation (LPO) results indicate the occurrence of apoptotic process activation and tissue damage after 28 days of exposure to CBZ. These findings suggest significant adverse effects of CBZ exposure to common carp at concentrations often found in surface waters.

The market for acquiring card payments from small and medium-sized Canadian merchants.

This article builds on industry data and a unique dataset of small and medium-sized merchants to provide insights into the acquirer-merchant market in Canada. Three main findings are presented. First, smaller merchants pay their acquirer more for every dollar of card payment than larger merchants. Second, this finding is mainly explained by high fixed costs. Third, the acquiring market in Canada is concentrated and has remained fairly stable since 2010. These findings could be relevant for other countries with mature card payments systems such as the US and Australia.

Automatic deep learning-driven label-free image-guided patch clamp system.

Patch clamp recording of neurons is a labor-intensive and time-consuming procedure. Here, we demonstrate a tool that fully automatically performs electrophysiological recordings in label-free tissue slices. The automation covers the detection of cells in label-free images, calibration of the micropipette movement, approach to the cell with the pipette, formation of the whole-cell configuration, and recording. The cell detection is based on deep learning. The model is trained on a new image database of neurons in unlabeled brain tissue slices. The pipette tip detection and approaching phase use image analysis techniques for precise movements. High-quality measurements are performed on hundreds of human and rodent neurons. We also demonstrate that further molecular and anatomical analysis can be performed on the recorded cells. The software has a diary module that automatically logs patch clamp events. Our tool can multiply the number of daily measurements to help brain research.

Comparison of manganese uptake and transport of maize seedlings by mini-PET camera.

Manganese is one of the most important essential micronutrients for the plants. To monitor its uptake and transport by radioactive tracking is a powerful method due to the no carrier added 52Mn in 10-12 moldm-3 concentration range. The generally used method is to measure the radioactivity of cut parts of plants by gamma-spectrometry. Only few studies reported about noninvasive measurement, using pairs of detectors connected in coincidence. We use a full ring MiniPET machine for this purpose to dynamically visualize the uptake and distribution of the radionuclide in 4D. The results are controlled with the conventional gamma spectroscopy after chopping the plants into six parts. The study of stress tolerance initiated by PEG 6000 in different hybrids of maize is also presented as possible application for the phenotyping of plants by PET camera.

3D-Cell-Annotator: an open-source active surface tool for single-cell segmentation in 3D microscopy images.

SUMMARY: Segmentation of single cells in microscopy images is one of the major challenges in computational biology. It is the first step of most bioimage analysis tasks, and essential to create training sets for more advanced deep learning approaches. Here, we propose 3D-Cell-Annotator to solve this task using 3D active surfaces together with shape descriptors as prior information in a semi-automated fashion. The software uses the convenient 3D interface of the widely used Medical Imaging Interaction Toolkit (MITK). Results on 3D biological structures (e.g. spheroids, organoids and embryos) show that the precision of the segmentation reaches the level of a human expert. AVAILABILITY AND IMPLEMENTATION: 3D-Cell-Annotator is implemented in CUDA/C++ as a patch for the segmentation module of MITK. The 3D-Cell-Annotator enabled MITK distribution can be downloaded at: www.3D-cell-annotator.org. It works under Windows 64-bit systems and recent Linux distributions even on a consumer level laptop with a CUDA-enabled video card using recent NVIDIA drivers. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

nucleAIzer: A Parameter-free Deep Learning Framework for Nucleus Segmentation Using Image Style Transfer.

Single-cell segmentation is typically a crucial task of image-based cellular analysis. We present nucleAIzer, a deep-learning approach aiming toward a truly general method for localizing 2D cell nuclei across a diverse range of assays and light microscopy modalities. We outperform the 739 methods submitted to the 2018 Data Science Bowl on images representing a variety of realistic conditions, some of which were not represented in the training data. The key to our approach is that during training nucleAIzer automatically adapts its nucleus-style model to unseen and unlabeled data using image style transfer to automatically generate augmented training samples. This allows the model to recognize nuclei in new and different experiments efficiently without requiring expert annotations, making deep learning for nucleus segmentation fairly simple and labor free for most biological light microscopy experiments. It can also be used online, integrated into CellProfiler and freely downloaded at www.nucleaizer.org. A record of this paper's transparent peer review process is included in the Supplemental Information.

