ALBERT

Description: The World Health Organization (WHO) defines infertility as the inability of a sexually active, non-contracepting couple to achieve spontaneous pregnancy within one year. Statistics show that the two sexes are equally at risk. Several causes may be responsible for male infertility; however, in 30–40% of cases a diagnosis of idiopathic male infertility is made in men with normal urogenital anatomy, no history of familial fertility-related diseases and a normal panel of values as for endocrine, genetic and biochemical markers. Idiopathic male infertility may be the result of gene/environment interactions, genetic and epigenetic abnormalities. Numerical and structural anomalies of the Y chromosome represent a minor yet significant proportion and are the topic discussed in this review. We searched the PubMed database and major search engines for reports about Y-linked male infertility. We present cases of Y-linked male infertility in terms of (i) anomalies of the Y chromosome structure/number; (ii) Y chromosome misbehavior in a normal genetic background; (iii) Y chromosome copy number variations (CNVs). We discuss possible explanations of male infertility caused by mutations, lower or higher number of copies of otherwise wild type, Y-linked sequences. Despite Y chromosome structural anomalies are not a major cause of male infertility, in case of negative results and of normal DNA sequencing of the ascertained genes causing infertility and mapping on this chromosome, we recommend an analysis of the karyotype integrity in all cases of idiopathic fertility impairment, with an emphasis on the structure and number of this chromosome.

Description: Prion diseases are fatal neurodegenerative diseases and are characterized by the accumulation of abnormal prion protein (PrPSc) in the brain. During the outbreak of the bovine spongiform encephalopathy (BSE) epidemic in the United Kingdom, prion diseases in several species were reported; however, horse prion disease has not been reported thus far. In previous studies, the shadow of prion protein (Sho) has contributed to an acceleration of conversion from normal prion protein (PrPC) to PrPSc, and the shadow of prion protein gene (SPRN) polymorphisms have been significantly associated with the susceptibility of prion diseases. We investigated the genotype, allele and haplotype frequencies of the SPRN gene using direct sequencing. In addition, we analyzed linkage disequilibrium (LD) and haplotypes among polymorphisms. We also investigated LD between PRNP and SPRN single nucleotide polymorphisms (SNPs). We compared the amino acid sequences of Sho protein between the horse and several prion disease-susceptible species using ClustalW2. To perform Sho protein modeling, we utilized SWISS-MODEL and Swiss-PdbViewer programs. We found a total of four polymorphisms in the equine SPRN gene; however, we did not observe an in/del polymorphism, which is correlated with the susceptibility of prion disease in prion disease-susceptible animals. The SPRN SNPs showed weak LD value with PRNP SNP. In addition, we found 12 horse-specific amino acids of Sho protein that can induce significantly distributional differences in the secondary structure and hydrogen bonds between the horse and several prion disease-susceptible species. To the best of our knowledge, this is the first report regarding the genetic and structural characteristics of the equine SPRN gene.

Description: The continuous cropping of sugar beet can result in soil degradation and a decrease in the sugar beet yield and quality. However, the role of continuous sugar beet (Beta vulgaris L. var. saccharifera) cropping in shaping the structure and function of the rhizosphere microbial community remains poorly investigated. In this study, we comparatively investigated the impact of different numbers of years of continuous sugar beet cropping on structural and functional changes in the microbial community of the rhizosphere using high-throughput sequencing and bioinformatics analysis. We collected rhizosphere soils from fields continuously cropped for one-year (T1), five-year (T5), and thirty-year (T30) periods, as well as one bulk soil (T0), in the Xinjiang Uygur Autonomous Region. The results demonstrated that continuous sugar beet cropping resulted in a significant decline in the community diversity of soil bacterial and fungal populations from T1 to T5. With continuous change in the structure of the microbial community, the Shannon diversity and observed species were increased in T30. With an abundance of pathogenic microbes, including Acidobacteria, Alternaria, and Fusarium, that were highly enriched in T30, soil-borne diseases could be accelerated, deduced by functional predictions based on 16S rRNA genes. Continuous sugar beet cropping also led to significant declines in beneficial bacteria, including Actinobacteria, Pseudomonas spp., and Bacillus spp. In addition, we profiled and analyzed predictive metabolic characteristics (metabolism and detoxification). The abundance of phenolic acid decarboxylase involved in the phenolic acid degradation pathway was significantly lower in groups T5 and T30 than that in T0 and T1, which could result in the phenolic compounds becoming excessive in long-term continuous cropping soil. Our results provide a deeper understanding of the rhizosphere soil microbial community’s response to continuous sugar beet cropping, which is important in evaluating the sustainability of this agricultural practice.

Description: Variants in more than 271 different genes have been linked to hereditary retinal diseases, making comprehensive genomic approaches mandatory for accurate diagnosis. We explored the genetic landscape of retinal disorders in consanguineous families from North-Western Pakistan, harboring a population of approximately 35 million inhabitants that remains relatively isolated and highly inbred (~50% consanguinity). We leveraged on the high degree of consanguinity by applying genome-wide high-density single-nucleotide polymorphism (SNP) genotyping followed by targeted Sanger sequencing of candidate gene(s) lying inside autozygous intervals. In addition, we performed whole-exome sequencing (WES) on at least one proband per family. We identified 7 known and 4 novel variants in a total of 10 genes (ABCA4, BBS2, CNGA1, CNGA3, CNGB3, MKKS, NMNAT1, PDE6B, RPE65, and TULP1) previously known to cause inherited retinal diseases. In spite of all families being consanguineous, compound heterozygosity was detected in one family. All homozygous pathogenic variants resided in autozygous intervals ≥2.0 Mb in size. Putative founder variants were observed in the ABCA4 (NM_000350.2:c.214G〉A; p.Gly72Arg; ten families) and NMNAT1 genes (NM_022787.3:c.25G〉A; p.Val9Met; two families). We conclude that geographic isolation and sociocultural tradition of intrafamilial mating in North-Western Pakistan favor both the clinical manifestation of rare “generic” variants and the prevalence of founder mutations.

Description: We previously demonstrated that 20 mM sucrose promotes the upper axillary bud outgrowth in two-node stems of Chrysanthemum morifolium. In this study, we aimed to screen for potential genes involved in this process. Quantitative reverse transcription (qRT)-PCR analysis of sugar-related genes in the upper axillary bud of plants treated with 20 mM sucrose revealed the specific expression of the gene CmSWEET17. Expression of this gene was increased in the bud, as well as the leaves of C. morifolium, following exogenous sucrose treatment. CmSWEET17 was isolated from C. morifolium and a subcellular localization assay confirmed that the protein product was localized in the cell membrane. Overexpression of CmSWEET17 promoted upper axillary bud growth in the two-node stems treatment as compared with the wild-type. In addition, the expression of auxin transporter genes CmAUX1, CmLAX2, CmPIN1, CmPIN2, and CmPIN4 was upregulated in the upper axillary bud of CmSWEET17 overexpression lines, while indole-3-acetic acid content decreased. The results suggest that CmSWEET17 could be involved in the process of sucrose-induced axillary bud outgrowth in C. morifolium, possibly via the auxin transport pathway.

Description: Adipogenesis is a complicated but precisely orchestrated process mediated by a series of transcription factors. Our previous study has identified a novel long noncoding RNA (lncRNA) that was differentially expressed during bovine adipocyte differentiation. Because this lncRNA overlaps with miR-221 in the genome, it was named miR-221 host gene (MIR221HG). The purpose of this study was to clone the full length of MIR221HG, detect its subcellular localization, and determine the effects of MIR221HG on bovine adipocyte differentiation. The 5′ rapid amplification of cDNA ends (RACE) and 3′ RACE analyses demonstrated that MIR221HG is a transcript of 1064 nucleotides, is located on the bovine X chromosome, and contains a single exon. Bioinformatics analyses suggested that MIR221HG is an lncRNA and the promoter of MIR221HG includes the binding consensus sequences of the forkhead box C1 (FOXC1) and krüppel-like factor5 (KLF5). The semi-quantitative PCR and quantitative real-time PCR (qRT-PCR) of nuclear and cytoplasmic fractions revealed that MIR221HG mainly resides in the nucleus. Inhibition of MIR221HG significantly increased adipocyte differentiation, as indicated by a dramatic increment in the number of mature adipocytes and in the expression of the respective adipogenic markers, peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα), and fatty acid binding protein 4 (FABP4). Our results provide a basis for elucidating the mechanism by which MIR221HG regulates adipocyte differentiation.

