Cereal leaf beetle-associated bacteria enhance the survival of their host upon insecticide treatments and respond differently to insecticides with different modes of action.

The cereal leaf beetle (CLB, Oulema melanopus) is one of the major cereal pests. The effect of insecticides belonging to different chemical classes, with different mechanisms of action and the active substances' concentrations on the CLB bacterial microbiome, was investigated. Targeted metagenomic analysis of the V3-V4 regions of the 16S ribosomal gene was used to determine the composition of the CLB bacterial microbiome. Each of the insecticides caused a decrease in the abundance of bacteria of the genus Pantoea, and an increase in the abundance of bacteria of the genus Stenotrophomonas, Acinetobacter, compared to untreated insects. After cypermethrin application, a decrease in the relative abundance of bacteria of the genus Pseudomonas was noted. The dominant bacterial genera in cypermethrin-treated larvae were Lactococcus, Pantoea, while in insects exposed to chlorpyrifos or flonicamid it was Pseudomonas. Insecticide-treated larvae were characterized, on average, by higher biodiversity and richness of bacterial genera, compared to untreated insects. The depletion of CLB-associated bacteria resulted in a decrease in larval survival, especially after cypermethrin and chlorpyrifos treatments. The use of a metagenome-based functional prediction approach revealed a higher predicted function of bacterial acetyl-CoA C-acetyltransferase in flonicamid and chlorpyrifos-treated larvae and tRNA dimethyltransferase in cypermethrin-treated insects than in untreated insects.

A massive alteration of gene expression in undescended testicles of dogs and the association of KAT6A variants with cryptorchidism.

Cryptorchidism is the most common form of disorder of sex development in male dogs, but its hereditary predisposition is poorly elucidated. The gonadal transcriptome of nine unilaterally cryptorchid dogs and seven control dogs was analyzed using RNA-seq. Comparison between the scrotal and inguinal gonads of unilateral cryptorchid dogs revealed 8,028 differentially expressed genes (DEGs) (3,377 up-regulated and 4,651 down-regulated). A similar number of DEGs (7,619) was found by comparing the undescended testicles with the descended testicles of the control dogs. The methylation status of the selected DEGs was also analyzed, with three out of nine studied DEGs showing altered patterns. Bioinformatic analysis of the cDNA sequences revealed 20,366 SNP variants, six of which showed significant differences in allelic counts between cryptorchid and control dogs. Validation studies in larger cohorts of cryptorchid (n = 122) and control (n = 173) dogs showed that the TT genotype (rs850666472, p.Ala1230Val) and the AA genotype in 3'UTR (16:23716202G>A) in KATA6, responsible for acetylation of lysine 9 in histone H3, are associated with cryptorchidism (P = 0.0383). Both the transcript level of KAT6A and H3K9 acetylation were lower in undescended testes, and additionally, the acetylation depended on the genotypes in exon 17 and the 3'UTR. Our study showed that the massive alteration of the transcriptome in undescended testicles is not caused by germinal DNA variants in DEG regulatory sequences but is partly associated with an aberrant DNA methylation and H3K9 acetylation patterns. Moreover, variants of KAT6A can be considered markers associated with the risk of this disorder.

Ambiguous genes due to aligners and their impact on RNA-seq data analysis.

The main scope of the study is ambiguous genes, i.e. genes whose expression is difficult to estimate from the data produced by next-generation sequencing technologies. We focused on the RNA sequencing (RNA-Seq) type of experiment performed on the Illumina platform. It is crucial to identify such genes and understand the cause of their difficulty, as these genes may be involved in some diseases. By giving misleading results, they could contribute to a misunderstanding of the cause of certain diseases, which could lead to inappropriate treatment. We thought that the ambiguous genes would be difficult to map because of their complex structure. So we looked at RNA-seq analysis using different mappers to find genes that would have different measurements from the aligners. We were able to identify such genes using a generalized linear model with two factors: mappers and groups introduced by the experiment. A large proportion of ambiguous genes are pseudogenes. High sequence similarity of pseudogenes to functional genes may indicate problems in alignment procedures. In addition, predictive analysis verified the performance of difficult genes in classification. The effectiveness of classifying samples into specific groups was compared, including the expression of difficult and not difficult genes as covariates. In almost all cases considered, ambiguous genes have less predictive power.

