Inhalable Textile Microplastic Fibers Impair Airway Epithelial Differentiation.

Rationale: Microplastics are a pressing global concern, and inhalation of microplastic fibers has been associated with interstitial and bronchial inflammation in flock workers. However, how microplastic fibers affect the lungs is unknown. Objectives: Our aim was to assess the effects of 12 × 31 µm nylon 6,6 (nylon) and 15 × 52 µm polyethylene terephthalate (polyester) textile microplastic fibers on lung epithelial growth and differentiation. Methods: We used human and murine alveolar and airway-type organoids as well as air-liquid interface cultures derived from primary lung epithelial progenitor cells and incubated these with either nylon or polyester fibers or nylon leachate. In addition, mice received one dose of nylon fibers or nylon leachate, and, 7 days later, organoid-forming capacity of isolated epithelial cells was investigated. Measurements and Main Results: We observed that nylon microfibers, more than polyester, inhibited developing airway organoids and not established ones. This effect was mediated by components leaching from nylon. Epithelial cells isolated from mice exposed to nylon fibers or leachate also formed fewer airway organoids, suggesting long-lasting effects of nylon components on epithelial cells. Part of these effects was recapitulated in human air-liquid interface cultures. Transcriptomic analysis revealed upregulation of Hoxa5 after exposure to nylon fibers. Inhibiting Hoxa5 during nylon exposure restored airway organoid formation, confirming Hoxa5's pivotal role in the effects of nylon. Conclusions: These results suggest that components leaching from nylon 6,6 may especially harm developing airways and/or airways undergoing repair, and we strongly encourage characterization in more detail of both the hazard of and the exposure to microplastic fibers.

Cigarette smoking prolongs inflammation associated with influenza infection and delays its clearance in mice.

Epidemiological studies have shown that smoking is associated with increased incidence of severe viral infections leading to hospitalization. Moreover, studies in experimental models have identified impaired antiviral responses and altered inflammatory responses, yet it is unclear how the effects of smoke exposure and influenza A infection interact and how this varies over the course of infection. We hypothesized that smoking would exacerbate innate immune responses against influenza. To test this, female BALB/c mice were exposed to cigarette smoke or air twice a day for 24-28 days and (mock) infected with H3N2 influenza A on day 21 while smoking continued. Three and seven days after infection, changes in immune cell populations, the transcriptome, and viral clearance in lung tissue were analyzed. After influenza A infection, smoke-exposed mice lost significantly more weight than air-exposed controls, indicating that smoking resulted in more severe disease. Immune cell and lung tissue transcriptome analysis revealed that neutrophil infiltration was prolonged and macrophage activation dysregulated after infection of smoke-exposed mice compared to air-exposed controls. Expression of genes in IL-6 and interferon pathways was similarly longer active. In parallel, we observed lower clearance of influenza virus in smoke-exposed mice after infection compared to air-exposed controls, indicating ineffective antiviral responses. Altogether, the data from our mouse model indicate that cigarette smoke exposure prolongs innate immune responses against influenza A. The results from this study help to explain the susceptibility of current smokers to severe influenza A disease.

Osteoprotegerin is an Early Marker of the Fibrotic Process and of Antifibrotic Treatment Responses in Ex Vivo Lung Fibrosis.

BACKGROUND: Lung fibrosis is a chronic lung disease with a high mortality rate with only two approved drugs (pirfenidone and nintedanib) to attenuate its progression. To date, there are no reliable biomarkers to assess fibrosis development and/or treatment effects for these two drugs. Osteoprotegerin (OPG) is used as a serum marker to diagnose liver fibrosis and we have previously shown it associates with lung fibrosis as well. METHODS: Here we used murine and human precision-cut lung slices to investigate the regulation of OPG in lung tissue to elucidate whether it tracks with (early) fibrosis development and responds to antifibrotic treatment to assess its potential use as a biomarker. RESULTS: OPG mRNA expression in murine lung slices was higher after treatment with profibrotic cytokines TGFß1 or IL13, and closely correlated with Fn and PAI1 mRNA expression. More OPG protein was released from fibrotic human lung slices than from the control human slices and from TGFß1 and IL13-stimulated murine lung slices compared to control murine slices. This OPG release was inhibited when murine slices were treated with pirfenidone or nintedanib. OPG release from human fibrotic lung slices was inhibited by pirfenidone treatment. CONCLUSION: OPG can already be detected during the early stages of fibrosis development and responds, both in early- and late-stage fibrosis, to treatment with antifibrotic drugs currently on the market for lung fibrosis. Therefore, OPG should be further investigated as a potential biomarker for lung fibrosis and a potential surrogate marker for treatment effect.