Description: Staphylococcus aureus is a major human pathogen and an important cause of livestock infections. More than 20 staphylococcal enterotoxins with emetic activity can be produced by specific strains responsible for staphylococcal food poisoning, one of the most common food-borne diseases. Whole genome sequencing provides a comprehensive view of the genome structure and gene content that have largely been applied in outbreak investigations and genomic comparisons. In this study, six enterotoxigenic S. aureus strains were characterised using a combination of molecular, phenotypical and computational methods. The genomes were analysed for the presence of virulence factors (VFs), where we identified 110 genes and classified them into five categories: adherence (n = 31), exoenzymes (n = 28), genes involved in host immune system evasion (n = 7); iron uptake regulatory system (n = 8); secretion machinery factors and toxins’ genes (n = 36), and 39 genes coding for transcriptional regulators related to staphylococcal VFs. Each group of VFs revealed correlations among the six enterotoxigenic strains, and further analysis revealed their accessory genomic content, including mobile genetic elements. The plasmids pLUH02 and pSK67 were detected in the strain ProNaCC1 and ProNaCC7, respectively, carrying out the genes sed, ser, and selj. The genes carried out by prophages were detected in the strain ProNaCC2 (see), ProNaCC4, and ProNaCC7 (both positive for sea). The strain ProNaCC5 resulted positive for the genes seg, sei, sem, sen, seo grouped in an exotoxin gene cluster, and the strain ProNaCC6 resulted positive for seh, a transposon-associated gene. The six strains were used for the production of naturally contaminated cheeses which were tested with the European Screening Method for staphylococcal enterotoxins. The results obtained from the analysis of toxins produced in cheese, combined with the genomic features represent a portrait of the strains that can be used for the production of staphylococcal enterotoxin-positive cheese as reference material.

Description: The FANCJ helicase unfolds G-quadruplexes (G4s) in human cells to support DNA replication. This action is coupled to the recruitment of REV1 polymerase to synthesize DNA across from a guanine template. The precise mechanisms of these reactions remain unclear. While FANCJ binds to G4s with an AKKQ motif, it is not known whether this site recognizes damaged G4 structures. FANCJ also has a PIP-like (PCNA Interacting Protein) region that may recruit REV1 to G4s either directly or through interactions mediated by PCNA protein. In this work, we measured the affinities of a FANCJ AKKQ peptide for G4s formed by (TTAGGG)4 and (GGGT)4 using fluorescence spectroscopy and biolayer interferometry (BLI). The effects of 8-oxoguanine (8oxoG) on these interactions were tested at different positions. BLI assays were then performed with a FANCJ PIP to examine its recruitment of REV1 and PCNA. FANCJ AKKQ bound tightly to a TTA loop and was sequestered away from the 8oxoG. Reducing the loop length between guanine tetrads increased the affinity of the peptide for 8oxoG4s. FANCJ PIP targeted both REV1 and PCNA but favored interactions with the REV1 polymerase. The impact of these results on the remodeling of damaged G4 DNA is discussed herein.

Description: Pathogen fitness landscapes change when transmission cycles establish in non-native environments or spill over into new vectors and hosts. The introduction of Leishmania infantum in the Americas into the Neotropics during European colonization represents a unique case study to investigate the mechanisms of ecological adaptation of this important parasite. Defining the evolutionary trajectories that drive L. infantum fitness in this new environment are of great public health importance as they will allow unique insight into pathways of host/pathogen co-evolution and their consequences for region-specific changes in disease manifestation. This review summarizes current knowledge on L. infantum genetic and phenotypic diversity in the Americas and its possible role in the unique epidemiology of visceral leishmaniasis (VL) in the New World. We highlight the importance of appreciating adaptive molecular mechanisms in L. infantum to understand the parasites’ successful establishment on the continent.

Description: Viral vector use is wide-spread in the field of gene therapy, with new clinical trials starting every year for different human pathologies and a growing number of agents being approved by regulatory agencies. However, preclinical testing is long and expensive, especially during the early stages of development. Nowadays, the model organism par excellence is the mouse (Mus musculus), and there are few investigations in which alternative models are used. Here, we assess the possibility of using zebrafish (Danio rerio) as an in vivo model for adenoviral vectors. We describe how E1/E3-deleted adenoviral vectors achieve efficient transduction when they are administered to zebrafish embryos via intracranial injection. In addition, helper-dependent (high-capacity) adenoviral vectors allow sustained transgene expression in this organism. Taking into account the wide repertoire of genetically modified zebrafish lines, the ethical aspects, and the affordability of this model, we conclude that zebrafish could be an efficient alternative for the early-stage preclinical evaluation of adenoviral vectors.

Description: The importance of genome organization at the supranucleosomal scale in the control of gene expression is increasingly recognized today. In mammals, Topologically Associating Domains (TADs) and the active/inactive chromosomal compartments are two of the main nuclear structures that contribute to this organization level. However, recent works reviewed here indicate that, at specific loci, chromatin interactions with nuclear bodies could also be crucial to regulate genome functions, in particular transcription. They moreover suggest that these nuclear bodies are membrane-less organelles dynamically self-assembled and disassembled through mechanisms of phase separation. We have recently developed a novel genome-wide experimental method, High-salt Recovered Sequences sequencing (HRS-seq), which allows the identification of chromatin regions associated with large ribonucleoprotein (RNP) complexes and nuclear bodies. We argue that the physical nature of such RNP complexes and nuclear bodies appears to be central in their ability to promote efficient interactions between distant genomic regions. The development of novel experimental approaches, including our HRS-seq method, is opening new avenues to understand how self-assembly of phase-separated nuclear bodies possibly contributes to mammalian genome organization and gene expression.

Description: Fragile X syndrome (FXS) is the most common inherited cause of intellectual disability and autism spectrum disorder, and among those with fragile X syndrome, approximately 1/3rd meet a threshold for an autism spectrum disorder (ASD) diagnosis. Previous functional imaging studies of fragile X syndrome have typically focused on those with fragile X syndrome compared to either neurotypical or autism spectrum disorder control groups. Further, the majority of previous studies have tended to focus on those who are more intellectually able than is typical for fragile X syndrome. In this study, we examine the impact of autistic traits in individuals with fragile X syndrome on a paradigm looking at facial emotion processing. The study included 17 individuals with fragile X syndrome, of whom 10 met criteria for autism as measured by the Autism Diagnostic Observation Schedule (ADOS). Prior to the scan, participants rehearsed on a mock scanner to help acclimatize to the scanner environment and thus allow more severely affected individuals to participate. The task examined the blood-oxygen-level-dependent (BOLD) response to fearful and neutral faces taken from the Ekman faces series. Individuals in the autism group had a region of significantly reduced activity centered on the left superior temporal gyrus, compared to those with FXS alone, in response to the fearful faces. We suggest that autism in individuals with fragile X syndrome is associated with similar changes in the neurobiology of facial emotion processing as seen in idiopathic autism.