The Epidermal Transcriptome Analysis of a Novel c.639_642dup LORICRIN Variant-Delineation of the Loricrin Keratoderma Pathology.

Loricrin keratoderma (LK) is a rare autosomal dominant genodermatosis caused by LORICRIN gene mutations. The pathogenesis of the disease is not yet fully understood. So far, only 10 pathogenic variants in LORICRIN have been described, with all of them but one being deletions or insertions. The significance of rare nonsense variants remains unclear. Furthermore, no data regarding the RNA expression in affected patients are available. The aim of this study is to describe the two variants in the LORICRIN gene found in two distinct families: the novel pathogenic variant c.639_642dup and a rare c.10C > T (p.Gln4Ter) of unknown significance. We also present the results of the transcriptome analysis of the lesional loricrin keratoderma epidermis of a patient with c.639_642dup. We show that in the LK lesion, the genes associated with epidermis development and keratocyte differentiation are upregulated, while genes engaged in cell adhesion, differentiation developmental processes, ion homeostasis and transport, signaling and cell communication are downregulated. In the context of the p.Gln4Ter clinical significance evaluation, we provide data indicating that LORICRIN haploinsufficiency has no skin consequences. Our results give further insight into the pathogenesis of LK, which may have therapeutic implications in the future and important significance in the context of genetic counseling.

Insect Gut Bacteria Promoting the Growth of Tomato Plants (Solanum lycopersicum L.).

We investigated gut bacteria from three insect species for the presence of plant growth properties (PGP). Out of 146 bacterial strains obtained from 20 adult specimens of Scolytidae sp., 50 specimens of Oulema melanopus, and 150 specimens of Diabrotica virgifera, we selected 11 strains displaying the following: PGP, phosphate solubility, production of cellulase, siderophore, lipase, protease, and hydrogen cyanide. The strains were tested for growth promotion ability on tomato (Lycopersicon esculentum) plants. Each strain was tested individually, and all strains were tested together as a bacterial consortium. Tomato fruit yield was compared with the negative control. The plants treated with bacterial consortium showed a significant increase in fruit yield, in both number of fruits (+41%) and weight of fruits (+44%). The second highest yield was obtained for treatment with Serratia liquefaciens Dv032 strain, where the number and weight of yielded fruits increased by 35% and 30%, respectively. All selected 11 strains were obtained from Western Corn Rootworm (WCR), Diabrotica virgifera. The consortium comprised: Ewingella americana, Lactococcus garvieae, L. lactis, Pseudomonas putida, Serratia liquefaciens, and S. plymuthica. To our knowledge, this is the first successful application of D. virgifera gut bacteria for tomato plant growth stimulation that has been described.

Whole genome sequencing identifies a missense polymorphism in PADI6 associated with testicular/ovotesticular XX disorder of sex development in dogs.

Disorders of sex development (DSDs) are congenital malformations defined as discrepancies between sex chromosomes and phenotypical sex. Testicular or ovotesticular XX DSDs are frequently observed in female dogs, while monogenic XY DSDs are less frequent. Here, we applied whole genome sequencing (WGS) to search for causative mutations in XX DSD females in French Bulldogs (FB) and American Staffordshire Terries (AST) and in XY DSD Yorkshire Terries (YT). The WGS results were validated by Sanger sequencing and ddPCR. It was shown that a missense SNP of the PADI6 gene, is significantly associated with the XX DSD (SRY-negative) phenotype in AST (P = 0.0051) and FB (P = 0.0306). On the contrary, we did not find any associated variant with XY DSD in YTs. Our study suggests that the genetic background of the XX DSD may be more complex and breed-specific.