New insight into social relationships in dairy cows and how time of birth, parity, and relatedness affect spatial interactions later in life.

Social interactions between cows play a fundamental role in the daily activities of dairy cattle. Real-time location systems provide on a continuous and automated basis information about the position of individual cows inside barns, offering a valuable opportunity to monitor dyadic social contacts. Understanding dyadic social interactions could be applied to enhance the stability of the social structure promoting animal welfare and to model disease transmission in dairy cattle. This study aimed to identify the effect of different cow characteristics on the likelihood of the formation and persistence of social contacts in dairy cattle. The individual position of the lactating cows was automatically collected once per second for 2 wk, using an ultra-wideband system on a Swedish commercial farm consisting of almost 200 dairy cows inside a freestall barn. Social networks were constructed using the position data of 149 cows with available information on all characteristics during the study period. Social contacts were considered as a binary variable indicating whether a cow pair was within 2.5 m of each other for at least 10 min per day. The role of cow characteristics in social networks was studied by applying separable temporal exponential random graph models. Our results revealed that cows of the same parity interacted more consistently, as well as those born within 7 d of each other or closely related by pedigree. The repeatability of the topological parameters indicated a consistent short-term stability of the individual animal roles within the social network structure. Additional research is required to elucidate the underlying mechanisms governing the long-term evolution of social contacts among dairy cattle and to investigate the relationship between these networks and the transmission of diseases in the dairy cattle population.

Assessment of milk metabolites as biomarkers for predicting feed efficiency in dairy sheep.

Estimating feed efficiency (FE) in dairy sheep is challenging due to the high cost of systems that measure individual feed intake. Identifying proxies that can serve as effective predictors of FE could make it possible to introduce FE into breeding programs. Here, 39 Assaf ewes in first lactation were evaluated regarding their FE by 2 metrics, residual feed intake (RFI) and feed conversion ratio (FCR). The ewes were classified into high, medium and low groups for each metric. Milk samples of the 39 ewes were subjected to untargeted metabolomics analysis. The complete milk metabolomic signature was used to discriminate the FE groups using partial least squares discriminant analysis. A total of 41 and 26 features were selected as the most relevant features for the discrimination of RFI and FCR groups, respectively. The predictive ability when utilizing the complete milk metabolomic signature and the reduced data sets were investigated using 4 machine learning (ML) algorithms and a multivariate regression method. The orthogonal partial least squares algorithm outperformed other ML algorithms for FCR prediction in the scenarios using the complete milk metabolite signature (R2 = 0.62 ± 0.06) and the 26 selected features (R2 = 0.62 ± 0.15). Regarding RFI predictions, the scenarios using the 41 selected features outperformed the scenario with the complete milk metabolite signature, where the multilayer feedforward artificial neural network (R2 = 0.18 ± 0.14) and extreme gradient boosting (R2 = 0.17 ± 0.15) outperformed other algorithms. The functionality of the selected metabolites implied that the metabolism of glucose, galactose, fructose, sphingolipids, amino acids, insulin, and thyroid hormones was at play. Compared with the use of traditional methods, practical applications of these biomarkers might simplify and reduce costs in selecting feed-efficient ewes.

Unraveling Dynamic Transcriptomic Changes in Sheep's Lactating Mammary Gland Following Escherichia coli Lipopolysaccharide Exposure.