Description: Osteoarthritis (OA) is a widely prevalent disease worldwide, and with an increasingly ageing society, it has become a challenge for the field of regenerative medicine. OA is a disease process involving multiple joint tissues, including those not visible on radiography, and is a complex disease process with multiple phenotypes that require evaluation by a multimodality imaging assessment. The purpose of this study was to evaluate the effect of micro-fragmented fat tissue intra-articular injection 24 months after application in two ways: Indirectly using functional magnetic resonance imaging (MRI) assessment analyzing the glycosaminoglycans (GAG) content in cartilage by means of delayed gadolinium (Gd)-enhanced magnetic resonance imaging of cartilage (dGEMRIC), as well as clinical outcome on observed level of GAG using standard orthopedic physical examination including VAS assessment. In our previous study assessing comprehensive results after 12 months, the dGEMRIC results have drawn attention. The present study explores the long-term effect of intra-articular injection of autologous microfragmented adipose tissue to host chondrocytes and cartilage proteoglycans in patients with knee OA. A prospective, non-randomized, interventional, single-center, open-label clinical trial was conducted from January 2016 to April 2018. A total of 17 patients were enrolled in the study, and 32 knees were assessed in a 12-month follow-up, but only 10 patients of them with 18 knees are included in a 24-month follow-up. The rest of the seven patients dropped out of the study 12 months after follow-up: three patients underwent knee arthroplasty, and the remaining four did not fulfil the basic criteria of 24 months involvement in the study. Surgical intervention (lipoaspiration), followed by tissue processing and intra-articular injection of the final microfragmented adipose tissue product into the affected knee(s), was performed in all patients. Patients were assessed for a visual analog scale (VAS), dGEMRIC at the baseline, three, six, 12 and 24 months after the treatment. A magnetic resonance sequence in dGEMRIC due to infiltration of the anionic, negatively-charged contrast gadopentetate dimeglumine (Gd-DTPA2) into the cartilage indicated that the contents of cartilage glycosaminoglycans significantly increased in specific areas of the treated knee joint. Our results suggest that this method of single intra-articular injection of autologous microfragmented adipose tissue improves GAG content on a significant scale, with over half of the measurements suggesting relevant improvement 24 months after intra-articular injection opposed to the expected GAG decrease over the natural course of the disease.

Description: Summer mortality, caused by thermal conditions, is the biggest threat to abalone aquaculture production industries. Various measures have been taken to mitigate this issue by adjusting the environment; however, the cellular processes of Pacific abalone (Haliotis discus hannai) have been overlooked due to the paucity of genetic information. The draft genome of H. discus hannai has recently been reported, prompting exploration of the genes responsible for thermal regulation in Pacific abalone. In this study, 413 proteins were systematically annotated as members of the heat shock protein (HSP) super families, and among them 26 HSP genes from four Pacific abalone tissues (hemocytes, gill, mantle, and muscle) were differentially expressed under cold and heat stress conditions. The co-expression network revealed that HSP expression patterns were tissue-specific and similar to those of other shellfish inhabiting intertidal zones. Finally, representative HSPs were selected at random and their expression patterns were identified by RNA sequencing and validated by qRT-PCR to assess expression significance. The HSPs expressed in hemocytes were highly similar in both analyses, suggesting that hemocytes could be more reliable samples for validating thermal condition markers compared to other tissues.

Description: This special issue of Genes demonstrates clearly that research in epigenetics has proceeded at a very rapid pace in the last decade. [...]

Description: Glutathione S-transferases (GSTs)—an especially plant-specific tau class of GSTs—are key enzymes involved in biotic and abiotic stress responses. To improve the stress resistance of crops via the genetic modification of GSTs, we predicted the amino acids present in the GSH binding site (G-site) and hydrophobic substrate-binding site (H-site) of OsGSTU17, a tau class GST in rice. We then examined the enzyme activity, substrate specificity, enzyme kinetics and thermodynamic stability of the mutant enzymes. Our results showed that the hydrogen bonds between Lys42, Val56, Glu68, and Ser69 of the G-site and glutathione were essential for enzyme activity and thermal stability. The hydrophobic side chains of amino acids of the H-site contributed to enzyme activity toward 4-nitrobenzyl chloride but had an inhibitory effect on enzyme activity toward 1-chloro-2,4-dinitrobenzene and cumene hydroperoxide. Different amino acids of the H-site had different effects on enzyme activity toward a different substrate, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. Moreover, Leu112 and Phe162 were found to inhibit the catalytic efficiency of OsGSTU17 to 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, while Pro16, Leu112, and Trp165 contributed to structural stability. The results of this research enhance the understanding of the relationship between the structure and function of tau class GSTs to improve the abiotic stress resistance of crops.

Description: MicroRNAs (miRNAs) are short noncoding RNA molecules that regulate gene expression at the posttranscriptional level. Reverse transcription-quantitative PCR (RT-qPCR) is one of the most common methods used for quantification of miRNA expression, and the levels of expression are normalized by comparing with reference genes. Thus, the selection of reference genes is critically important for accurate quantification. The present study was intended to identify appropriate miRNA reference genes for normalizing the level of miRNA expression in Citrus sinensis L. Osbeck and Citrus reticulata Blanco infected by Xanthomonas citri subsp. citri, which caused citrus canker disease. Five algorithms (Delta Ct, geNorm, NormFinder, BestKeeper and RefFinder) were used for screening reference genes, and two quantification approaches, poly(A) extension RT-qPCR and stem-loop RT-qPCR, were used to determine the most appropriate method for detecting expression patterns of miRNA. An overall comprehensive ranking output derived from the multi-algorithms showed that poly(A)-tailed miR162-3p/miR472 were the best reference gene combination for miRNA RT-qPCR normalization in citrus canker research. Candidate reference gene expression profiles determined by poly(A) RT-qPCR were more consistent in the two citrus species. To the best of our knowledge, this is the first systematic comparison of two miRNA quantification methods for evaluating reference genes. These results highlight the importance of rigorously assessing candidate reference genes and clarify some contradictory results in miRNA research on citrus.

Description: Vulpinic acid, a naturally occurring methyl ester of pulvinic acid, has been reported to exert anti-fungal, anti-cancer, and anti-oxidative effects. However, its metabolic action has not been implicated yet. Here, we show that vulpinic acid derived from a mushroom, Pulveroboletus ravenelii controls the cell fate of mesenchymal stem cells and preadipocytes by inducing the acetylation of histone H3 and α-tubulin, respectively. The treatment of 10T1/2 mesenchymal stem cells with vulpinic acid increased the expression of Wnt6, Wnt10a, and Wnt10b, which led to osteogenesis inhibiting the adipogenic lineage commitment, through the upregulation of H3 acetylation. By contrast, treatment with vulpinic acid promoted the terminal differentiation of 3T3-L1 preadipocytes into mature adipocytes. In this process, the increase in acetylated tubulin was accompanied, while acetylated H3 was not altered. As excessive generation of adipocytes occurs, the accumulation of lipid drops was not concentrated, but dispersed into a number of adipocytes. Consistently, the expressions of lipolytic genes were upregulated and inflammatory factors were downregulated in adipocytes exposed to vulpinic acid during adipogenesis. These findings reveal the multiple actions of vulpinic acid in two stages of differentiation, promoting the osteogenesis of mesenchymal stem cells and decreasing hypertrophic adipocytes, which can provide experimental evidence for the novel metabolic advantages of vulpinic acid.

Description: The human papillomaviruses (HPV) are a group of double-stranded DNA viruses that exhibit an exclusive tropism for squamous epithelia. HPV can either be low- or high-risk depending on its ability to cause benign lesions or cancer, respectively. Unsurprisingly, the majority of epigenetic research has focused on the high-risk HPV types, neglecting the low-risk types in the process. Therefore, the main objective of this study is to better understand the epigenetics of wart formation by investigating the differences in methylation between HPV-induced cutaneous warts and normal skin. A number of clear and very significant differences in methylation patterns were found between cutaneous warts and normal skin. Around 55% of the top-ranking 100 differentially methylated genes in warts were protein coding, including the EXOC4, KCNU, RTN1, LGI1, IRF2, and NRG1 genes. Additionally, non-coding RNA genes, such as the AZIN1-AS1, LINC02008, and MGC27382 genes, constituted 11% of the top-ranking 100 differentially methylated genes. Warts exhibited a unique pattern of methylation that is a possible explanation for their transient nature. Since the genetics of cutaneous wart formation are not completely known, the findings of the present study could contribute to a better understanding of how HPV infection modulates host methylation to give rise to warts in the skin.

