Fine-Tuning of DADA2 Parameters for Multiregional Metabarcoding Analysis of 16S rRNA Genes from Activated Sludge and Comparison of Taxonomy Classification Power and Taxonomy Databases.

Taxonomic classification using metabarcoding is a commonly used method in microbiological studies of environmental samples and during monitoring of biotechnological processes. However, it is difficult to compare results from different laboratories, due to the variety of bioinformatics tools that have been developed and used for data analysis. This problem is compounded by different choices regarding which variable region of the 16S rRNA gene and which database is used for taxonomic identification. Therefore, this study employed the DADA2 algorithm to optimize the preprocessing of raw data obtained from the sequencing of activated sludge samples, using simultaneous analysis of three frequently used regions of 16S rRNA (V1-V3, V3-V4, V4-V5). Additionally, the study evaluated which variable region and which of the frequently used microbial databases for taxonomic classification (Greengenes2, Silva, RefSeq) more accurately classify OTUs into taxa. Adjusting the values of selected parameters of the DADA2 algorithm, we obtained the highest possible numbers of OTUs for each region. Regarding biodiversity within regions, the V3-V4 region had the highest Simpson and Shannon indexes, and the Chao1 index was similar to that of the V1-V3 region. Beta-biodiversity analysis revealed statistically significant differences between regions. When comparing databases for each of the regions studied, the highest numbers of taxonomic groups were obtained using the SILVA database. These results suggest that standardization of metabarcoding of short amplicons may be possible.

Paternal-effect-genes revealed through sperm cryopreservation in Perca fluviatilis.

Knowledge about paternal-effect-genes (PEGs) (genes whose expression in the progeny is influenced by paternal factors present in the sperm) in fish is very limited. To explore this issue, we used milt cryopreservation as a specific challenge test for sperm cells, thus enabling selection amidst cryo-sensitivity. We created two groups of Eurasian perch (Perca fluviatilis) as a model - eggs fertilized either with fresh (Fresh group) or cryopreserved (Cryo group) milt from the same male followed by phenotypic-transcriptomic examination of consequences of cryopreservation in obtained progeny (at larval stages). Most of the phenotypical observations were similar in both groups, except the final weight which was higher in the Cryo group. Milt cryopreservation appeared to act as a "positive selection" factor, upregulating most PEGs in the Cryo group. Transcriptomic profile of freshly hatched larvae sourced genes involved in the development of visual perception and we identified them as PEGs. Consequently, larvae from the Cryo group exhibited enhanced eyesight, potentially contributing to more efficient foraging and weight gain compared to the Fresh group. This study unveils, for the first time, the significant influence of the paternal genome on the development of the visual system in fish, highlighting pde6g, opn1lw1, and rbp4l as novel PEGs.

Comparative transcriptomics of two Salvia subg. Perovskia species contribute towards molecular background of abietane-type diterpenoid biosynthesis.