Mammary gland infections constitute a significant challenge in dairy sheep, impacting productivity and welfare. Temporal RNA-Seq provide a valuable approach to evaluate the evolution of the host defensive molecular mechanisms triggered by mastitis caused by external agents or events. This study aimed to characterize the transcriptomic response of sheep mammary glands to an intramammary inflammation induced with an Escherichia coli lipopolysaccharide (LPS) inoculation based on RNA-Seq samples generated from milk somatic cells collected at 3 time points: pre-inoculation (0 h), and 6 h and 24 h post-LPS inoculation. The differential expression analyses between the analyzed time points were performed using 2 statistical approaches: one parametric (DESeq2) and one non-parametric (Wilcoxon rank sum test). The differentially expressed genes (DEGs) commonly identified by both approaches encompass 5,872 for the 0 h versus 6 h comparison, 4,063 for the 0 h versus 24 h comparison, and 1,034 for the 6 h versus 24 h comparison. At both 6 h and 24 h, transcriptomic data highlighted a significant decrease in the expression of genes linked to metabolic processes crucial for milk protein and lipid synthesis within the mammary gland. Concurrently, increased expression of genes related to the neutrophil attraction was observed for 6 and 24 h, with differences in gene expression between DEGs with the highest expression at 6 h, related to T cell activation, type I interferon-mediated signaling pathway, and 24 h, related to cell-cell neutrophil adhesion extravasation or epithelial cell proliferation. In summary, this study reveals how the sheep mammary gland transcriptome responds dynamically to an LPS inoculation, providing a comprehensive understanding of how gene expression patterns evolve over time and shedding light on the molecular mechanisms driving the initial defensive response of the mammary gland against potential inflammatory challenges.

Epigenetic regulation of functional candidate genes for milk production traits in dairy sheep subjected to protein restriction in the prepubertal stage.

BACKGROUND: As the prepubertal stage is a crucial point for the proper development of the mammary gland and milk production, this study aims to evaluate how protein restriction at this stage can affect methylation marks in milk somatic cells. Here, 28 Assaf ewes were subjected to 42.3% nutritional protein restriction (14 animals, NPR) or fed standard diets (14 animals, C) during the prepubertal stage. During the second lactation, the milk somatic cells of these ewes were sampled, and the extracted DNA was subjected to whole-genome bisulfite sequencing. RESULTS: A total of 1154 differentially methylated regions (DMRs) were identified between the NPR and C groups. Indeed, the results of functional enrichment analyses of the genes harboring these DMRs suggested their relevant effects on the development of the mammary gland and lipid metabolism in sheep. The additional analysis of the correlations of the mean methylation levels within these DMRs with fat, protein, and dry extract percentages in the milk and milk somatic cell counts suggested associations between several DMRs and milk production traits. However, there were no phenotypic differences in these traits between the NPR and C groups. CONCLUSION: In light of the above, the results obtained in the current study might suggest potential candidate genes for the regulation of milk production traits in the sheep mammary gland. Further studies focusing on elucidating the genetic mechanisms affected by the identified DMRs may help to better understand the biological mechanisms modified in the mammary gland of dairy sheep as a response to nutritional challenges and their potential effects on milk production.

Temperature and exudativory as drivers of the marmoset (Callithrix spp.) daily activity period.

Primates are affected by fluctuations in ambient temperatures, mostly through thermoregulatory costs and changes in the availability of food. In the present study, we investigate whether the ambient temperature and proxies of food availability affect the activity period of marmosets (Callithrix spp.). We predicted that: (i) at colder sites, marmosets would spend more time at sleeping sites; (ii) midday resting bouts would be longer at hotter sites; (iii) the onset/cessation of activity and resting behavior at midday would be more closely related to temperature than food availability, and (iv) highly exudativorous groups would have higher total levels of resting. We compiled data on the onset and cessation of activity and the time spent resting at midday from seven marmoset studies from sites with a wide range of temperatures. We used generalized linear mixed models to verify the relationship between the dependent variables (lag between dawn and the onset of activities, lag between cessation of activities and dusk, and proportion of resting during midday) and the minimum and maximum temperatures at the respective study sites, together with proxies of food availability (exudativory rates, the amount of habitat available per individual, and net primary productivity) using each sample month as a sampling unit and the identity of the study as a categorical random factor. At colder sites and during colder months, the marmosets left sleeping trees later in the morning and ceased their activities earlier, while at hotter sites and during hotter months, they spent more time resting during midday. More exudativorous groups become active later in the morning, but also ceased their activities later. The abundance of food did not affect the timing of activities. We provide evidence that both low and high temperatures affect marmosets' activities, and that their activity period appears to be more influenced by the thermal environment than food availability.