Description: One of the primary aims of the Functional Annotation of ANimal Genomes (FAANG) initiative is to characterize tissue-specific regulation within animal genomes. To this end, we used chromatin immunoprecipitation followed by sequencing (ChIP-Seq) to map four histone modifications (H3K4me1, H3K4me3, H3K27ac, and H3K27me3) in eight prioritized tissues collected as part of the FAANG equine biobank from two thoroughbred mares. Data were generated according to optimized experimental parameters developed during quality control testing. To ensure that we obtained sufficient ChIP and successful peak-calling, data and peak-calls were assessed using six quality metrics, replicate comparisons, and site-specific evaluations. Tissue specificity was explored by identifying binding motifs within unique active regions, and motifs were further characterized by gene ontology (GO) and protein–protein interaction analyses. The histone marks identified in this study represent some of the first resources for tissue-specific regulation within the equine genome. As such, these publicly available annotation data can be used to advance equine studies investigating health, performance, reproduction, and other traits of economic interest in the horse.

Description: The giant freshwater prawn (Macrobrachium rosenbergii) exhibits sex dimorphism between the male and female individuals. To date, the molecular mechanism governing gonadal development was unclear, and limited data were available on the gonad transcriptome of M. rosenbergii. Here, we conducted comprehensive gonadal transcriptomic analysis of female (ZW), super female (WW), and male (ZZ) M. rosenbergii for gene discovery. A total of 70.33 gigabases (Gb) of sequences were generated. There were 115,338 unigenes assembled with a mean size of 1196 base pair (bp) and N50 of 2195 bp. Alignment against the National Center for Biotechnology Information (NCBI) non-redundant nucleotide/protein sequence database (NR and NT), the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, SwissProt database, Protein family (Pfam), Gene ontology (GO), and the eukaryotic orthologous group (KOG) database, 36,282 unigenes were annotated at least in one database. Comparative transcriptome analysis observed that 10,641, 16,903, and 3393 genes were significantly differentially expressed in ZW vs. ZZ, WW vs. ZZ, and WW vs. ZW samples, respectively. Enrichment analysis of differentially expressed genes (DEGs) resulted in 268, 153, and 42 significantly enriched GO terms, respectively, and a total of 56 significantly enriched KEGG pathways. Additionally, 23 putative sex-related genes, including Gtsf1, IR, HSP21, MRPINK, Mrr, and other potentially promising candidate genes were identified. Moreover, 56,241 simple sequence repeats (SSRs) were identified. Our findings provide a valuable archive for further functional analyses of sex-related genes and future discoveries of underlying molecular mechanisms of gonadal development and sex determination.

Description: Hearing loss is a genetically heterogeneous disorder affecting approximately 360 million people worldwide and is among the most common sensorineural disorders. Here, we report a genetic analysis of seven large consanguineous families segregating prelingual sensorineural hearing loss. Whole-exome sequencing (WES) revealed seven different pathogenic variants segregating with hearing loss in these families, three novel variants (c.1204G〉A, c.322G〉T, and c.5587C〉T) in TMPRSS3, ESRRB, and OTOF, and four previously reported variants (c.208C〉T, c.6371G〉A, c.226G〉A, and c.494C〉T) in LRTOMT, MYO15A, KCNE1, and LHFPL5, respectively. All identified variants had very low frequencies in the control databases and were predicted to have pathogenic effects on the encoded proteins. In addition to being familial, we also found intersibship locus heterogeneity in the evaluated families. The known pathogenic c.226C〉T variant identified in KCNE1 only segregates with the hearing loss phenotype in a subset of affected members of the family GCNF21. This study further highlights the challenges of identifying disease-causing variants for highly heterogeneous disorders and reports the identification of three novel and four previously reported variants in seven known deafness genes.

Description: To compare in vivo the infection process of Monilinia fructicola on nectarines and apples using confocal microscopy it is necessary to transform a pathogenic strain with a construct expressing a fluorescent chromophore such as GFP. Thus, germinated conidia of the pathogen were transformed with Agrobacterium tumefaciens carrying the plasmid pPK2-hphgfp that allowed the expression of a fluorescent Hph-GFP chimera. The transformants were selected according to their resistance to hygromycin B, provided by the constitutive expression of the hph-gfp gene driven by the glyceraldehyde 3P dehydrogenase promoter of Aspergillus nidulans. The presence of T-DNA construct in the genomic DNA was confirmed by PCR using a range of specific primers. Subsequent PCR-mediated analyses proved integration of the transgene at a different genomic location in each transformant and the existence of structural reorganizations at these insertion points. The expression of Hph-GFP in three independent M. fructicola transformants was monitored by immunodetection and epifluorescence and confocal microscopy. The Atd9-M. fructicola transformant displayed no morphological defects and showed growth and pathogenic characteristics similar to the wild type. Microscopy analysis of the Atd9 transformant evidenced that nectarine infection by M. fructicola was at least three times faster than on apples.

Description: Retinoblastoma (RB) is an inherited retinal disorder (IRD) caused by the mutation in the RB1 gene or, rarely, by alterations in the MYCN gene. In recent years, new treatment advances have increased ocular and visual preservation in the developed world. The management of RB has improved significantly in recent decades, from the use of external beam radiation to recently, more localized treatments. Determining the underlying genetic cause of RB is critical for timely management decisions. The advent of next-generation sequencing technologies have assisted in understanding the molecular pathology of RB. Liquid biopsy of the aqueous humor has also had significant potential implications for tumor management. Currently, patients’ genotypic information, along with RB phenotypic presentation, are considered carefully when making treatment decisions aimed at globe preservation. Advances in molecular testing that improve our understanding of the molecular pathology of RB, together with multiple directed treatment options, are critical for developing precision medicine strategies to treat this disease.

Description: Quinoa (Chenopodium quinoa Willd.), a model halophytic crop species, was used to shed light on salt tolerance mechanisms at the transcriptomic level. An RNA-sequencing analysis of genotype R49 at an early vegetative stage was performed by Illumina paired-ends method comparing high salinity and control conditions in a time-course pot experiment. Genome-wide transcriptional salt-induced changes and expression profiling of relevant salt-responsive genes in plants treated or not with 300 mM NaCl were analyzed after 1 h and 5 days. We obtained up to 49 million pairs of short reads with an average length of 101 bp, identifying a total of 2416 differentially expressed genes (DEGs) based on the treatment and time of sampling. In salt-treated vs. control plants, the total number of up-regulated and down-regulated genes was 945 and 1471, respectively. The number of DEGs was higher at 5 days than at 1 h after salt treatment, as reflected in the number of transcription factors, which increased with time. We report a strong transcriptional reprogramming of genes involved in biological processes like oxidation-reduction, response to stress and response to abscisic acid (ABA), and cell wall organization. Transcript analyses by real-time RT- qPCR supported the RNA-seq results and shed light on the contribution of roots and shoots to the overall transcriptional response. In addition, it revealed a time-dependent response in the expression of the analyzed DEGs, including a quick (within 1 h) response for some genes, suggesting a “stress-anticipatory preparedness” in this highly salt-tolerant genotype.

Description: Women with BRCA variants have a high lifetime risk of developing breast and ovarian cancer. The aim of this study was to investigate the standard incidence ratios (SIR) for breast and ovarian cancer and standard mortality ratios (SMR) in a population-based cohort of women in Western Sweden, under surveillance and after risk reducing surgery. Women who tested positive for a BRCA variant between 1995–2016 (n = 489) were prospectively registered and followed up for cancer incidence, risk reducing surgery and mortality. The Swedish Cancer Register was used to compare breast and ovarian cancer incidence and mortality with and without risk reducing surgery for women with BRCA variants in comparison to women in the general population. SIR for breast cancer under surveillance until risk-reducing mastectomy (RRM) was 14.0 (95% CI 9.42–20.7) and decreased to 1.93 (95% CI 0.48–7.7) after RRM. The SIR for ovarian cancer was 124.6 (95% CI 59.4–261.3) under surveillance until risk reducing salpingo-oophorectomy (RRSO) and decreased to 13.5 (95% CI 4.34–41.8) after RRSO. The SMR under surveillance before any risk reducing surgery was 5.56 (95% 2.09–14.8) and after both RRM and RRSO 4.32 (95% CI 1.62–11.5). Women with cancer diagnoses from the pathology report after risk reducing surgery were excluded from the analyses. Risk reducing surgery reduced the incidence of breast and ovarian cancer in women with BRCA variants. However, overall mortality was significantly increased in comparison to the women in the general population and remained elevated even after risk reducing surgery. These findings warrant further research regarding additional measures for these women.