Tanshinones, are a group of diterpenoid red pigments present in Danshen - an important herbal drug of Traditional Chinese Medicine which is a dried root of Salvia miltiorrhiza Bunge. Some of the tanshinones are sought after as pharmacologically active natural products. To date, the biosynthetic pathway of tanshinones has been only partially elucidated. These compounds are also present in some of the other Salvia species, i.a. from subgenus Perovskia, such as S. abrotanoides (Kar.) Sytsma and S. yangii B.T. Drew. Despite of the close genetic relationship between these species, significant qualitative differences in their diterpenoid profile have been discovered. In this work, we have used the Liquid Chromatography-Mass Spectrometry analysis to follow the content of diterpenoids during the vegetation season, which confirmed our previous observations of a diverse diterpenoid profile. As metabolic differences are reflected in different transcript profile of a species or tissues, we used metabolomics-guided transcriptomic approach to select candidate genes, which expression possibly led to observed chemical differences. Using an RNA-sequencing technology we have sequenced and de novo assembled transcriptomes of leaves and roots of S. abrotanoides and S. yangii. As a result, 134,443 transcripts were annotated by UniProt and 56,693 of them were assigned as Viridiplantae. In order to seek for differences, the differential expression analysis was performed, which revealed that 463, 362, 922 and 835 genes indicated changes in expression in four comparisons. GO enrichment analysis and KEGG functional analysis of selected DEGs were performed. The homology and expression of two gene families, associated with downstream steps of tanshinone and carnosic acid biosynthesis were studied, namely: cytochromes P-450 and 2-oxoglutarate-dependend dioxygenases. Additionally, BLAST analysis revealed existence of 39 different transcripts related to abietane diterpenoid biosynthesis in transcriptomes of S. abrotanoides and S. yangii. We have used quantitative real-time RT-PCR analysis of selected candidate genes, to follow their expression levels over the vegetative season. A hypothesis of an existence of a multifunctional CYP76AH89 in transcriptomes of S. abrotanoides and S. yangii is discussed and potential roles of other CYP450 homologs are speculated. By using the comparative transcriptomic approach, we have generated a dataset of candidate genes which provides a valuable resource for further elucidation of tanshinone biosynthesis. In a long run, our investigation may lead to optimization of diterpenoid profile in S. abrotanoides and S. yangii, which may become an alternative source of tanshinones for further research on their bioactivity and pharmacological therapy.

Complete chloroplast genomes of Cerastium alpinum, C. arcticum and C. nigrescens: genome structures, comparative and phylogenetic analysis.

The genus Cerastium includes about 200 species that are mostly found in the temperate climates of the Northern Hemisphere. Here we report the complete chloroplast genomes of Cerastium alpinum, C. arcticum and C. nigrescens. The length of cp genomes ranged from 147,940 to 148,722 bp. Their quadripartite circular structure had the same gene organization and content, containing 79 protein-coding genes, 30 tRNA genes, and four rRNA genes. Repeat sequences varied from 16 to 23 per species, with palindromic repeats being the most frequent. The number of identified SSRs ranged from 20 to 23 per species and they were mainly composed of mononucleotide repeats containing A/T units. Based on Ka/Ks ratio values, most genes were subjected to purifying selection. The newly sequenced chloroplast genomes were characterized by a high frequency of RNA editing, including both C to U and U to C conversion. The phylogenetic relationships within the genus Cerastium and family Caryophyllaceae were reconstructed based on the sequences of 71 protein-coding genes. The topology of the phylogenetic tree was consistent with the systematic position of the studied species. All representatives of the genus Cerastium were gathered in a single clade with C. glomeratum sharing the least similarity with the others.

miRNA Expression Signatures Induced by Chicken Astrovirus Infection in Chickens.

miRNAs represent ubiquitous regulators of gene expression and play an important and pivotal regulatory role in viral disease pathogenesis and virus-host interactions. Although previous studies have provided basic data for understanding the role of miRNAs in the molecular mechanisms of viral infection in birds, the role of miRNAs in the regulation of host responses to chicken astrovirus (CAstV) infection in chickens is not yet understood. In our study, we applied next-generation sequencing to profile miRNA expression in CAstV-infected chickens and to decipher miRNA-targeted specific signaling pathways engaged in potentially vital virus-infection biological processes. Among the 1354 detected miRNAs, we identified 58 mature miRNAs that were significantly differentially expressed in infected birds. Target prediction resulted in 4741 target genes. GO and KEGG pathway enrichment analyses showed that the target genes were mainly involved in the regulation of cellular processes and immune responses.

IncRna: The R Package for Optimizing lncRNA Identification Processes.

In silico identification of long noncoding RNAs (lncRNAs) is a multistage process including filtering of transcripts according to their physical characteristics (e.g., length, exon-intron structure) and determination of the coding potential of the sequence. A common issue within this process is the choice of the most suitable method of coding potential analysis for the conducted research. Selection of tools on the sole basis of their single performance may not provide the most effective choice for a specific problem. To overcome these limitations, we developed the R library lncRna, which provides functions to easily carry out the entire lncRNA identification process. For example, the package prepares the data files for coding potential analysis to perform error analysis. Moreover, the package gives the opportunity to analyze the effectiveness of various combinations of the lncRNA prediction methods to select the optimal configuration of the entire process.