The Role of Efflux Pumps and Environmental pH in Bacterial Multidrug Resistance.

BACKGROUND/AIM: One of the most studied bacterial resistance mechanisms is the resistance related to multidrug efflux pumps. In our study the pump activity of the Escherichia coli K-12 AG100 strain expressing the AcrAB-TolC pump system was investigated at pH 7 and pH 5 in the presence of the efflux pump inhibitor (EPI) promethazine (PMZ). MATERIALS AND METHODS: The EPI activity was assessed by real-time fluorimetry. The influence of PMZ treatment on the relative expression of the pump genes acrA, acrB and their regulators marA, marB, marR, the stress genes soxS, rob, as well as the bacterial growth control genes ftsI, and sdiA were determined by RT-qPCR. RESULTS: The EPI activity of PMZ was more effective at neutral pH. The PMZ treatment induced a significant stress response in the bacterium at acidic pH by the up-regulation of genes. CONCLUSION: The genetic system that regulates the activity of the main efflux pump is pH-dependent.

Antifibrotic effect of mitomycin-C on human vocal cord fibroblasts.

OBJECTIVE: Acquired laryngotracheal stenosis is a potentially life-threatening situation and a very difficult and challenging problem in laryngology. Therefore, new trends and innovative approaches based on antifibrotic drugs and minimally invasive regimens are being developed to attenuate laryngotracheal fibrosis and scarring. The purpose of this study was to examine the efficacy of mitomycin-C (MMC) to reverse the transforming growth factor (TGF)-ß-induced differentiation of MRC-5 fibroblast and human primary vocal cord fibroblasts to reveal the possible applicability of MMC to laryngotracheal fibrotic conditions. METHODS: Human primary fibroblast cells were isolated from vocal cord specimens of patients undergoing total laryngectomy. The established primary vocal cord fibroblast cell cultures as well as the MRC-5 human fibroblast cells were treated with 5 ng/mL TGF-ß alone and then with 0.5 µg/mL MMC for 24 hours. Differentiation of fibroblasts was characterized by α-smooth muscle actin (α-SMA) immunhistochemistry, Western blot analysis, and real-time polymerase chain reaction. Cell motility was assessed by wound-healing assay. RESULTS: Elevated α-SMA mRNA and protein expression as well as increased cell motility were observed upon TGF-ß exposures. However, after MMC treatments the TGF-ß-induced fibroblasts exhibited a significant decrease in α-SMA expression and wound-healing activity. Therefore, TGF-ß-stimulated fibroblast-myofibroblast transformation was reversed at least in part by MMC treatment. Histopathological examinations of tissue specimens of a laryngotracheal stenosis patient supported these findings. CONCLUSION: Antifibrotic effects of MMC were demonstrated on the human MRC-5 cell line and on primary vocal cord fibroblast cultures. These results verify that MMC can be used with success to reverse upper airway stenosis by reverting the myofibroblast phenotype. LEVEL OF EVIDENCE: NA Laryngoscope, 129:E255-E262, 2019.

The comparison of two different extenders for the improvement of large-scale sperm cryopreservation in common carp (Cyprinus carpio).