The structure of the cereal leaf beetle (Oulema melanopus) microbiome depends on the insect's developmental stage, host plant, and origin.

Cereal leaf beetle (CLB, Oulema melanopus, Coleoptera, Chrysomelidae) is a serious agricultural pest that causes considerable damages to agricultural production. The aim of this study was to characterize the bacterial communities associated with larvae and imagoes of CLB collected from various cereal host species and locations. The bacterial profile was characterized by 16S rRNA gene sequencing at the V3-V4 hypervariable region. Using taxonomy-based analysis, the bacterial community of CLB containing 16 phyla, 26 classes, 49 orders, 78 families, 94 genera, and 63 species of bacteria was identified. The abundance of Wolbachia, Rickettsia, and Lactococcus genus was significantly higher in CLB imagoes than in larvae. Statistical analysis confirmed that the bacterial community of the larvae is more diverse in comparison to imagoes and that insects collected from spring barley and wheat are characterized by a much higher biodiversity level of bacterial genera and species than insects collected from other cereals. Obtained results indicated that the developmental stage, the host plant, and the insect's sampling location affected the CLB's microbiome. Additionally, the CLB core microbiome was determined. It consists of 2 genera (Wolbachia and Rickettsia) shared by at least 90% tested CLB insects, regardless of the variables analysed.

The Effect of Feeding Management and Culling of Cows on the Lactation Curves and Milk Production of Primiparous Dairy Cows.

The study attempted to estimate the lactation curves of primiparous dairy cows in relation to their feeding management. Therefore, the first aim of the study was to determine and compare the lactation curves of primiparous dairy cows using Wood's model and to estimate the association between the lactation curves and feeding management. The second objective was to investigate the effect of the culling rate on improvement in the milk yield of primiparous dairy herds. The study was conducted on four commercial dairy farms of Polish Holstein-Friesian cows using different feeding systems (TMR-total mixed ration and PMR-partial mixed ration) and management (T1-one TMR throughout lactation; P1-one PMR throughout lactation; T2 and T3-three feed periods such as FRESH, TMR I and TMR II according to days in milk). The data used for the study were obtained from monthly milk performance evaluations of 1662 primiparous cows conducted by the Polish Federation of Cattle Breeding and Dairy Farmers throughout the year 2015. Wood's lactation model was used to plot curves for milk yield, fat and protein content, lactose content, and milk urea contents. The highest milk yield for the whole lactation and in the peak lactation phase was recorded for cows in herd T1. This herd reached peak lactation on day 105 of milking, with an average milk yield of 42.1 kg, which was about 5 kg more milk than in the other herds. The study showed that the culling of primiparous cows in herd T1 after 30, 60 and 90 days of lactation prevented a significant reduction in milk yield in a 305-day lactation. It also increased average milk production by 1586.9 kg per primiparous dairy cow.

Identification of a Stable, Non-Canonically Regulated Nrf2 Form in Lung Cancer Cells.

Nrf2 (nuclear factor erythroid 2 (NF-E2)-related factor 2) transcription factor is recognized for its pro-survival and cell protective role upon exposure to oxidative, chemical, or metabolic stresses. Nrf2 controls a number of cellular processes such as proliferation, differentiation, apoptosis, autophagy, lipid synthesis, and metabolism and glucose metabolism and is a target of activation in chronic diseases like diabetes, neurodegenerative, and inflammatory diseases. The dark side of Nrf2 is revealed when its regulation is imbalanced (e.g., via oncogene activation or mutations) and under such conditions constitutively active Nrf2 promotes cancerogenesis, metastasis, and radio- and chemoresistance. When there is no stress, Nrf2 is instantly degraded via Keap1-Cullin 3 (Cul3) pathway but despite this, cells exhibit a basal activation of Nrf2 target genes. It is yet not clear how Nrf2 maintains the expression of its targets under homeostatic conditions. Here, we found a stable 105 kDa Nrf2 form that is resistant to Keap1-Cul3-mediated degradation and translocates to the nucleus of lung cancer cells. RNA-Seq analysis indicate that it might originate from the exon 2 or exon 3-truncated transcripts. This stable 105 kDa Nrf2 form might help explain the constitutive activity of Nrf2 under normal cellular conditions.