Description: Resolving the origin of endangered taxa is an essential component of conservation. This information can be used to guide efforts of bolstering genetic diversity, and also enables species recovery and future evolutionary studies. Here, we used low-coverage whole genome sequencing to clarify the origin of Helianthus schweinitzii, an endangered tetraploid sunflower that is endemic to the Piedmont Plateau in the eastern United States. We surveyed four accessions representing four populations of H. schweinitzii and 38 accessions of six purported parental species. Using de novo approaches, we assembled 87,004 bp of the chloroplast genome and 6770 bp of the nuclear 35S rDNA. Phylogenetic reconstructions based on the chloroplast genome revealed no reciprocal monophyly of taxa. In contrast, nuclear rDNA data strongly supported the currently accepted sections of the genus Helianthus. Information from combined cpDNA and rDNA provided evidence that H. schweinitzii is likely an allo-tetraploid that formed as a result of hybridization between the diploids Helianthus giganteus and Helianthus microcephalus.

Description: The xerophyte Pugionium cornutum adapts to salt stress by accumulating inorganic ions (e.g., Cl−) for osmotic adjustment and enhancing the activity of antioxidant enzymes, but the associated molecular basis remains unclear. In this study, we first found that P. cornutum could also maintain cell membrane stability due to its prominent ROS-scavenging ability and exhibits efficient carbon assimilation capacity under salt stress. Then, the candidate genes associated with the important physiological traits of the salt tolerance of P. cornutum were identified through transcriptomic analysis. The results showed that after 50 mM NaCl treatment for 6 or 24 h, multiple genes encoding proteins facilitating Cl− accumulation and NO3− homeostasis, as well as the transport of other major inorganic osmoticums, were significantly upregulated in roots and shoots, which should be favorable for enhancing osmotic adjustment capacity and maintaining the uptake and transport of nutrient elements; a large number of genes related to ROS-scavenging pathways were also significantly upregulated, which might be beneficial for mitigating salt-induced oxidative damage to the cells. Meanwhile, many genes encoding components of the photosynthetic electron transport pathway and carbon fixation enzymes were significantly upregulated in shoots, possibly resulting in high carbon assimilation efficiency in P. cornutum. Additionally, numerous salt-inducible transcription factor genes that probably regulate the abovementioned processes were found. This work lays a preliminary foundation for clarifying the molecular mechanism underlying the adaptation of xerophytes to harsh environments.

Description: As a plant-specific transcription factor, the NAC (NAM, ATAF1/2 and CUC2) domain protein plays an important role in plant growth and development, as well as stress resistance. Based on the genomic data of the cacao tree, this study identified 102 cacao NAC genes and named them according to their location within the genome. The phylogeny of the protein sequence of the cacao tree NAC family was analyzed using various bioinformatic methods, and then divided into 12 subfamilies. Then, the amino-acid composition, physicochemical properties, genomic location, gene structure, conserved domains, and promoter cis-acting elements were analyzed. This study provides information on the evolution of the TcNAC gene and its possible functions, laying the foundation for further research on the NAC family.

Description: Gastropod molluscs, which have co-evolved with parasitic digenean trematodes for millions of years, utilize circulating heamocytes as the primary method of containing and killing these invading parasites. In order to do so, they must generate suitable amounts of haemocytes that are properly armed to kill parasitic worms. One method by which they generate the haemocytes required to initiate the appropriate cell mediated immune response is via the production and post-translational processing of granulins. Granulins are an evolutionarily conserved family of growth factors present in the majority of eukaryotic life forms. In their pro-granulin form, they can elicit cellular replication and differentiation. The pro-granulins can be further processed by elastase to generate smaller granulin fragments that have been shown to functionally differ from the pro-granulin precursor. In this study, we demonstrate that in vivo addition of Biomphalaria glabrata pro-granulin (BgGRN) can reduce Schistosoma mansoni infection success in numerous Biomphalaria sp. when challenged with different S. mansoni strains. We also demonstrate that cleavage of BgGRN into individual granulin subunits by elastase results in the stimulation of haemocytes to produce reactive oxygen species.

Description: Cave animals are a fascinating group of species often demonstrating characteristics including reduced eyes and pigmentation, metabolic efficiency, and enhanced sensory systems. Asellus aquaticus, an isopod crustacean, is an emerging model for cave biology. Cave and surface forms of this species differ in many characteristics, including eye size, pigmentation, and antennal length. Existing resources for this species include a linkage map, mapped regions responsible for eye and pigmentation traits, sequenced adult transcriptomes, and comparative embryological descriptions of the surface and cave forms. Our ultimate goal is to identify genes and mutations responsible for the differences between the cave and surface forms. To advance this goal, we decided to use a transcriptomic approach. Because many of these changes first appear during embryonic development, we sequenced embryonic transcriptomes of cave, surface, and hybrid individuals at the stage when eyes and pigment become evident in the surface form. We generated a cave, a surface, a hybrid, and an integrated transcriptome to identify differentially expressed genes in the cave and surface forms. Additionally, we identified genes with allele-specific expression in hybrid individuals. These embryonic transcriptomes are an important resource to assist in our ultimate goal of determining the genetic underpinnings of the divergence between the cave and surface forms.

Description: Enhancer-promoter interactions (EPIs) are crucial for transcriptional regulation. Mapping such interactions proves useful for understanding disease regulations and discovering risk genes in genome-wide association studies. Some previous studies showed that machine learning methods, as computational alternatives to costly experimental approaches, performed well in predicting EPIs from local sequence and/or local epigenomic data. In particular, deep learning methods were demonstrated to outperform traditional machine learning methods, and using DNA sequence data alone could perform either better than or almost as well as only utilizing epigenomic data. However, most, if not all, of these previous studies were based on randomly splitting enhancer-promoter pairs as training, tuning, and test data, which has recently been pointed out to be problematic; due to multiple and duplicating/overlapping enhancers (and promoters) in enhancer-promoter pairs in EPI data, such random splitting does not lead to independent training, tuning, and test data, thus resulting in model over-fitting and over-estimating predictive performance. Here, after correcting this design issue, we extensively studied the performance of various deep learning models with local sequence and epigenomic data around enhancer-promoter pairs. Our results confirmed much lower performance using either sequence or epigenomic data alone, or both, than reported previously. We also demonstrated that local epigenomic features were more informative than local sequence data. Our results were based on an extensive exploration of many convolutional neural network (CNN) and feed-forward neural network (FNN) structures, and of gradient boosting as a representative of traditional machine learning.

Description: Musculocontractural Ehlers–Danlos Syndome (mcEDS) is a type of EDS caused by biallelic pathogenic variants in the gene for carbohydrate sulfotransferase 14/dermatan 4-O-sulfotransferase 1 (CHST14/D4ST1, mcEDS-CHST14), or in the gene for dermatan sulfate epimerase (DSE, mcEDS-DSE). Thus far, 41 patients from 28 families with mcEDS-CHST14 and five patients from four families with mcEDS-DSE have been described in the literature. Clinical features comprise multisystem congenital malformations and progressive connective tissue fragility-related manifestations. This review outlines recent advances in understanding the pathophysiology of mcEDS. Pathogenic variants in CHST14 or DSE lead to reduced activities of relevant enzymes, resulting in a negligible amount of dermatan sulfate (DS) and an excessive amount of chondroitin sulfate. Connective tissue fragility is presumably attributable to a compositional change in the glycosaminoglycan chains of decorin, a major DS-proteoglycan in the skin that contributes to collagen fibril assembly. Collagen fibrils in affected skin are dispersed in the papillary to reticular dermis, whereas those in normal skin are regularly and tightly assembled. Glycosaminoglycan chains are linear in affected skin, stretching from the outer surface of collagen fibrils to adjacent fibrils; glycosaminoglycan chains are curved in normal skin, maintaining close contact with attached collagen fibrils. Homozygous (Chst14−/−) mice have been shown perinatal lethality, shorter fetal length and vessel-related placental abnormalities. Milder phenotypes in mcEDS-DSE might be related to a smaller fraction of decorin DS, potentially through residual DSE activity or compensation by DSE2 activity. These findings suggest critical roles of DS and DS-proteoglycans in the multisystem development and maintenance of connective tissues, and provide fundamental evidence to support future etiology-based therapies.

