High-throughput assay for effect screening of amphotericin B and bioactive components on filamentous Candida albicans

publishedVersio

Semen quality parameters including metabolites, sperm production traits and fertility in young Norwegian Red AI bulls

Genomic selection in cattle breeding has gradually allowed younger bulls to be recruited for semen production. In this study, sperm quality parameters, seminal plasma and sperm metabolites, semen production capacity and fertility in young Norwegian Red bulls were analysed. For in vitro analyses of sperm quality and metabolites, ejaculates were collected from the same 25 bulls at both 14 and 17 months of age. Semen production and fertility data were collected for all Norwegian Red bulls in production from December 2017 throughout 2019. Bull fertility was measured as 56 days non-return rate (NR56), for both age groups.acceptedVersionpublishedVersio

Semen quality parameters including metabolites, sperm production traits and fertility in young Norwegian Red AI bulls

Genomic selection in cattle breeding has gradually allowed younger bulls to be recruited for semen production. In this study, sperm quality parameters, seminal plasma and sperm metabolites, semen production capacity and fertility in young Norwegian Red bulls were analysed. For in vitro analyses of sperm quality and metabolites, ejaculates were collected from the same 25 bulls at both 14 and 17 months of age. Semen production and fertility data were collected for all Norwegian Red bulls in production from December 2017 throughout 2019. Bull fertility was measured as 56 days non-return rate (NR56), for both age groups. In both fresh and frozen-thawed semen samples, the proportion of hyperactive spermatozoa, average path velocity, curvilinear velocity and amplitude of lateral head displacement were higher in samples collected at 17 months of age compared to 14 months (PSemen quality parameters including metabolites, sperm production traits and fertility in young Norwegian Red AI bullsacceptedVersio

Sperm quality parameters, fertilizing potential, metabolites, and DNA methylation in cold-stored and cryopreserved milt from Atlantic salmon (Salmo salar L.)

Cold storage and freezing/thawing of milt may affect sperm functionality and the subsequent fertilization ability of milt. This study aimed to investigate sperm quality parameters and fertilization potential of Atlantic salmon milt, stored cold and subsequently cryopreserved, using different storage conditions. The objective was also to assess if analysis of milt metabolites and sperm DNA methylation signatures could be applicable to further elucidate sperm quality and fertilization following preservation. Milt samples were collected from eight mature Atlantic salmon males and stored for 4 days at 2°C and 8°C. Samples were taken on day one of storage at 2°C and on day four of storage at 2°C and 8°C. Storage for 4 days at 8°C is expected to be detrimental to sperm quality, and was included to create contrasts. Correspondingly, aliquots of cold-stored milt were prepared for cryopreservation, resulting in a total of six experimental conditions. Samples from all six experimental conditions were used in fertilization trials and analyzed for sperm viability, motility, ATP content, DNA fragmentation index, and High DNA stainability. In addition, milt samples from four of the males were analyzed for targeted metabolites and DNA methylation signatures by reduced representation bisulfite sequencing. The fertilization trials were performed using sperm:egg ratios of 75 × 103 and 500 × 103, respectively. Storage duration, temperature, and cryopreservation of cold-stored milt influenced several sperm quality parameters, metabolites, and DNA methylation signatures. The total motility, progressive motility, ATP, and velocity parameters were the sperm parameters with the strongest correlation to fertilization rates (p < 0.01). Several metabolites were correlated with fertility rates in both cold-stored and cryopreserved samples (p < 0.05). The fertilizing capacity of cold-stored milt was significantly reduced after 4 days of storage at 8°C, while corresponding cryopreserved milt showed reduced fertilization at both storage temperatures (2°C and 8°C) (p < 0.05). The results indicate that cryopreservation of milt stored for 1 day does not compromise either fertilization ability or DNA methylation signatures.publishedVersio

Synergistic effects of complex drug combinations in colorectal cancer cells predicted by logical modelling

Drug combinations have been proposed to combat drug resistance in cancer, but due to the large number of possible drug targets, in vitro testing of all possible combinations of drugs is challenging. Computational models of a disease hold great promise as tools for prediction of response to treatment, and here we constructed a logical model integrating signaling pathways frequently dysregulated in cancer, as well as pathways activated upon DNA damage, to study the effect of clinically relevant drug combinations. By fitting the model to a dataset of pairwise combinations of drugs targeting MEK, PI3K, and TAK1, as well as several clinically approved agents (palbociclib, olaparib, oxaliplatin, and 5FU), we were able to perform model simulations that allowed us to predict more complex drug combinations, encompassing sets of three and four drugs, with potentially stronger effects compared to pairwise drug combinations. All predicted third-order synergies, as well as a subset of non-synergies, were successfully confirmed by in vitro experiments in the colorectal cancer cell line HCT-116, highlighting the strength of using computational strategies to rationalize drug testing