Low-density SNP panel for efficient imputation and genomic selection of milk production and technological traits in dairy sheep.

The present study aimed to ascertain how different strategies for leveraging genomic information enhance the accuracy of estimated breeding values for milk and cheese-making traits and to evaluate the implementation of a low-density (LowD) SNP chip designed explicitly for that aim. Thus, milk samples from a total of 2,020 dairy ewes from 2 breeds (1,039 Spanish Assaf and 981 Churra) were collected and analyzed to determine 3 milk production and composition traits and 2 traits related to milk coagulation properties and cheese yield. The 2 studied populations were genotyped with a customized 50K Affymetrix SNP chip (Affymetrix Inc.) containing 55,627 SNP markers. The prediction accuracies were obtained using different multitrait methodologies, such as the BLUP model based on pedigree information, the genomic BLUP (GBLUP), and the BLUP at the SNP level (SNP-BLUP), which are based on genotypic data, and the single-step GBLUP (ssGBLUP), which combines both sources of information. All of these methods were analyzed by cross-validation, comparing predictions of the whole population with the test population sets. Additionally, we describe the design of a LowD SNP chip (3K) and its prediction accuracies through the different methods mentioned previously. Furthermore, the results obtained using the LowD SNP chip were compared with those based on the 50K SNP chip data sets. Finally, we conclude that implementing genomic selection through the ssGBLUP model in the current breeding programs would increase the accuracy of the estimated breeding values compared with the BLUP methodology in the Assaf (from 0.19 to 0.39) and Churra (from 0.27 to 0.44) dairy sheep populations. The LowD SNP chip is cost-effective and has proven to be an accurate tool for estimating genomic breeding values for milk and cheese-making traits, microsatellite imputation, and parentage verification. The results presented here suggest that the routine use of this LowD SNP chip could potentially increase the genetic gains of the breeding selection programs of the 2 Spanish dairy sheep breeds considered here.

Study on the concordance between different SNP-genotyping platforms in sheep.

Different SNP genotyping technologies are commonly used in multiple studies to perform QTL detection, genotype imputation, and genomic predictions. Therefore, genotyping errors cannot be ignored, as they can reduce the accuracy of different procedures applied in genomic selection, such as genomic imputation, genomic predictions, and false-positive results in genome-wide association studies. Currently, whole-genome resequencing (WGR) also offers the potential for variant calling analysis and high-throughput genotyping. WGR might overshadow array-based genotyping technologies due to the larger amount and precision of the genomic information provided; however, its comparatively higher price per individual still limits its use in larger populations. Thus, the objective of this work was to evaluate the accuracy of the two most popular SNP-chip technologies, namely, Affymetrix and Illumina, for high-throughput genotyping in sheep considering high-coverage WGR datasets as references. Analyses were performed using two reference sheep genome assemblies, the popular Oar_v3.1 reference genome and the latest available version Oar_rambouillet_v1.0. Our results demonstrate that the genotypes from both platforms are suggested to have high concordance rates with the genotypes determined from reference WGR datasets (96.59% and 99.51% for Affymetrix and Illumina technologies, respectively). The concordance results provided in the current study can pinpoint low reproducible markers across multiple platforms used for sheep genotyping data. Comparing results using two reference genome assemblies also informs how genome assembly quality can influence genotype concordance rates among different genotyping platforms. Moreover, we describe an efficient pipeline to test the reliability of markers included in sheep SNP-chip panels against WGR datasets available on public databases. This pipeline may be helpful for discarding low-reliability markers before exploiting genomic information for gene mapping analyses or genomic prediction.