Description: Seed vigor is a key factor that determines the quality of seeds, which is of great significance for agricultural production, with the potential to promote growth and productivity. However, the underlying molecular mechanisms and genetic basis for seed vigor remain unknown. High-density genetic linkage mapping is an effective method for genomic study and quantitative trait loci (QTL) mapping. In this study, a high-density genetic map was constructed from a 148 BC4F3 population cross between ‘M03’ and ‘M08’ strains based on specific-locus amplified fragment (SLAF) sequencing. The constructed high-density genetic linkage map (HDGM) included 3876 SNP markers on ten chromosomes covering 2413.25 cM in length, with a mean distance between markers of 0.62 cM. QTL analysis was performed on four sweet corn germination traits that are related to seed vigor under artificial aging conditions. A total of 18 QTLs were identified in two seasons. Interestingly, a stable QTL was detected in two seasons on chromosome 10—termed qGR10—within an interval of 1.37 Mb. Within this interval, combined with gene annotation, we found four candidate genes (GRMZM2G074309, GRMZM2G117319, GRMZM2G465812, and GRMZM2G343519) which may be related to seed vigor after artificial aging.

Description: Anti-Mullerian hormone (AMH) is an important reproductive marker of ovarian reserve produced by granulosa cells (GCs) of pre-antral and early-antral ovarian follicles in several species, including cattle. This hormone plays a vital role during the recruitment of primordial follicles and follicle stimulating hormone (FSH)-dependent follicular growth. However, the regulatory mechanism of AMH expression in follicles is still unclear. In this study, we compared the expression of AMH, AMHR-II, BMP2, BMP6, FSHR, and LHCGR genes during follicular development. In-vitro expression study was performed with and without FSH for AMH, AMHR-II, BMP2, and BMP6 genes in bovine GCs which were isolated from 3–8 mm follicles. Association among the mRNA expression and hormone level was estimated. GCs were collected from small (3–8 mm), medium (9–12 mm) and large size (13 to 24 mm) follicles before, during onset, and after deviation, respectively. Further, mRNA expression, hormones (AMH, FSH, and LH), apoptosis of GCs, and cell viability were detected by qRT-PCR, ELISA, flow cytometry, and spectrophotometry. AMH, AMHR-II, BMP2, and FSHR genes were highly expressed in small and medium follicles as compared to large ones. In addition, the highest level of AMH protein (84.14 ± 5.41 ng/mL) was found in medium-size follicles. Lower doses of FSH increased the viability of bovine GCs while higher doses repressed them. In-vitro cultured GCs treated with FSH significantly increased the AMH, AMHR-II, and BMP2 expression levels at lower doses, while expression levels decreased at higher doses. We found an optimum level of FSH (25 ng/mL) which can significantly enhance AMH and BMP2 abundance (p 〈 0.05). In summary, AMH, AMHR-II, and BMP2 genes showed a higher expression in follicles developed in the presence of FSH. However, lower doses of FSH demonstrated a stimulatory effect on AMH and BMP2 expression, while expression started to decline at the maximum dose. In this study, we have provided a better understanding of the mechanisms regulating AMH, AMHR II, and BMP2 signaling in GCs during folliculogenesis, which would improve the outcomes of conventional assisted reproductive technologies (ARTs), such as superovulation and oestrus synchronization in bovines.

Description: Drought stress is one of the main factors limiting crop production, which provokes a number of changes in plants at physiological, anatomical, biochemical and molecular level. To unravel the various mechanisms underpinning tobacco (Nicotiana tabacum L.) drought stress tolerance, we conducted a comprehensive physiological, anatomical, biochemical and transcriptome analyses of three tobacco cultivars (i.e., HongHuaDaJinYuan (H), NC55 (N) and Yun Yan-100 (Y)) seedlings that had been exposed to drought stress. As a result, H maintained higher growth in term of less reduction in plant fresh weight, dry weight and chlorophyll content as compared with N and Y. Anatomical studies unveiled that drought stress had little effect on H by maintaining proper leaf anatomy while there were significant changes in the leaf anatomy of N and Y. Similarly, H among the three varieties was the least affected variety under drought stress, with more proline content accumulation and a powerful antioxidant defense system, which mitigates the negative impacts of reactive oxygen species. The transcriptomic analysis showed that the differential genes expression between HongHuaDaJinYuan, NC55 and Yun Yan-100 were enriched in the functions of plant hormone signal transduction, starch and sucrose metabolism, and arginine and proline metabolism. Compared to N and Y, the differentially expressed genes of H displayed enhanced expression in the corresponding pathways under drought stress. Together, our findings offer insights that H was more tolerant than the other two varieties, as evidenced at physiological, biochemical, anatomical and molecular level. These findings can help us to enhance our understanding of the molecular mechanisms through the networks of various metabolic pathways mediating drought stress adaptation in tobacco.

Description: Aim: To identify disease-causing mutations in four Lebanese families: three families with Bardet–Biedl and one family with Usher syndrome (BBS and USH respectively), using next generation sequencing (NGS). Methods: We applied targeted NGS in two families and whole exome sequencing (WES) in two other families. Pathogenicity of candidate mutations was evaluated according to frequency, conservation, in silico prediction tools, segregation with disease, and compatibility with inheritance pattern. The presence of pathogenic variants was confirmed via Sanger sequencing followed by segregation analysis. Results: Most likely disease-causing mutations were identified in all included patients. In BBS patients, we found (M1): c.2258A 〉 T, p. (Glu753Val) in BBS9, (M2): c.68T 〉 C; p. (Leu23Pro) in ARL6, (M3): c.265_266delTT; p. (Leu89Valfs*11) and (M4): c.880T 〉 G; p. (Tyr294Asp) in BBS12. A previously known variant (M5): c.551A 〉 G; p. (Asp184Ser) was also detected in BBS5. In the USH patient, we found (M6): c.188A 〉 C, p. (Tyr63Ser) in CLRN1. M2, M3, M4, and M6 were novel. All of the candidate mutations were shown to be likely disease-causing through our bioinformatic analysis. They also segregated with the corresponding phenotype in available family members. Conclusion: This study expanded the mutational spectrum and showed the genetic diversity of BBS and USH. It also spotlighted the efficiency of NGS techniques in revealing mutations underlying clinically and genetically heterogeneous disorders.

Description: Notch is a key factor of a signaling cascade which regulates cell differentiation in all multicellular organisms. Numerous investigations have been directed mainly at studying the mechanism of Notch protein action; however, very little is known about the regulation of activity of the gene itself. Here, we provide the results of targeted 5’-end editing of the Drosophila Notch gene in its native environment and genetic and cytological effects of these changes. Using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9 (CRISPR/Cas9) system in combination with homologous recombination, we obtained a founder fly stock in which a 4-kb fragment, including the 5’ nontranscribed region, the first exon, and a part of the first intron of Notch, was replaced by an attachment Phage (attP) site. Then, fly lines carrying a set of six deletions within the 5’untranscribed region of the gene were obtained by ΦC31-mediated integration of transgenic constructs. Part of these deletions does not affect gene activity, but their combinations with transgenic construct in the first intron of the gene cause defects in the Notch target tissues. At the polytene chromosome level we defined a DNA segment (~250 bp) in the Notch5’-nontranscribed region which when deleted leads to disappearance of the 3C6/C7 interband and elimination of CTC-Factor (CTCF) and Chromator (CHRIZ) insulator proteins in this region.