Playing Peekaboo with a Master Manipulator: Metagenetic Detection and Phylogenetic Analysis of Wolbachia Supergroups in Freshwater Invertebrates.

The infamous "master manipulators"-intracellular bacteria of the genus Wolbachia-infect a broad range of phylogenetically diverse invertebrate hosts in terrestrial ecosystems. Wolbachia has an important impact on the ecology and evolution of their host with documented effects including induced parthenogenesis, male killing, feminization, and cytoplasmic incompatibility. Nonetheless, data on Wolbachia infections in non-terrestrial invertebrates are scarce. Sampling bias and methodological limitations are some of the reasons limiting the detection of these bacteria in aquatic organisms. In this study, we present a new metagenetic method for detecting the co-occurrence of different Wolbachia strains in freshwater invertebrates host species, i.e., freshwater Arthropoda (Crustacea), Mollusca (Bivalvia), and water bears (Tardigrada) by applying NGS primers designed by us and a Python script that allows the identification of Wolbachia target sequences from the microbiome communities. We also compare the results obtained using the commonly applied NGS primers and the Sanger sequencing approach. Finally, we describe three supergroups of Wolbachia: (i) a new supergroup V identified in Crustacea and Bivalvia hosts; (ii) supergroup A identified in Crustacea, Bivalvia, and Eutardigrada hosts, and (iii) supergroup E infection in the Crustacea host microbiome community.

A method for reducing the concentrations of Fusarium graminearum trichothecenes in durum wheat grain with the use of Debaryomyces hansenii.

Fusarium head blight (FHB), caused mainly by Fusarium graminearum, is one of the most dangerous diseases of durum wheat. This hemibiotrophic pathogen transitions from the biotrophic phase, during which it penetrates host tissues and secretes trichothecenes, to the necrotrophic phase which leads to the destruction of host tissues. Yeasts applied to spikes often reduce mycotoxin concentrations, but the underlying mechanisms have not been fully elucidated. Therefore, the aim of this study was to analyze the concentrations trichothecenes in durum wheat grain and changes in the F. graminearum transcriptome under the influence the Debaryomyces hansenii antagonistic yeast strain. Debaryomyces hansenii cells adhered to and formed cell aggregates/biofilm on the surface of spikes and pathogenic hyphae. Biological control suppressed the spread of F. graminearum by 90 % and decreased the content of deoxynivalenol (DON) in spikes by 31.2 %. Yeasts significantly reduced the expression of pathogen's genes encoding the rpaI subunit of RNA polymerase I and the activator of Hsp90 ATPase, but they had no effect on mRNA transcript levels of genes encoding the enzymes involved in the biosynthesis of trichothecenes. The yeast treatment reduced the number of F. graminearum operational taxonomic units (OTUs) nearly five-fold and increased the number of D. hansenii OTUs more than six-fold in the spike mycobiome. The mechanisms that suppress infections should be explored to develop effective biological methods for reducing the concentrations mycotoxins in wheat grain.

Assessment of consistency in aortic valve sizing between surgical and transcatheter aortic valve replacement.

Introduction: One of the crucial aspects of transcatheter aortic valve replacement (TAVR) and surgical aortic valve replacement (SAVR) is the valve prosthesis selection. Aim: To assess the consistency of the aortic valve sizing in SAVR and TAVR by comparing the sizes of aortic prostheses selected based on the intraprocedural annulus measurements and simulation of the TAVR planning. Material and methods: The study comprised of 167 patients with aortic stenosis treated with SAVR. Simulation of the prosthesis sizing blinded to the SAVR results was performed based on the assessment of cardiac computed tomography (CCT) images. Results: Based on the CCT images, the average value of the aortic annulus diameter was 25.4 ±3.0 mm. Aortic valve calcifications were mild in 29 cases, moderate in 78 cases, and severe in 53 cases. The sizes of the valves recommended by the simulations were larger than valves surgically implanted in 98.6% of patients for self-expanding and in 91.7% of patients for balloon-expandable prostheses. The average difference for self-expanding prostheses was 6.4 mm and 4.5 mm for balloon expandable valves. Additionally, a negative correlation was observed for the difference in prosthesis size and size of the valve used by surgeons. Conclusions: There is a systematic difference between sizes of aortic prostheses used in SAVR and TAVR. Further studies are needed to evaluate if the difference in prosthesis size selection contributes to the frequency of prosthesis-patient mismatch phenomenon and burden of high postoperative mean transaortic gradient.