Estimation of genetic parameters for cheese-making traits in Spanish Churra sheep.

The global production of sheep milk is growing, and the main industrial use of sheep milk is cheese making. The Spanish Churra sheep breed is one of the most important native dairy breeds in Spain. The present study aimed to estimate genetic parameters for a wide range of traits influencing the cheese-making ability of Churra sheep milk. Using a total of 1,049 Churra ewes, we studied the following cheese-making traits: 4 traits related to milk coagulation properties (rennet coagulation time, curd-firming time, and curd firmness at 30 and 60 min after addition of rennet), 2 traits related to cheese yield (individual laboratory cheese yield and individual laboratory dried curd yield), and 3 traits measuring curd firmness over time (maximum curd firmness, time to attain maximum curd firmness, and syneresis). In addition, a list of milk traits, including the native pH of the milk and several milk production and composition traits (milk yield; the fat, protein, and dried extract percentages; and the somatic cell count), were also analyzed for the studied animals. After discarding the noncoagulating samples (only 3.7%), data of 1,010 ewes were analyzed with multiple-trait animal models by using the restricted maximum likelihood method to estimate (co)variance components, heritabilities, and genetic correlations. In general, the heritability estimates were low to moderate, ranging from 0.08 (for the individual laboratory dried curd yield trait) to 0.42 (for the fat percentage trait). High genetic correlations were found within pairs of related traits (i.e., 0.93 between fat and dried extract percentages, -0.93 between the log of the curd-firming time and curd firmness at 30 min, 0.70 between individual laboratory cheese yield and individual laboratory dried curd yield, and -0.94 between time to attain maximum curd firmness and syneresis). Considering all the information provided here, we suggest that in addition to the current consideration of the protein percentage trait for improving cheese yield traits, the inclusion of the pH of milk as a measured trait in the Churra dairy breeding program would represent an efficient strategy for improving the cheese-making ability of milk from this breed.

Genome-wide association studies (GWAS) and post-GWAS analyses for technological traits in Assaf and Churra dairy breeds.

This study aimed to perform a GWAS to identify genomic regions associated with milk and cheese-making traits in Assaf and Churra dairy sheep breeds; second, it aimed to identify possible positional and functional candidate genes and their interactions through post-GWAS studies. For 2,020 dairy ewes from 2 breeds (1,039 Spanish Assaf and 981 Churra), milk samples were collected and analyzed to determine 6 milk production and composition traits and 6 traits related to milk coagulation properties and cheese yield. The genetic profiles of the ewes were obtained using a genotyping chip array that included 50,934 SNP markers. For both milk and cheese-making traits, separate single-breed GWAS were performed using GCTA software. The set of positional candidate genes identified via GWAS was subjected to guilt-by-association-based prioritization analysis with ToppGene software. Totals of 84 and 139 chromosome-wise significant associations for the 6 milk traits and the 6 cheese-making traits were identified in this study. No significant SNPs were found in common between the 2 studied breeds, possibly due to their genetic heterogeneity of the phenotypes under study. Additionally, 63 and 176 positional candidate genes were located in the genomic intervals defined as confidence regions in relation to the significant SNPs identified for the analyzed traits for Assaf and Churra breeds. After the functional prioritization analysis, 71 genes were identified as promising positional and functional candidate genes and proposed as targets of future research to identify putative causative variants in relation to the traits under examination. In addition, this multitrait study allowed us to identify variants that have a pleiotropic effect on both milk production and cheese-related traits. The incorporation of variants among the proposed functional and positional candidate genes into genomic selection strategies represent an interesting approach for achieving rapid genetic gains, specifically for those traits difficult to measure, such as cheese-making traits.

Effects of mining truck traffic on cricket calling activity.