Description: ICEpMERPH, the first integrative conjugative element (ICE) of the SXT/R391 familyisolated in the United Kingdom and Europe, was analyzed to determine the nature of its adaptivefunctions, its genetic structure, and its homology to related elements normally found in pathogenicVibrio or Proteus species. Whole genome sequencing of Escherichia coli (E. coli) isolate K802 (whichcontains the ICEpMERPH) was carried out using Illumina sequencing technology. ICEpMERPH hasa size of 110 Kb and 112 putative open reading frames (ORFs). The “hotspot regions” of the elementwere found to contain putative restriction digestion systems, insertion sequences, and heavy metalresistance genes that encoded resistance to mercury, as previously reported, but also surprisinglyto arsenate. A novel arsenate resistance system was identified in hotspot 4 of the element, unrelatedto other SXT/R391 elements. This arsenate resistance system was potentially linked to two genes:orf69, encoding an organoarsenical efflux major facilitator superfamily (MFS) transporter‐likeprotein related to ArsJ, and orf70, encoding nicotinamide adenine dinucleotide (NAD)‐dependentglyceraldehyde‐3‐phosphate dehydrogenase. Phenotypic analysis using isogenic strains ofEscherichia coli strain AB1157 with and without the ICEpMERPH revealed resistance to low levels ofarsenate in the range of 1–5 mM. This novel, low‐level resistance may have an important adaptivefunction in polluted environments, which often contain low levels of arsenate contamination. Abioinformatic analysis on the novel determinant and the phylogeny of ICEpMERPH was presented.

Description: This study was designed to establish a real-time quantitative polymerase chain reaction (qPCR) method to rapidly and reliably analyze the hypoglycemic polypeptide-P gene expression pattern in Momordica charantia (MC) and to examine its expression changes in different MC accessions, harvesting seasons, and tissue types. The qPCR results were further verified by using Western blotting (WB). A total of 10 MCs with different accessions were collected and tested in this study. Among the tested accessions, RU5H showed the highest expression level of the polypeptide-P gene. The expression level of the polypeptide-P gene was not only season-related (with the highest in early July) but also tissue-related (with the highest in the seed tissue). In addition, the expression characteristic of the polypeptide-P gene was maturity-related, with the highest expression level in the tender MC. The WB results show that the transcription level of this gene shows an almost similar trend to the corresponding protein expression level. In conclusion, the established qPCR method can rapidly and effectively detect the expression levels of the polypeptide-P gene in MCs with different accessions; furthermore, various factors, including the accessions, harvesting seasons, and tissue types can affect the expression level.

Description: Background: Staphylococcus aureus is a highly prevalent respiratory pathogen in cystic fibrosis (CF). It is unclear how this organism establishes chronic infections in CF airways. We hypothesized that S. aureus isolates from patients with CF would share common virulence properties that enable chronic infection. Methods: 77 S. aureus isolates were obtained from 45 de-identified patients with CF at the University of Iowa. We assessed isolates phenotypically and used genotyping assays to determine the presence or absence of 18 superantigens (SAgs). Results: We observed phenotypic diversity among S. aureus isolates from patients with CF. Genotypic analysis for SAgs revealed 79.8% of CF clinical isolates carried all six members of the enterotoxin gene cluster (EGC). MRSA and MSSA isolates had similar prevalence of SAgs. We additionally observed that EGC SAgs were prevalent in S. aureus isolated from two geographically distinct CF centers. Conclusions: S. aureus SAgs belonging to the EGC are highly prevalent in CF clinical isolates. The greater prevalence in these SAgs in CF airway specimens compared to skin isolates suggests that these toxins confer selective advantage in the CF airway.

Description: De novo shoot regeneration is one of the important manifestations of cell totipotency in organogenesis, which reflects a survival strategy organism evolved when facing natural selection. Compared with tissue regeneration, and somatic embryogenesis, de novo shoot regeneration denotes a shoot regeneration process directly from detatched or injured tissues of plant. Studies on plant shoot regeneration had identified key genes mediating shoot regeneration. However, knowledge was derived from Arabidopsis; the regeneration capacity is hugely distinct among species. To achieve a comprehensive understanding of the shoot regeneration mechanism from tree species, we select four genetic lines of Populus euphratica from a natural population to be sequenced at transcriptome level. On the basis of the large difference of differentiation capacity, between the highly differentiated (HD) and low differentiated (LD) groups, the analysis of differential expression identified 4920 differentially expressed genes (DEGs), which were revealed in five groups of expression patterns by clustering analysis. Enrichment showed crucial pathways involved in regulation of regeneration difference, including “plant hormone signal transduction”, “cell differentiation”, "cellular response to auxin stimulus", and “auxin-activated signaling pathway”. The expression of nine genes reported to be associated with shoot regeneration was validated using quantitative real-time PCR (qRT-PCR). For the specificity of regeneration mechanism with P. euphratica, large amount of DEGs involved in "plant-pathogen interaction", ubiquitin-26S proteosome mediated proteolysis pathway, stress-responsive DEGs, and senescence-associated DEGs were summarized to possibly account for the differentiation difference with distinct genotypes of P. euphratica. The result in this study helps screening of key regulators in mediating the shoot differentiation. The transcriptomic characteristic in P. euphratica further enhances our understanding of key processes affecting the regeneration capacity of de novo shoots among distinct species.

Description: Wing dimorphism is considered as an adaptive trait of insects. Brown planthoppers (BPHs) Nilaparvata lugens, a serious pest of rice, are either macropterous or brachypterous. Genetic and environmental factors are both likely to control wing morph determination in BPHs, but the hereditary law and genes network are still unknown. Here, we investigated changes in gene expression levels between macropterous and brachypterous BPHs by creating artificially bred morphotype lines. The nearly pure-bred strains of macropterous and brachypterous BPHs were established, and their transcriptomes and gene expression levels were compared. Over ten-thousand differentially expressed genes (DEGs) between macropterous and brachypterous strains were found in the egg, nymph, and adult stages, and the three stages shared 6523 DEGs. The regulation of actin cytoskeleton, focal adhesion, tight junction, and adherens junction pathways were consistently enriched with DEGs across the three stages, whereas insulin signaling pathway, metabolic pathways, vascular smooth muscle contraction, platelet activation, oxytocin signaling pathway, sugar metabolism, and glycolysis/gluconeogenesis were significantly enriched by DEGs in a specific stage. Gene expression trend profiles across three stages were different between the two strains. Eggs, nymphs, and adults from the macropterous strain were distinguishable from the brachypterous based on gene expression levels, and genes that were related to wing morphs were differentially expressed between wing strains or strain × stage. A proposed mode based on genes and environments to modulate the wing dimorphism of BPHs was provided.

Description: NAC proteins are one of the largest families of plant-specific transcription factors (TFs). They regulate diverse complex biological processes, including secondary xylem differentiation and wood formation. Recent genomic and transcriptomic studies of Tectona grandis L.f. (teak), one of the most valuable hardwood trees in the world, have allowed identification and analysis of developmental genes. In the present work, T. grandis NAC genes were identified and analyzed regarding to their evolution and expression profile during wood formation. We analyzed the recently published T. grandis genome, and identified 130 NAC proteins that are coded by 107 gene loci. These proteins were classified into 23 clades of the NAC family, together with Populus, Eucalyptus, and Arabidopsis. Data on transcript expression revealed specific temporal and spatial expression patterns for the majority of teak NAC genes. RT-PCR indicated expression of VND genes (Tg11g04450-VND2 and Tg15g08390-VND4) related to secondary cell wall formation in xylem vessels of 16-year-old juvenile trees. Our findings open a way to further understanding of NAC transcription factor genes in T. grandis wood biosynthesis, while they are potentially useful for future studies aiming to improve biomass and wood quality using biotechnological approaches.

Description: Two of the most economically important plants in the Artocarpus genus are jackfruit (A. heterophyllus Lam.) and breadfruit (A. altilis (Parkinson) Fosberg). Both species are long-lived trees that have been cultivated for thousands of years in their native regions. Today they are grown throughout tropical to subtropical areas as an important source of starch and other valuable nutrients. There are hundreds of breadfruit varieties that are native to Oceania, of which the most commonly distributed types are seedless triploids. Jackfruit is likely native to the Western Ghats of India and produces one of the largest tree-borne fruit structures (reaching up to 45 kg). To-date, there is limited genomic information for these two economically important species. Here, we generated 273 Gb and 227 Gb of raw data from jackfruit and breadfruit, respectively. The high-quality reads from jackfruit were assembled into 162,440 scaffolds totaling 982 Mb with 35,858 genes. Similarly, the breadfruit reads were assembled into 180,971 scaffolds totaling 833 Mb with 34,010 genes. A total of 2822 and 2034 expanded gene families were found in jackfruit and breadfruit, respectively, enriched in pathways including starch and sucrose metabolism, photosynthesis, and others. The copy number of several starch synthesis-related genes were found to be increased in jackfruit and breadfruit compared to closely-related species, and the tissue-specific expression might imply their sugar-rich and starch-rich characteristics. Overall, the publication of high-quality genomes for jackfruit and breadfruit provides information about their specific composition and the underlying genes involved in sugar and starch metabolism.