Molecular structure, comparative and phylogenetic analysis of the complete chloroplast genome sequences of weedy rye Secale cereale ssp. segetale.

The complete chloroplast genome of Secale cereale ssp. segetale (Zhuk.) Roshev. (Poaceae: Triticeae) was sequenced and analyzed to better use its genetic resources to enrich rye and wheat breeding. The study was carried out using the following methods: DNA extraction, sequencing, assembly and annotation, comparison with other complete chloroplast genomes of the five Secale species, and multigene phylogeny. As a result of the study, it was determined that the chloroplast genome is 137,042 base pair (bp) long and contains 137 genes, including 113 unique genes and 24 genes which are duplicated in the IRs. Moreover, a total of 29 SSRs were detected in the Secale cereale ssp. segetale chloroplast genome. The phylogenetic analysis showed that Secale cereale ssp. segetale appeared to share the highest degree of similarity with S. cereale and S. strictum. Intraspecific diversity has been observed between the published chloroplast genome sequences of S. cereale ssp. segetale. The genome can be accessed on GenBank with the accession number (OL688773).

Adaptation of the Porcine Pituitary Transcriptome, Spliceosome and Editome during Early Pregnancy.

The physiological mechanisms of the porcine reproduction are relatively well-known. However, transcriptomic changes and the mechanisms accompanying transcription and translation processes in various reproductive organs, as well as their dependence on hormonal status, are still poorly understood. The aim of this study was to gain a principal understanding of alterations within the transcriptome, spliceosome and editome occurring in the pituitary of the domestic pig (Sus scrofa domestica L.), which controls basic physiological processes in the reproductive system. In this investigation, we performed extensive analyses of data obtained by high-throughput sequencing of RNA from the gilts' pituitary anterior lobes during embryo implantation and the mid-luteal phase of the estrous cycle. During analyses, we obtained detailed information on expression changes of 147 genes and 43 long noncoding RNAs, observed 784 alternative splicing events and also found the occurrence of 8729 allele-specific expression sites and 122 RNA editing events. The expression profiles of the selected 16 phenomena were confirmed by PCR or qPCR techniques. As a final result of functional meta-analysis, we acquired knowledge regarding intracellular pathways that induce changes in the processes accompanying transcription and translation regulation, which may induce modifications in the secretory activity of the porcine adenohypophyseal cells.

Specific expression of alternatively spliced genes in the turkey (Meleagris gallopavo) reproductive tract revealed their function in spermatogenesis and post-testicular sperm maturation.

The tissue-specific profile of alternatively spliced genes (ASGs) and their involvement in reproduction processes characteristic of turkey testis, epididymis, and ductus deferens were investigated for the first time in birds. Deep sequencing of male turkey reproductive tissue RNA samples (n = 6) was performed using Illumina RNA-Seq with 2 independent methods, rMATs and SUPPA2, for differential alternative splicing (DAS) event prediction. The expression of selected ASGs was validated using quantitative real-time reverse transcriptase-polymerase chain reaction. The testis was found to be the site of the highest number of posttranscriptional splicing events within the reproductive tract, and skipping exons were the most frequently occurring class of alternative splicing (AS) among the reproductive tract. Statistical analysis revealed 86, 229, and 6 DAS events in the testis/epididymis, testis/ductus deferens, and epididymis/ductus deferens comparison, respectively. Alternative splicing was found to be a mechanism of gene expression regulation within the turkey reproduction tract. In testis, modification was observed for spermatogenesis specific genes; the changes in 5' UTR could act as regulator of MEIG1 expression (a player during spermatocytes meiosis), and modification of 3' UTR led to diversification of CREM mRNA (modulator of gene expression related to the structuring of mature spermatozoa). Sperm tail formation can be regulated by changes in the 5' UTR of testicular SLC9A3R1 and gene silencing by producing dysfunctional variants of ODF2 in the testis and ATP1B3 in the epididymis. Predicted differentially ASGs in the turkey reproductive tract seem to be involved in the regulation of spermatogenesis, including acrosome formation and sperm tail formation and binding of sperm to the zona pellucida. Several ASGs were classified as cilia by actin and microtubule cytoskeleton organization. Such genes may play a role in the organization of sperm flagellum and post-testicular motility development. To our knowledge, this is the first functional investigation of alternatively spliced genes associated with tissue-specific processes in the turkey reproductive tract.