In our study, a traditionally used (Grayling, already used in cyprinid species) and a newly tested (Pike) extender was tested to avoid sperm agglutination phenomenon following thawing during carp sperm cryopreservation. A large-scale (elevated volume of sperm) freezing method in a controlled-rate freezer using 5 ml straw and 10 ml cryotube was also systematically established. In all experiments, the sperm cryopreserved in using Grayling extender (except only one sample) showed an agglutination phenomenon (damaged and intact cells adhered to each other) after thawing where Pike extender resulted the regular cell suspension. No significant difference was observed between the two cryopreserved groups (Pike and Grayling extender) in all motility parameters using the 0.5 ml straw and the polystyrene box. Similarly, motility parameters did not show a significant difference in the two frozen groups with the 5 ml straw, also in the polystyrene box. A significantly higher progressive motility (pMOT, Grayling: 54% ± 8%, Pike: 37% ± 5%), straight line velocity (VSL, Grayling: 50 ± 5 µm/s, Pike: 39 ± 4 µm/s) and beat cross frequency (BCF, Grayling: 20 ± 1 Hz, Pike: 17 ± 1 Hz) was observed in the case of the grayling extender by the 5 ml straw cryopreserved in a controlled-rate freezer (CRF) compare to the pike extender. A significantly higher VSL (Grayling: 45 ± 3 µm/s, Pike: 38 ± 4 µm/s) was observed by the grayling extender using the 10 ml cryotube than with the pike extender. Despite the randomly occurring differences in a few parameters, our new controlled freezing method using the newly tested Pike extender, the 5 ml straw or the 10 ml cryotube can be a good solution for the preservation of elevated volume of carp sperm.

DIC image reconstruction using an energy minimization framework to visualize optical path length distribution.

Label-free microscopy techniques have numerous advantages such as low phototoxicity, simple setup and no need for fluorophores or other contrast materials. Despite their advantages, most label-free techniques cannot visualize specific cellular compartments or the location of proteins and the image formation limits quantitative evaluation. Differential interference contrast (DIC) is a qualitative microscopy technique that shows the optical path length differences within a specimen. We propose a variational framework for DIC image reconstruction. The proposed method largely outperforms state-of-the-art methods on synthetic, artificial and real tests and turns DIC microscopy into an automated high-content imaging tool. Image sets and the source code of the examined algorithms are made publicly available.

Silver nanoparticles modulate ABC transporter activity and enhance chemotherapy in multidrug resistant cancer.

The emergence of multidrug resistant (MDR) cancer phenotypes dramatically attenuates the efficiency of antineoplastic drug treatments often leading to the failure of chemotherapy. Therefore there is an urgent need to engineer new therapeutically useful agents and propose innovative approaches able to defeat resistant cancer cells. Although the remarkable anti-cancer features of silver nanoparticles (AgNPs) have already been delineated their impact on MDR cancer has never been investigated. Herein, we report that AgNPs have notable anti-proliferative effect and induce apoptosis mediated cell death both in drug sensitive and in MDR cancer cells. Furthermore we show evidence that AgNPs exert an inhibitory action on the efflux activity of MDR cancer cells which feature could be exploited to enhance drug accumulation. We verified synergistic interactions of AgNPs with six different antineoplastic agents on drug resistant cells which emphasizes the excellent potential of AgNPs as combinational partners in the chemotherapy of MDR cancer. FROM THE CLINICAL EDITOR: The treatment of cancer often fails due to the development of multidrug resistant (MDR) cancer cells. Hence, novel approaches are being investigated to combat drug resistant cancer cells. One particular method studied here uses silver nanoparticles (AgNPs). The authors showed that AgNPs had anti-proliferative effect and ?exerted an inhibitory action on ABC transporter. The findings could suggest the possible use of AgNPs in combination with other chemotherapeutic agents in the clinical setting.

Molecular Docking Analysis of Steroid-based Copper Transporter 1 Inhibitors.

Copper transporter 1 (CTR1) represents an important determinant of cisplatin resistance. A series of 35 semi-substituted steroids were recently investigated on cisplatin-resistant CTR1-expressing A2780cis ovarian carcinoma cells as well as their parental sensitive counterparts regarding their cytotoxic and resistance-reversing features. In the present investigation, three compounds ( 4: , 5: , 25: ) were selected for molecular docking analysis on the homology-modelled human CTR1 transmembrane domain. Steroids 4: , 5: and 25: interacted with CTR1 at a similar docking pose and with even higher binding affinities than the known CTR1 inhibitor, cimetidine. Applying the defined docking mode, the binding energies were found to be -7.15±<0.001 kcal/mol (compound 4: ), -8.71±0.06 kcal/mol (compound 5: ), -7.63±0.01 kcal/mol (compound 25: ), and -5.05±0.02 kcal/mol (for cimetidine). These steroids have the potential for further development as CTR1 inhibitors overcoming cisplatin resistance.