Anthropogenic noise is a global pollutant and several studies have identified its impact on wildlife. This research shows how the noise produced by mining affects crickets' acoustic communication. Two passive acoustic monitoring devices (SMII) were installed in a forest fragment located at 500 m from the Brucutu Mine in Brazil. Another two SMII were installed distant 2500 from the mine. The equipment was configured to record from 17:00 to 05:00 h during seven days in April 2013. The authors analyzed the spectral characteristics of acoustic activity of three species of crickets (Anaxipha sp., Gryllus sp., and a Podoscirtinae species) before, during, and after the passing of mine trucks. For comparison the authors analyzed the acoustic characteristics for Anaxipha sp. and Gryllus sp. found in the distant site. Results showed a calling interruption for all the species during truck transit. Gryllus sp. emitted calls with higher maximum frequencies, average power, and larger bandwidth in the site close to the mine. Podoscirtinae species emitted calls with lower minimum frequencies, higher average power, and large bandwidth in the close site. The authors show that insect acoustic behavior varies between areas with different levels of noise. The disruption of this behavior may have negative consequences for their reproductive success.

Regional Differences in Human Intestinal Drug Metabolism.

The intestines are key for the absorption of nutrients and water as well as drug metabolism, and it is well known that there are clear differences in the expression profile of drug metabolism enzymes along the intestinal tract. Yet only a few studies have thoroughly investigated regional differences in human intestinal drug metabolism. In this study, we evaluated phase I and phase II metabolism in matched human ileum and colon precision-cut intestinal slices (PCIS). To this end, human PCIS were incubated for 3 hours with testosterone and 7-hydroxycoumarin (7-HC) to examine phase I and phase II metabolism, respectively. Metabolite formation was assessed by high-performance liquid chromatography analysis. Our results demonstrated that androstenedione, 6ß-hydroxytestosterone, 2ß-hydroxytestosterone, and 7-HC sulfate were predominantly formed in the ileum, while 15α-hydroxytestosterone and 7-HC glucuronide were mainly produced in the colon. Moreover, we also observed sex differences in phase II metabolite formation, which appeared to be higher in men compared with women. Taken together, we demonstrated that phase I metabolism predominantly occurs in ileum PCIS, while phase II metabolism mostly takes place in colon PCIS. Moreover, we revealed that human PCIS can be used to study both regional and sex differences in intestinal metabolism.

P-gp activity and inhibition in the different regions of human intestine ex vivo.

Although intestinal P-glycoprotein (P-gp) has been extensively studied in vitro and in animals, its activity and the consequences of P-gp inhibition for drug disposition and toxicity in humans are still difficult to accurately extrapolate from these studies. Moreover, existing in vitro models do not take into consideration that the intestine is heterogeneous with respect to P-gp expression. Recently, we reported rat precision-cut intestinal slices (PCIS) as a physiological ex vivo model to study the regional gradient of P-gp activity and inhibition. Here we extended the application of PCIS to the human intestine. For this purpose rhodamine 123 (R123) accumulation in the presence or absence of the P-gp inhibitors verapamil, cyclosporine A, quinidine, ketoconazole, PSC833 and CP100356 was measured in PCIS of human duodenum, jejunum, ileum and colon. R123 accumulation in the presence of the P-gp inhibitors appeared to be most enhanced in the ileum compared to the other regions. Moreover, the regional differences in accumulation are in line with published differences in abundance of P-gp. The rank order of the potency of the P-gp inhibitors, reflected by their IC50 , was comparable to that in rat PCIS. However, the increase in accumulation of the P-gp substrate R123 by the inhibitors was larger in human ileum PCIS than in rat PCIS, indicating species difference in P-gp abundance. These data show that human PCIS are an appropriate ex vivo model to study the activity of intestinal P-gp and predict the inhibitory effect of drugs and of transporter-mediated drug-drug interactions in the human intestine. Copyright © 2016 John Wiley & Sons, Ltd.

Chemical Composition, Antibacterial, Schistosomicidal, and Cytotoxic Activities of the Essential Oil of Dysphania ambrosioides (L.) Mosyakin & Clemants (Chenopodiaceae).