Transcriptome Remodeling in Gradual Development of Inverse Resistance between Paclitaxel and Cisplatin in Ovarian Cancer Cells.

Resistance to anti-cancer drugs is the main challenge in oncology. In pre-clinical studies, established cancer cell lines are primary tools in deciphering molecular mechanisms of this phenomenon. In this study, we proposed a new, transcriptome-focused approach, utilizing a model of isogenic cancer cell lines with gradually changing resistance. We analyzed trends in gene expression in the aim to find out a scaffold of resistance development process. The ovarian cancer cell line A2780 was treated with stepwise increased concentrations of paclitaxel (PTX) to generate a series of drug resistant sublines. To monitor transcriptome changes we submitted them to mRNA-sequencing, followed by the identification of differentially expressed genes (DEGs), principal component analysis (PCA), and hierarchical clustering. Functional interactions of proteins, encoded by DEGs, were analyzed by building protein-protein interaction (PPI) networks. We obtained human ovarian cancer cell lines with gradually developed resistance to PTX and collateral sensitivity to cisplatin (CDDP) (inverse resistance). In their transcriptomes, we identified two groups of DEGs: (1) With fluctuations in expression in the course of resistance acquiring; and (2) with a consistently changed expression at each stage of resistance development, constituting a scaffold of the process. In the scaffold PPI network, the cell cycle regulator-polo-like kinase 2 (PLK2); proteins belonging to the tumor necrosis factor (TNF) ligand and receptor family, as well as to the ephrin receptor family were found, and moreover, proteins linked to osteo- and chondrogenesis and the nervous system development. Our cellular model of drug resistance allowed for keeping track of trends in gene expression and studying this phenomenon as a process of evolution, reflected by global transcriptome remodeling. This approach enabled us to explore novel candidate genes and surmise that abrogation of the osteomimic phenotype in ovarian cancer cells might occur during the development of inverse resistance between PTX and CDDP.

Genes sharing the protein family domain decrease the performance of classification with RNA-seq genomic signatures.

BACKGROUND: The experience with running various types of classification on the CAMDA neuroblastoma dataset have led us to the conclusion that the results are not always obvious and may differ depending on type of analysis and selection of genes used for classification. This paper aims in pointing out several factors that may influence the downstream machine learning analysis. In particular those factors are: type of the primary analysis, type of the classifier and increased correlation between the genes sharing a protein domain. They influence the analysis directly, but also interplay between them may be important. We have compiled the gene-domain database and used it for analysis to see the differences between the genes that share a domain versus the rest of the genes in the datasets. RESULTS: The major findings are: pairs of genes that share a domain have an increased Spearman's correlation coefficients of counts; genes sharing a domain are expected to have a lower predictive power due to increased correlation. For most of the cases it can be seen with the higher number of misclassified samples; classifiers performance may vary depending on a method, still in most cases using genes sharing a domain in the training set results in a higher misclassification rate; increased correlation in genes sharing a domain results most often in worse performance of the classifiers regardless of the primary analysis tools used, even if the primary analysis alignment yield varies. CONCLUSIONS: The effect of sharing a domain is likely more a results of real biological co-expression than just sequence similarity and artifacts of mapping and counting. Still, this is more difficult to conclude and needs further research. The effect is interesting itself, but we also point out some practical aspects in which it may influence the RNA sequencing analysis and RNA biomarker use. In particular it means that a gene signature biomarker set build out of RNA-sequencing results should be depleted for genes sharing common domains. It may cause to perform better when applying classification. REVIEWERS: This article was reviewed by Dimitar Vassiliev and Susmita Datta.