A Promising Future: Comparable Imaging Capability of MRI-Compatible Silicon Photomultiplier and Conventional Photosensor Preclinical PET Systems.

UNLABELLED: We recently completed construction of a small-animal PET system-the MiniPET-3-that uses state-of-the-art silicon photomultiplier (SiPM) photosensors, making possible dual-modality imaging with MRI. In this article, we compare the MiniPET-3 with the MiniPET-2, a system with the same crystal geometry but conventional photomultiplier tubes (PMTs). METHODS: The standard measurements proposed by the National Electrical Manufacturers Association NU 4 protocols were performed on both systems. These measurements included spatial resolution, system sensitivity, energy resolution, counting rate performance, scatter fraction, spillover ratio for air and water, recovery coefficient, and image uniformity. The energy windows were set to 350-650 keV on the MiniPET-2 and 360-662 keV on the MiniPET-3. RESULTS: Spatial resolution was approximately 17% better on average for the MiniPET-3 than the MiniPET-2. The systems performed similarly in terms of peak absolute sensitivity (∼1.37%), spillover ratio for air (∼0.15), spillover ratio for water (∼0.25), and recovery coefficient (∼0.33, 0.59, 0.81, 0.89, and 0.94). Uniformity was 5.59% for the MiniPET-2 and 6.49% for the MiniPET-3. Minor differences were found in scatter fraction. With the ratlike phantom, the peak noise-equivalent counting rate was 14 kcps on the MiniPET-2 but 24 kcps on the MiniPET-3. However, with the mouselike phantom, these values were 55 and 91 kcps, respectively. The optimal coincidence time window was 6 ns for the MiniPET-2 and 8 ns for the MiniPET-3. CONCLUSION: Images obtained with the SiPM-based MiniPET-3 small-animal PET system are similar in quality to those obtained with the conventional PMT-based MiniPET-2.

Reversal of ABCB1-related Multidrug Resistance of Colonic Adenocarcinoma Cells by Phenothiazines.

BACKGROUND: The most common mechanism that reduces the efficacy of anticancer agents is overexpression of ATP-binding cassette (ABC) drug transporters. Phenothiazines and structurally-related compounds can sensitize multidrug-resistant (MDR) cells to chemotherapeutics. MATERIALS AND METHODS: Phenothiazine derivatives were investigated regarding their anticancer and MDR-reversing effect on colonic adenocarcinoma cells. The anti-proliferative and cytotoxic effects of the derivatives were assessed by the thiazolyl blue tetrazolium bromide (MTT) method, the modulation of the ABCB1 activity was measured by rhodamine 123 accumulation assay using flow cytometry. RESULTS: All phenothiazines exhibited potent cytotoxic effect on the sensitive and MDR colon adenocarcinoma cell lines. The inhibition of the ABCB1 transporter was greater in the presence of the phenothiazine derivatives than for the known ABCB1 inhibitor verapamil. CONCLUSION: It can be concluded that these derivatives show synergism in the presence of doxorubicin and could have potential as ABCB1 inhibitors.

Efflux pump inhibiting properties of racemic phenothiazine derivatives and their enantiomers on the bacterial AcrAB-TolC system.

BACKGROUND/AIM: Bacterial resistance to antibiotics has become a serious problem in antibacterial chemotherapy and resistance of bacteria to chemically-unrelated anti-microbial agents can be associated with the over-expression of efflux pumps. The simultaneous therapy with efflux pump inhibitors (EPIs) could be a solution to improve the effectiveness of antibiotics. The response of an organism to an EPI often depends on how that molecule fits a particular site of a protein. Because enantiomers of a given compound rotate plane-polarized light in a solution by the same angle but in opposite directions, the rational drug design should take the chirality into account if there is a difference between the racemic compound and its enantiomers. MATERIALS AND METHODS: The main goal of the present study was to elucidate the role of chirality of N-hydroxyalkyl-2-aminophenothiazines as effective EPIs by an automated method that uses the general efflux pump substrate ethidium bromide (EB) for the assessment of AcrAB-TolC system of wild-type Escherichia coli K-12 AG100. It has been shown that the most active EPIs among the N-hydroxyalkyl-2-aminophenothiazines were the compounds rac-3i, (+)-3i, and (-)-3i by modulating the AcrAB-TolC efflux pump. CONCLUSION: Comparison of effects of enantiomeric pairs revealed that their activities were similar to that of racemic derivatives. Moreover, there was no significant difference between the racemic compounds and their enantiomers related to their antibacterial and efflux pump inhibiting effects.