We have investigated the chemical composition and the antibacterial activity of the essential oil of Dysphania ambrosioides (L.) Mosyakin & Clemants (Chenopodiaceae) (DA-EO) against a representative panel of cariogenic bacteria. We have also assessed the in vitro schistosomicidal effects of DA-EO on Schistosoma mansoni and its cytotoxicity to GM07492-A cells in vitro. Gas chromatography (GC) and gas chromatography-mass spectrometry (GC/MS) revealed that the monoterpenes cis-piperitone oxide (35.2%), p-cymene (14.5%), isoascaridole (14.1%), and α-terpinene (11.6%) were identified by as the major constituents of DA-EO. DA-EO displayed weak activity against Streptococcus sobrinus and Enterococcus faecalis (minimum inhibitory concentration (MIC) = 1000 µg/ml). On the other hand, DA-EO at 25 and 12.5 µg/ml presented remarkable schistosomicidal action in vitro and killed 100% of adult worm pairs within 24 and 72 h, respectively. The LC50 values of DA-EO were 6.50 ± 0.38, 3.66 ± 1.06, and 3.65 ± 0.76 µg/ml at 24, 48, and 72 h, respectively. However, DA-EO at concentrations higher than 312.5 µg/ml significantly reduced the viability of GM07492-A cells (IC50  = 207.1 ± 4.4 µg/ml). The selectivity index showed that DA-EO was 31.8 times more toxic to the adult S. mansoni worms than GM07492-A cells. Taken together, these results demonstrate the promising schistosomicidal potential of the essential oil of Dysphania ambrosioides.

Replication of a genetic variant in ACYP2 associated with cisplatin-induced hearing loss in patients with osteosarcoma.

OBJECTIVE: Irreversible hearing loss is a frequent side effect of the chemotherapeutic agent cisplatin and shows considerable interpatient variability. The variant rs1872328 in the ACYP2 gene was recently identified as a risk factor for the development of cisplatin-induced ototoxicity in children with brain tumors. We aimed to replicate this finding in patients with osteosarcoma. METHODS: An independent cohort of 156 patients was genotyped for the rs1872328 variant and evaluated for the presence of cisplatin-induced ototoxicity. RESULTS: A significant association was observed between carriership of the A allele and cisplatin-induced ototoxicity after the end of treatment (P=0.027). CONCLUSION: This is the first study replicating the association of ACYP2 variant rs1872328 with cisplatin-induced ototoxicity in patients with osteosarcoma who did not receive potentially ototoxic cranial irradiation. Hence, the ACYP2 variant should be considered a predictive pharmacogenetic marker for hearing loss, which may be used to guide therapies for patients treated with cisplatin.

The Consequence of Drug-Drug Interactions Influencing the Interplay between P-Glycoprotein and Cytochrome P450 3a: An Ex Vivo Study with Rat Precision-Cut Intestinal Slices.

P-glycoprotein (P-gp) and cytochrome P450 3A (CYP3A) are differentially expressed along the intestine and work coordinately to reduce the intracellular concentration of xenobiotics and the absorption of orally taken drugs. Drug-drug interactions (DDIs) based on P-gp/CYP3A interplay are of clinical importance and require preclinical investigation. We investigated the P-gp/Cyp3a interplay and related DDIs with different P-gp inhibitors in the various regions of the rat intestine ex vivo using precision-cut intestinal slices (PCIS) with quinidine (Qi), a dual substrate of P-gp and Cyp3a, as the probe. The results showed that P-gp efflux was the main factor limiting the intracellular Qi content at concentrations below 5µM, whereas both efflux and metabolism were saturated at [Qi] > 50µM. The selective P-gp inhibitors CP100356 [N-(3,4-dimethoxyphenethyl)-4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2[1H]-yl)-6,7-dimethoxyquinazolin-2-amine] and PSC833 [valspodar, 6-[(2S,4R,6E)-4-methyl-2-(methylamino)-3-oxo-6-octenoic acid]-7-l-valine-cyclosporin A] enhanced the Qi accumulation in slices in line with the different P-gp expression in the intestinal regions and, as a result, also enhanced metabolism in the jejunum and ileum. Dual inhibitors of both P-gp and Cyp3a (verapamil and ketoconazole) increased the concentration of Qi in the jejunum and ileum, but less 3-hydroxy-quinidine was produced due to inhibition of Cyp3a. The results indicate that the P-gp/Cyp3a interplay depends on the concentration of the drug and on the intestinal region under study. Furthermore, due to the P-gp/Cyp3a interplay, DDIs can lead to remarkable changes in the intracellular concentration of both the parent drug and the metabolite, which varies among the intestinal regions and depends on the selectivity of the inhibitors, with potentially important implications for disposition and toxicity of drugs and their metabolites.