Pulmonary artery embolism: comprehensive transcriptomic analysis in understanding the pathogenic mechanisms of the disease.

BACKGROUND: Pulmonary embolism (PE) is a severe disease that usually originates from deep vein thrombosis (DVT) of the lower extremities. This study set out to investigate the changes in the transcriptome of the pulmonary artery (PA) in the course of the PE in the porcine model. METHODS: The study was performed on 11 male pigs: a thrombus was formed in each right femoral vein in six animals, and then was released to induce PE, the remaining five animals served as a control group. In the experimental animals total RNA was isolated from the PA where the blood clot lodged, and in the control group, from the corresponding PA segments. High-throughput RNA sequencing was used to analyse the global changes in the transcriptome of PA with induced PE (PA-E). RESULTS: Applied multistep bioinformatics revealed 473 differentially expressed genes (DEGs): 198 upregulated and 275 downregulated. Functional Gene Ontology annotated 347 DEGs into 27 biological processes, 324 to the 11 cellular components and 346 to the 2 molecular functions categories. In the signaling pathway analysis, KEGG 'protein processing in endoplasmic reticulum' was identified for the mRNAs modulated during PE. The same KEGG pathway was also exposed by 8 differentially alternative splicing genes. Within single nucleotide variants, the 61 allele-specific expression variants were localised in the vicinity of the genes that belong to the cellular components of the 'endoplasmic reticulum'. The discovered allele-specific genes were also classified as signatures of the cardiovascular system. CONCLUSIONS: The findings of this research provide the first thorough investigation of the changes in the gene expression profile of PA affected by an embolus. Evidence from this study suggests that the disturbed homeostasis in the biosynthesis of proteins in the endoplasmic reticulum plays a major role in the pathogenesis of PE.

Graph Mapping: A novel and simple test to validly assess fluid reasoning.

We present Graph Mapping - a simple and effective computerized test of fluid intelligence (reasoning ability). The test requires structure mapping - a key component of the reasoning process. Participants are asked to map a pair of corresponding nodes across two mathematically isomorphic but visually different graphs. The test difficulty can be easily manipulated - the more complex structurally and dissimilar visually the graphs, the higher response error rate. Graph Mapping offers high flexibility in item generation, ranging from trivial to extremally difficult items, supporting progressive item sequences suitable for correlational studies. It also allows multiple item instances (clones) at a fixed difficulty level as well as full item randomization, both particularly suitable for within-subject experimental designs, longitudinal studies, and adaptive testing. The test has short administration times and is unfamiliar to participants, yielding practical advantages. Graph Mapping has excellent psychometric properties: Its convergent validity and reliability is comparable to the three leading traditional fluid reasoning tests. The convenient software allows a researcher to design the optimal test variant for a given study and sample. Graph Mapping can be downloaded from: https://osf.io/wh7zv/.

In Silico Identification of lncRNAs Regulating Sperm Motility in the Turkey (Meleagris gallopavo L.).

