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).

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.

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.

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.

Synthesis and pharmacological investigation of new N-hydroxyalkyl-2-aminophenothiazines exhibiting marked MDR inhibitory effect.

Novel N-hydroxyalkyl-2-aminophenothiazines implying a tetrazole moiety at the alkyl chain have been synthesized by hydroboration-oxidation of dienes followed by Buchwald-Hartwig cross-coupling reaction. Also, some sulfoxide and sulfone derivatives have been prepared by selective oxidations. MDR inhibition studies on rat hepatocyte cell culture revealed that some derivatives exhibit marked biological efficacy exceeding that of the standard verapamil (e.g., 3h, 4h, 16). Selected derivatives were subjected to chemical resolution to provide both enantiomers which were shown of similar activity on P-gp interaction measurements. The new compounds exhibited no toxicity.

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.

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.

Selective hydroboration of dieneamines. Formation of hydroxyalkylphenothiazines as MDR modulators.

N-dienylphenothiazines synthesized from tetrazolo[1,5-a]pyridinium salts by treatment with phenothiazine were subjected to catalytic hydrogenation to yield N-butylphenothiazines, whereas transformation of these dienes with borane dimethyl sulfide (BH(3) × Me(2)S) resulted in selective hydroboration of one double bond and full reduction of the other double bond to give 2-hydroxybutylphenothiazines. Position of the hydroxyl group was supported by NMR spectroscopy and verified by X-ray analysis. Comparison of MDR modulatory activity of the new derivatives revealed that the hydroxybutyl compounds are promising candidates for development of novel MDR inhibitors.

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.

Inhibition of quorum-sensing signals by essential oils.

The role of quorum sensing (QS) is well known in microbial pathogenicity and antibiotic resistance. QS is responsible for motility, swarming, and biofilm production based on the signal molecules, e.g., acylated homoserine lactones (AHLs) produced by micro-organisms above certain population density. The inhibition of QS may reduce pathogenicity, antibiotic resistance and biofilm formation in systemic and local infections. The homoserine lactones and other transmitters contribute to antibiotic resistance and pathogenicity of several bacteria; consequently the inhibition of QS signals reduces the problem of resistance and virulence. Due to the increasing number of persistent non-treatable infections, there is an urgent need to develop new strategies to combat infections that destabilize bacterial communities in the host. The effect of essential oils on bacterial growth and QS were evaluated using the sensor strain Chromobacterium violaceum CV026 and N-acyl homoserine lactone (AHL) producing Escherichia coli ATTC 31298 and the grapevine colonizing Ezf 10-17 strains. Of the tested oils, rose, geranium, lavender and rosemary oils were the most potent QS inhibitors. Eucalyptus and citrus oils moderately reduced pigment production by CV026, whereas the chamomile, orange and juniper oils were ineffective.

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.

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.

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.

Demonstration of the activity of P-glycoprotein by a semi-automated fluorometric method.

BACKGROUND: We have developed a semi-automated fluorometric method that utilizes ethidium bromide (EB), a common substrate of bacterial efflux pumps. The method is sufficiently sensitive to characterize the efflux pump systems of bacteria. Because EB is also recognized and extruded by ATP-binding cassette (ABC) transporters and these have similarity to P-glycoprotein (P-gp), the method has been extended for the evaluation of agents that can inhibit the extrusion of EB on a real-time basis by mouse lymphoma cells containing the human ABCB1 (mdr1) gene. MATERIALS AND METHODS: Monitoring of uptake and extrusion of EB was assessed using the Rotor-Gene 3000 (Corbett Research) under different conditions. RESULTS: Whereas extrusion of EB took place readily, the addition of known inhibitors of efflux pumps (verapamil, reserpine) caused retention of EB. CONCLUSION: This method is inexpensive and allows the detection of neoplastic cells with increased efflux activity as well as the screening of large numbers of compounds for inhibition of the P-gp.

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.

Reversal of multidrug resistance by synthetic and natural compounds in drug-resistant MCF-7 cell lines.