Cold wall effect eliminating method to determine the contrast recovery coefficient for small animal PET scanners using the NEMA NU-4 image quality phantom.

The contrast recovery coefficients (CRC) were evaluated for five different small animal PET scanners: GE Explore Vista, Genisys4, MiniPET-2, nanoScan PC and Siemens Inveon. The NEMA NU-4 2008 performance test with the suggested image quality phantom (NU4IQ) does not allow the determination of the CRC values for the hot regions in the phantom. This drawback of NU4IQ phantom motivated us to develop a new method for this purpose. The method includes special acquisition and reconstruction protocols using the original phantom, and results in an artificially merged image enabling the evaluation of CRC values. An advantageous feature of this method is that it stops the cold wall effect from distorting the CRC calculation. Our suggested protocol results in a set of CRC values contributing to the characterization of small animal PET scanners. GATE simulations were also performed to validate the new method and verify the evaluated CRC values. We also demonstrated that the numerical values of this parameter depend on the actual object contrast of the hot region(s) and this mainly comes from the spillover effect. This effect was also studied while analysing the background activity level around the hot rods. We revealed that the calculated background mean values depended on the target contrast in a scanner specific manner. Performing the artificially merged imaging procedure and additional simulations using the micro hollow sphere (MHS) phantom geometry, we also proved that the inactive wall around the hot spheres can have a remarkable impact on the calculated CRC. In conclusion, we have shown that the proposed artificial merging procedure and the commonly used NU4IQ phantom prescribed by the NEMA NU-4 can easily deliver reliable CRC data otherwise unavailable for the NU4IQ phantom in the conventional protocol or the MHS phantom.

Multidrug resistance reversing activity of newly developed phenothiazines on P-glycoprotein (ABCB1)-related resistance of mouse T-lymphoma cells.

BACKGROUND: Phenothiazines have anticancer properties and are able to reverse the multidrug resistance of neoplastic cells by inhibiting the ATP-binding cassette, sub-family B (MDR/TAP), member 1 protein (ABCB1 or P-glycoprotein) activity. MATERIALS AND METHODS: A series of new phenothiazine derivatives was investigated regarding their ABCB1-modulating effect on multidrug resistant mouse T-lymphoma cells by rhodamine 123 accumulation assay and real-time ethidium bromide accumulation assay. RESULTS: The phenothiazine derivatives exhibited a potent anticancer effect on the parental cell line and on its multidrug-resistant mouse T-lymphoma subline overexpressing the ABCB1 transporter. The inhibition of the ABCB1 transporter in the presence of the newly-developed phenothiazines was greater than that for the known ABCB1 inhibitors thioridazine and verapamil. CONCLUSION: Based on the chemical structures and biological activity, compounds with bivalent sulfur atom in the phenothiazine ring demonstrated marked ABCB1-modulating effect, however, other derivatives with halogen or amide substitutions were ineffective.

Synthesis and structure-activity relationships of novel ecdysteroid dioxolanes as MDR modulators in cancer.

Ecdysteroids, molting hormones of insects, can exert several mild, non-hormonal bioactivities in mammals, including humans. In a previous study, we have found a significant effect of certain derivatives on the ABCB1 transporter mediated multi-drug resistance of a transfected murine leukemia cell line. In this paper, we present a structure-activity relationship study focused on the apolar dioxolane derivatives of 20-hydroxyecdysone. Semi-synthesis and bioactivity of a total of 32 ecdysteroids, including 20 new compounds, is presented, supplemented with their complete 1H- and 13C-NMR signal assignment.