Long non-coding RNAs (lncRNAs) are transcripts not translated into proteins with a length of more than 200 bp. LncRNAs are considered an important factor in the regulation of countless biological processes, mainly through the regulation of gene expression and interactions with proteins. However, the detailed mechanism of interaction as well as functions of lncRNAs are still unclear and therefore constitute a serious research challenge. In this study, for the first time, potential mechanisms of lncRNA regulation of processes related to sperm motility in turkey were investigated and described. Customized bioinformatics analysis was used to detect and identify lncRNAs, and their correlations with differentially expressed genes and proteins were also investigated. Results revealed the expression of 863 new/unknown lncRNAs in ductus deferens, testes and epididymis of turkeys. Moreover, potential relationships of the lncRNAs with the coding mRNAs and their products were identified in turkey reproductive tissues. The results obtained from the OMICS study may be useful in describing and characterizing the way that lncRNAs regulate genes and proteins as well as signaling pathways related to sperm motility.

Molecular Diversity and Phylogeny Reconstruction of Genus Colobanthus (Caryophyllaceae) Based on Mitochondrial Gene Sequences.

Mitochondrial genomes have become an interesting object of evolutionary and systematic study both for animals and plants, including angiosperms. Although the framework of the angiosperm phylogeny was built on the information derived from chloroplast and nuclear genes, mitochondrial sequences also revealed their usefulness in solving the phylogenetic issues at different levels of plant systematics. Here, we report for the first time the complete sequences of 26 protein-coding genes of eight Colobanthus species (Caryophyllaceae). Of these, 23 of them represented core mitochondrial genes, which are directly associated with the primary function of that organelle, and the remaining three genes represented a facultative set of mitochondrial genes. Comparative analysis of the identified genes revealed a generally high degree of sequence conservation. The Ka/Ks ratio was <1 for most of the genes, which indicated purifying selection. Only for rps12 was Ka/Ks > 1 in all studied species, suggesting positive selection. We identified 146−165 potential RNA editing sites in genes of the studied species, which is lower than in most angiosperms. The reconstructed phylogeny based on mitochondrial genes was consistent with the taxonomic position of the studied species, showing the separate character of the family Caryophyllaceae and close relationships between all studied Colobanthus species, with C. lycopodioides sharing less similarity.

Tandem Mass Tagging (TMT) Reveals Tissue-Specific Proteome of L4 Larvae of Anisakis simplex s. s.: Enzymes of Energy and/or Carbohydrate Metabolism as Potential Drug Targets in Anisakiasis.

Anisakis simplex s. s. is a parasitic nematode of marine mammals and causative agent of anisakiasis in humans. The cuticle and intestine of the larvae are the tissues most responsible for direct and indirect contact, respectively, of the parasite with the host. At the L4 larval stage, tissues, such as the cuticle and intestine, are fully developed and functional, in contrast to the L3 stage. As such, this work provides for the first time the tissue-specific proteome of A. simplex s. s. larvae in the L4 stage. Statistical analysis (FC ≥ 2; p-value ≤ 0.01) showed that 107 proteins were differentially regulated (DRPs) between the cuticle and the rest of the larval body. In the comparison between the intestine and the rest of the larval body at the L4 stage, 123 proteins were identified as DRPs. Comparison of the individual tissues examined revealed a total of 272 DRPs, with 133 proteins more abundant in the cuticle and 139 proteins more abundant in the intestine. Detailed functional analysis of the identified proteins was performed using bioinformatics tools. Glycolysis and the tricarboxylic acid cycle were the most enriched metabolic pathways by cuticular and intestinal proteins, respectively, in the L4 stage of A. simplex s. s. The presence of two proteins, folliculin (FLCN) and oxoglutarate dehydrogenase (OGDH), was confirmed by Western blot, and their tertiary structure was predicted and compared with other species. In addition, host-pathogen interactions were identified, and potential new allergens were predicted. The result of this manuscript shows the largest number of protein identifications to our knowledge using proteomics tools for different tissues of L4 larvae of A. simplex s. s. The identified tissue-specific proteins could serve as targets for new drugs against anisakiasis.

Chemerin effect on transcriptome of the porcine endometrium during implantation determined by RNA-sequencing†.