Identification of novel cyanoacrylate monomers for use in nanoparticle drug delivery systems prepared by miniemulsion polymerisation – A multistep screening approach

Poly (alkyl cyanoacrylate) (PACA) polymeric nanoparticles (NPs) are promising drug carriers in drug delivery. However, the selection of commercially available alkyl cyanoacrylate (ACA) monomers is limited, because most monomers were designed for use in medical and industrial glues and later repurposed for drug encapsulation. This study therefore aimed to seek out novel ACA materials for use in NP systems using a toxicity led screening approach. A multistep strategy, including cytotoxicity screening of alcohols as degradation products of PACA (44 alcohols), NPs (14 polymers), and a final in vivo study (2 polymers) gave poly (2-ethylhexyl cyanoacrylate) PEHCA as a promising novel PACA candidate. For the first time, this work presents cytotoxicity data on several novel ACAs, PEHCA in vivo toxicity data, and miniemulsion polymerisation-based encapsulation of the cabazitaxel and NR688 in novel PACA candidates. Furthermore, several of the ACA candidates were compatible with a wider selection of lipophilic active pharmaceutical ingredients (APIs) versus commercially available controls. Combined, this work demonstrates the potential benefits of expanding the array of available ACA materials in drug delivery. Novel ACAs have the potential to encapsulate a wider range of APIs in miniemulsion polymerisation processes and may also broaden PACA applicability in other fields.publishedVersio

Use of immobilized cryopreserved bovine semen in a blind artificial insemination trial

AbstractTo make timing of artificial insemination (AI) relative to ovulation less critical, methods for prolonging shelf life of spermatozoa in vivo after AI have been attempted to be developed. Encapsulation of sperm cells is a documented technology, and recently, a technology in which sperm cells are embedded in alginate gel has been introduced and commercialized. In this study, standard processed semen with the Biladyl extender (control) was compared with semen processed by sperm immobilization technology developed by SpermVital AS in a blind field trial. Moreover, in vitro acrosome and plasma membrane integrity was assessed and compared with AI fertility data for possible correlation. Semen from 16 Norwegian Red young bulls with unknown fertility was collected and processed after splitting the semen in two aliquots. These aliquots were processed with the standard Biladyl extender or the SpermVital extender to a final number of 12 × 106 and 25 × 106 spermatozoa/dose, respectively. In total, 2000 semen doses were produced from each bull, divided equally by treatment. Artificial insemination doses were set up to design a blinded AI regime; 5 + 5 straws from each extender within ejaculates in ten-straw goblets were distributed to AI technicians and veterinarians all over Norway. Outcomes of the inseminations were measured as 56-day nonreturn rate (NRR). Postthaw sperm quality was assessed by flow cytometry using propidium iodide and Alexa 488–conjugated peanut agglutinin to assess the proportion of plasma membrane and acrosome-intact sperm cells, respectively. In total, data from 14,125 first inseminations performed over a 12-month period, 7081 with Biladyl and 7044 with SpermVital semen, were used in the statistical analyses. There was no significant difference in 56-day NRR for the two semen categories, overall NRR being 72.5% and 72.7% for Biladyl and SpermVital, respectively. The flow cytometric results revealed a significant higher level of acrosome-intact live spermatozoa in Biladyl-processed semen compared to SpermVital semen. The results indicate that the level of acrosome-intact live spermatozoa in the AI dose did not affect the 56-day NRR for the two semen processing methods. In conclusion, this study has showed that immobilized spermatozoa provide equal fertility results as standard processed semen when AI is performed in a blinded field trial, although the immobilization procedure caused increased sperm damage evaluated in vitro compared to standard semen processing procedure

Streptomyces tardus sp. nov.: A Slow-Growing Actinobacterium Producing Candicidin, Isolated From Sediments of the Trondheim Fjord