BACKGROUND: Ineffectiveness of anticancer drugs is frequently observed in cancer chemotherapy. The resistance of tumor cells to various cytotoxic drugs is defined as multidrug resistance (MDR). The purpose of this study is to investigate the potential reversal effect of some synthetic and natural chemicals on drug-resistant MCF-7 cell lines. The effects of potential MDR modulators combined with some anticancer drugs were also studied. METHODS: Flow cytometry, MTT cytotoxicity assays and checkerboard combination assays were performed to study the reversal of drug resistance and to investigate the antiproliferative effects of the combination of anticancer drugs and the potential modulators. The results indicated that verapamil, capsanthin, zeaxanthin and promethazine inhibited P-gp effectively, but chrysin was not effective at reversing the resistance in MCF-7 sublines. Four selective anticancer drugs (paclitaxel, docetaxel, doxorubicin and vincristine) and 4 effective MDR modulators (verapamil, capsanthin, zeaxanthin and promethazine) were applied to the sublines in combination. RESULTS AND CONCLUSION: Fractional inhibitory indices show that verapamil and zeaxanthin seem to be the most effective MDR reversal agents that may be used together with paclitaxel, docetaxel, vincristine and doxorubicin in drug-resistant mammary carcinoma sublines. In conclusion, this report represents the importance to find out active and efficient drug resistance modulators for improving the efficacy of chemotherapy.

Review. Molecular background of chemoresistance in pancreatic cancer.

The survival data of patients with ductal pancreatic adenocarcinoma are rather poor, partly because the disease is frequently diagnosed at an advanced stage, partly because it is characterized by a chemoresistant phenotype. Even first-line chemotherapeutic drugs result in a modest objective response. This drug resistance is attributed to many different, unrelated mechanisms, including abnormal membrane receptor transport, ineffective metabolic drug conversion or enhanced metabolite inactivation, increased DNA repair and alterations in the apoptotic pathways. The role of NF-kappaB, cyclin D1 and stromal factors is also emphasized by many groups. The involvement of the ABC-transporters is not a universal feature, their alterations are important only in the resistance against specific cytostatics. Although several well-known molecular mechanisms have been elucidated, our understanding of drug insensitivity is still fragmentary, especially because recent microarray studies revealed that hundreds of genes are up- or down-regulated in resistant tumor cells, but their exact significance is still unclear. The reversal of the drug resistance is an area of intensive investigation, but to date, the compounds investigated are effective mainly in experimental systems and prospective studies are needed to validate their clinical applicability.

Development of rational in vitro models for drug resistance in breast cancer and modulation of MDR by selected compounds.

BACKGROUND: [corrected] The effectiveness of chemotherapy is limited by the emergence of multidrug resistance (MDR). MDR is caused by the activity of various ATP binding cassette (ABC) transporters that pump anticancer drugs out of the cells in an ATP-dependent manner. Additionally some other cellular mechanisms of MDR have been reported. The purpose of this study was to investigate mechanisms of MDR in drug resistant MCF-7 cell lines and to modulate P-glycoprotein (P-gp) and MRP1-based MDR. MATERIALS AND METHODS: Paclitaxel (MCF-7/Pac), docetaxel (MCF-7/Doc), doxorubicin (MCF-7/Dox) and vincristine (MCF-7/Vinc) resistant sublines were developed from the parent MCF-7 cell line (MCF-7/S) by stepwise selection in dose increments over two years. Flow cytometry, MTT cytotoxicity assay, RT-PCR, caspase-3 activity assay and checkerboard combination assay were performed to investigate the degree of resistance developed in sublines and to reverse drug resistance phenotype. RESULTS: The flow cytometry histograms of drug accumulation assays demonstrated that the drug-resistant cell lines are P-gp and MRPI positive. RT-PCR results showed that the resistant sublines express both MDR1 and MRP1 genes. Resistance indices of each subline to each anticancer drug were determined using the MTT cytotoxicity assay and it was found that all the sublines were resistant to their respective agents. Caspase-3 activities of the cell lines were also determined. Caspase-3 activity is an important indicator of apoptosis in the cell. The reversal of MDR was attempted by two cinnamylidene ketone and two organosilicon compounds. The results indicated that these compounds modulated P-gp effectively, but they were not very effective at reversing MRP1 activity in the MCF-7 sublines. Four selective anticancer drugs (paclitaxel, docetaxel, doxorubicin and vincristine) and four synthetic MDR modulators [2-(2-methoxycinnamylidene) indan-1-one (cinnamylidene-1), 2-(2- methoxycinnamylidene)-3, 4-dihydro-2H-naphthelen-1-one) (cinnamylidene-2), ALIS 409 and ALIS 421] were applied to the sublines in combination. The fractional inhibitory indices illustrated that combined applications of cinnamylidene ketones and organosilicon compounds with paclitaxel, docetaxel or vincristine exerted significant antiproliferative effects on the resistant sublines. CONCLUSION: This report demonstrates the development of rational models for drug resistance MCF-7 cell lines and reversal of acquired drug resistance.