It is well known that the body's metabolism and reproduction are closely related. Chemerin (CHEM) is one of many biologically active proteins secreted by the adipose tissue involved in the regulation of the energy homeostasis of the organism. In the present study, RNA-sequencing was performed to investigate the differentially expressed genes (DEGs), long non-coding RNAs (lncRNAs), and alternatively spliced (AS) transcripts in the cultured porcine endometrium exposed to chemerin for 24 hours (CHEM; 400 ng/mL) collected during the implantation period (15-16 days of gestation). High-throughput sequencing of transcriptomes was performed on the Illumina NovaSeq 6000 platform (Illumina, USA). In the current study, among all 130 DEGs, 58 were upregulated and 72 were downregulated in the CHEM-treated group. DEGs were assigned to 73 functional annotations. Twelve identified lncRNAs indicated a difference in the expression profile after CHEM administration. Additionally, we detected 386 differentially AS events encompassed 274 protein-coding genes and 2 lncRNAs. All AS events were divided into five alternative splicing types: alternative 3' splice site (A3SS), 5' splice site (A5SS), mutually exclusive exons (MXE), retention intron (RI), and skipping exon (SE). Within all AS events, we identified 42 A3SS, 43 A5SS, 53 MXE, 9 RI, and 239 SE. In summary, CHEM affects the transcriptomic profile of the porcine endometrium, controlling the expression of numerous genes, including those involved in the cell migration and adhesion, angiogenesis, inflammation, and steroidogenesis. It can be assumed that CHEM may be an important factor for a proper course of gestation and embryo development.

Effects of neonatal methoxychlor exposure on the ovarian transcriptome in piglets.

Methoxychlor (MXC) is a man-made organochlorine insecticide capable of disrupting endocrine functions due to its mixed steroidal properties (estrogenic, anti-estrogenic and/or anti-androgenic). Retarded follicle development was recently reported in neonatal pigs treated with MXC. The goal of the current study was to better understand the mechanism of MXC action in the ovary of newborn piglets. By employing RNA-Seq we studied the expression of protein coding (mRNA) and long non-coding RNA (lncRNA) transcripts in the ovary of the MXC-treated piglets. Piglets were injected (sc) daily with MXC (100 mg/kg body weight) or corn oil (controls) between postnatal Days 1 and 10 (n = 3 piglets/group). The ovaries excised from 11-day-old piglets were processed for total RNA isolation and subsequent RNA sequencing. Four hundred sixty differentially expressed genes (DEGs) and 143 differentially expressed lncRNAs (DELs) were identified in the ovaries of MXC-treated piglets (P-adjusted < 0.05; abs(log2FC) > 1). Functional enrichment analysis showed that MXC altered the expression of genes associated with intracellular and membrane transport, intra-ovarian signaling as well as cell-cell junction and communication. Moreover, positive and negative correlations determined between the identified DEGs and DELs suggest that some lncRNAs may mediate the MXC action in the ovary. The results support the hypothesis that MXC-induced changes in the expression of genes involved in neonatal ovarian folliculogenesis increase the risk of fertility problems in adults.

PPARγ regulates the expression of genes involved in the DNA damage response in an inflamed endometrium.

Inflammation is a biological response of the immune system, which can be triggered by many factors, including pathogens. These factors may induce acute or chronic inflammation in various organs, including the reproductive system, leading to tissue damage or disease. In this study, the RNA-Seq technique was used to determine the in vitro effects of peroxisome proliferator-activated receptor gamma (PPARγ) ligands on the expression of genes and long non-coding RNA, and alternative splicing events (ASEs) in LPS-induced inflammation of the porcine endometrium during the follicular phase of the estrous cycle. Endometrial slices were incubated in the presence of LPS and PPARγ agonists (PGJ2 or pioglitazone) and a PPARγ antagonist (T0070907). We identified 169, 200, 599 and 557 differentially expressed genes after LPS, PGJ2, pioglitazone or T0070907 treatment, respectively. Moreover, changes in differentially expressed long non-coding RNA and differential alternative splicing events were described after the treatments. The study revealed that PPARγ ligands influence the LPS-triggered expression of genes controlling the DNA damage response (GADD45ß, CDK1, CCNA1, CCNG1, ATM). Pioglitazone treatment exerted a considerable effect on the expression of genes regulating the DNA damage response.