Marine environments are home to an extensive number of microorganisms, many of which remain unexplored for taxonomic novelty and functional capabilities. In this study, a slow-growing Streptomyces strain expressing unique genomic and phenotypic characteristics, P38-E01T , was described using a polyphasic taxonomic approach. This strain is part of a collection of over 8,000 marine Actinobacteria isolates collected in the Trondheim fjord of Norway by SINTEF Industry (Trondheim, Norway) and the Norwegian University of Science and Technology (NTNU, Trondheim, Norway). Strain P38-E01T was isolated from the sediments of the Trondheim fjord, and phylogenetic analyses affiliated this strain with the genus Streptomyces, but it was not closely affiliated with other described species. The closest related type strains were Streptomyces daliensis YIM 31724T (98.6%), Streptomyces rimosus subsp. rimosus ATCC 10970T (98.4%), and Streptomyces sclerotialus NRRL ISP-5269T (98.3%). Predominant fatty acids were C16V0 iso, C16V0, and Summed Feature 3, and the predominant respiratory quinones were MK-10(H6), MK-10(H4), and MK9(H4). The main polar lipids were identified as diphosphatidylglycerol, phosphatidylethanolamine, hosphatidylglycerol, and phosphoglycolipid. The whole-cell sugars were glucose, ribose, and in minor amounts, mannose. The cell wall peptidoglycan contained LL-diaminopimelic acid. The draft genome has a size of 6.16 Mb, with a %G C C content of 71.4% and is predicted to contain at least 19 biosynthetic gene clusters encoding diverse secondary metabolites. Strain P38-E01T was found to inhibit the growth of the pathogenic yeast Candida albicans ATCC 90028 and a number of Gram-positive bacterial human and plant pathogens. Metabolites extracted from cultures of P38-E01T were analyzed by mass spectrometry, and it was found that the isolate produced the antifungal compound candicidin. Phenotypic and chemotaxonomic signatures, along with phylogenetic analyses, distinguished isolate P38-E01T from its closest neighbors; thus, this isolate represents a novel species of the genus Streptomyces for which the name Streptomyces tardus sp. nov. (P38-E01T D CCM 9049T D DSM 111582T ) is proposed.publishedVersio

Interlaboratory evaluation of a digital holographic microscopy–based assay for label-free in vitro cytotoxicity testing of polymeric nanocarriers

State-of-the-art in vitro test systems for nanomaterial toxicity assessment are based on dyes and several staining steps which can be affected by nanomaterial interference. Digital holographic microscopy (DHM), an interferometry-based variant of quantitative phase imaging (QPI), facilitates reliable proliferation quantification of native cell populations and the extraction of morphological features in a fast and label- and interference-free manner by biophysical parameters. DHM therefore has been identified as versatile tool for cytotoxicity testing in biomedical nanotechnology. In a comparative study performed at two collaborating laboratories, we investigated the interlaboratory variability and performance of DHM in nanomaterial toxicity testing, utilizing complementary standard operating procedures (SOPs). Two identical custom-built off-axis DHM systems, developed for usage in biomedical laboratories, equipped with stage-top incubation chambers were applied at different locations in Europe. Temporal dry mass development, 12-h dry mass increments and morphology changes of A549 human lung epithelial cell populations upon incubation with two variants of poly(alkyl cyanoacrylate) (PACA) nanoparticles were observed in comparison to digitonin and cell culture medium controls. Digitonin as cytotoxicity control, as well as empty and cabazitaxel-loaded PACA nanocarriers, similarly impacted 12-h dry mass development and increments as well as morphology of A549 cells at both participating laboratories. The obtained DHM data reflected the cytotoxic potential of the tested nanomaterials and are in agreement with corresponding literature on biophysical and chemical assays. Our results confirm DHM as label-free cytotoxicity assay for polymeric nanocarriers as well as the repeatability and reproducibility of the technology. In summary, the evaluated DHM assay could be efficiently implemented at different locations and facilitates interlaboratory in vitro toxicity testing of nanoparticles with prospects for application in regulatory science.publishedVersio

Biosynthesis of Macrolactam BE-14106 Involves Two Distinct PKS Systems and Amino Acid Processing Enzymes for Generation of the Aminoacyl Starter Unit

SummaryBE-14106 is a macrocyclic lactam with an acyl side chain previously identified in a marine-derived Streptomyces sp. The gene cluster for BE-14106 biosynthesis was cloned from a Streptomyces strain newly isolated from marine sediments collected in the Trondheimsfjord (Norway). Bioinformatics and experimental analyses of the genes in the cluster suggested an unusual mechanism for assembly of the molecule. Biosynthesis of the aminoacyl starter apparently involves the concerted action of a distinct polyketide synthase (PKS) system and several enzymes that activate and process an amino acid. The resulting starter unit is loaded onto a second PKS complex, which completes the synthesis of the macrolactam ring. Gene inactivation experiments, enzyme assays with heterologously expressed proteins, and feeding studies supported the proposed model for the biosynthesis and provided new insights into the assembly of macrolactams with acyl side chain