Caffeine-based gold(I) N-heterocyclic carbenes as possible anticancer agents: synthesis and biological properties.

A new series of gold(I) N-heterocyclic carbene (NHC) complexes based on xanthine ligands have been synthesized and characterized by mass spectrometry, NMR, and X-ray diffraction. The compounds have been tested for their antiproliferative properties in human cancer cells and nontumorigenic cells in vitro, as well as for their toxicity in healthy tissues ex vivo. The bis-carbene complex [Au(caffein-2-ylidene)2][BF4] (complex 4) appeared to be selective for human ovarian cancer cell lines and poorly toxic in healthy organs. To gain preliminary insights into their actual mechanism of action, two biologically relevant in cellulo targets were studied, namely, DNA (more precisely a higher-order DNA structure termed G-quadruplex DNA that plays key roles in oncogenetic regulation) and a pivotal enzyme of the DNA damage response (DDR) machinery (poly-(adenosine diphosphate (ADP)-ribose) polymerase 1 (PARP-1), strongly involved in the cancer resistance mechanism). Our results indicate that complex 4 acts as an efficient and selective G-quadruplex ligand while being a modest PARP-1 inhibitor (i.e., poor DDR impairing agent) and thus provide preliminary insights into the molecular mechanism that underlies its antiproliferative behavior.

Milk transcriptome biomarker identification to enhance feed efficiency and reduce nutritional costs in dairy ewes.

In recent years, rising prices for high-quality protein-based feeds have significantly increased nutrition costs. Consequently, investigating strategies to reduce these expenses and improve feed efficiency (FE) have become increasingly important for the dairy sheep industry. This research investigates the impact of nutritional protein restriction (NPR) during prepuberty and FE on the milk transcriptome of dairy Assaf ewes (sampled during the first lactation). To this end, we first compared transcriptomic differences between NPR and control ewes. Subsequently, we evaluated gene expression differences between ewes with divergent FE, using feed conversion ratio (FCR), residual feed intake (RFI), and consensus classifications of high- and low-FE animals for both indices. Lastly, we assess milk gene expression as a predictor of FE phenotype using random forest. No effect was found for the prepubertal NPR on milk performance or FE. Moreover, at the milk transcriptome level, only one gene, HBB, was differentially expressed between the NPR (n = 14) and the control group (n = 14). Further, the transcriptomic analysis between divergent FE sheep revealed 114 differentially expressed genes (DEGs) for RFI index (high-FERFI = 10 vs low-FERFI = 10), 244 for FCR (high-FEFCR = 10 vs low-FEFCR = 10), and 1 016 DEGs between divergent consensus ewes for both indices (high-FEconsensus = 8 vs low-FEconsensus = 8). These results underscore the critical role of selected FE indices for RNA-Seq analyses, revealing that consensus divergent animals for both indices maximise differences in transcriptomic responses. Genes overexpressed in high-FEconsensus ewes were associated with milk production and mammary gland development, while low-FEconsensus genes were linked to higher metabolic expenditure for tissue organisation and repair. The best prediction accuracy for FE phenotype using random forest was obtained for a set of 44 genes consistently differentially expressed across lactations, with Spearman correlations of 0.37 and 0.22 for FCR and RFI, respectively. These findings provide insights into potential sustainability strategies for dairy sheep, highlighting the utility of transcriptomic markers as FE proxies.