Description: A challenge in molecular diagnosis and genetic counseling is the interpretation of variants of uncertain significance. Proper pathogenicity classification of new variants is important for the conclusion of molecular diagnosis and the medical management of patient treatments. The purpose of this study was to reclassify two RPE65 missense variants, c.247T〉C (p.Phe83Leu) and c.560G〉A (p.Gly187Glu), found in Brazilian families. To achieve this aim, we reviewed the sequencing data of a 224-gene retinopathy panel from 556 patients (513 families) with inherited retinal dystrophies. Five patients with p.Phe83Leu and seven with p.Gly187Glu were selected and their families investigated. To comprehend the pathogenicity of these variants, we evaluated them based on the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) classification guidelines. Initially, these RPE65 variants met only three pathogenic criteria: (i) absence or low frequency in the population, (ii) several missense pathogenic RPE65 variants, and (iii) 15 out of 16 lines of computational evidence supporting them as damaging, which together allowed the variants to be classified as uncertain significance. Two other pieces of evidence were accepted after further analysis of these Brazilian families: (i) p.Phe83Leu and p.Gly187Glu segregate with childhood retinal dystrophy within families, and (ii) their prevalence in Leber congenital amaurosis (LCA)/early-onset retinal dystrophy (EORD) patients can be considered higher than in other inherited retinal dystrophy patients. Therefore, these variants can now be classified as likely pathogenic according to ACMG/AMP classification guidelines.

Description: Alfalfa (Medicago sativa L.) is a high quality leguminous forage. Drought stress is one of the main factors that restrict the development of the alfalfa industry. High-throughput sequencing was used to analyze the microRNA (miRNA) profiles of alfalfa plants treated with CK (normal water), PEG (polyethylene glycol-6000; drought stress), and PEG + SNP (sodium nitroprusside; nitric oxide (NO) sprayed externally under drought stress). We identified 90 known miRNAs belonging to 46 families and predicted 177 new miRNAs. Real-time quantitative fluorescent PCR (qRT-PCR) was used to validate high-throughput expression analysis data. A total of 32 (14 known miRNAs and 18 new miRNAs) and 55 (24 known miRNAs and 31 new miRNAs) differentially expressed miRNAs were identified in PEG and PEG + SNP samples. This suggested that exogenous NO can induce more new miRNAs. The differentially expressed miRNA maturation sequences in the two treatment groups were targeted by 86 and 157 potential target genes, separately. The function of target genes was annotated by gene ontology (GO) enrichment and kyoto encyclopedia of genes and genomes (KEGG) analysis. The expression profiles of nine selected miRNAs and their target genes verified that their expression patterns were opposite. This study has documented that analysis of miRNA under PEG and PEG + SNP conditions provides important insights into the improvement of drought resistance of alfalfa by exogenous NO at the molecular level. This has important scientific value and practical significance for the improvement of plant drought resistance by exogenous NO.

Description: Background: Hosta plantaginea (Lam.) Aschers (HPA), a species in the family Liliaceae, is an important landscaping plant and herbaceous ornamental flower. However, because the flower has only two colors, white and purple, color matching applications are extremely limited. To date, the mechanism underlying flower color regulation remains unclear. Methods: In this study, the transcriptomes of three cultivars—H. plantaginea (HP, white flower), H. Cathayana (HC, purple flower), and H. plantaginea ‘Summer Fragrance’ (HS, purple flower)—at three flowering stages (bud stage, initial stage, and late flowering stage) were sequenced with the Illumina HiSeq 2000 (San Diego, CA, USA). The RNA-Seq results were validated by qRT-PCR of eight differentially expressed genes (DEGs). Then, we further analyzed the relationship between anthocyanidin synthase (ANS), chalcone synthase (CHS), and P450 and the flower color regulation by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Eukaryotic Orthologous Groups (KOG) network and pathway enrichment analyses. The overexpression of CHS and ANS in transgenic tobacco petals was verified using qRT-PCR, and the petal colors associated with the overexpression lines were confirmed using absorbance values. Results: Over 434,349 transcripts were isolated, and 302,832 unigenes were identified. Additionally, through transcriptome comparisons, 2098, 722, and 606 DEGs between the different stages were found for HP, HC, and HS, respectively. Furthermore, GO and KEGG pathway analyses showed that 84 color-related DEGs were enriched in 22 pathways. In particular, the flavonoid biosynthetic pathway, regulated by CHS, ANS, and the cytochrome P450-type monooxygenase gene, was upregulated in both purple flower varieties in the late flowering stage. In contrast, this gene was hardly expressed in the white flower variety, which was verified in the CHS and ANS overexpression transgenic tobacco petals. Conclusions: The results suggest that CHS, ANS, and the cytochrome P450s-regulated flavonoid biosynthetic pathway might play key roles in the regulation of flower color in HPA. These insights into the mechanism of flower color regulation could be used to guide artificial breeding of polychrome varieties of ornamental flowers.

Description: Porcine epidemic diarrhea (PED) is a major gastrointestinal disease afflicting suckling pigs that causes huge industrial economic losses. In this study, we investigated microbiota from the colonic mucosa and content in healthy and PED piglets. High-throughput 16S rRNA gene sequencing was performed to identify inter-group differences. Firmicutes, Fusobacteria, Proteobacteria, and Bacteroidetes were the top four affected phyla. The proportion of Proteobacteria was higher in infected than in healthy piglets, and the opposite was observed for Bacteroidetes (more than four-fold higher in the healthy group). In the infected group, Fusobacterium accounted for 36.56% and 21.61% in the colonic mucosa and contents, respectively, while in the healthy group, they comprised 22.53% and 12.67%, respectively. The percentage of Lactobacillus in healthy colons (15.63%) was considerably higher than that in the disease group (

Description: Antioxidants play an important role in animal and plant life owing to their involvement in complex metabolic and signaling mechanisms, hence uncovering the genetic basis associated with antioxidant activity is very important for the development of improved varieties. Here, a total of 182 common bean (Phaseolus vulgaris) landraces and six commercial cultivars collected from 19 provinces of Turkey were evaluated for seed antioxidant activity under four environments and two locations. Antioxidant activity was measured using ABTS radical scavenging capacity and mean antioxidant activity in common bean landraces was 20.03 µmol TE/g. Analysis of variance reflected that genotype by environment interaction was statistically non-significant and heritability analysis showed higher heritability of antioxidant activity. Variations in seed color were observed, and a higher antioxidant activity was present in seeds having colored seed as compared to those having white seeds. A negative correlation was found between white-colored seeds and antioxidant activity. A total of 7900 DArTseq markers were used to explore the population structure that grouped the studied germplasm into two sub-populations on the basis of their geographical origins and trolox equivalent antioxidant capacity contents. Mean linkage disequilibrium (LD) was 54%, and mean LD decay was 1.15 Mb. Mixed linear model i.e., the Q + K model demonstrated that four DArTseq markers had significant association (p 〈 0.01) for antioxidant activity. Three of these markers were present on chromosome Pv07, while the fourth marker was located on chromosome Pv03. Among the identified markers, DArT-3369938 marker showed maximum (14.61%) variation. A total of four putative candidate genes were predicted from sequences reflecting homology to identified DArTseq markers. This is a pioneering study involving the identification of association for antioxidant activity in common bean seeds. We envisage that this study will be very helpful for global common bean breeding community in order to develop cultivars with higher antioxidant activity.

Description: In stock enhancement and sea-ranching procedures, the adipose fin of hundreds of millions of salmonids is removed for marking purposes annually. However, recent studies proved the significance of the adipose fin as a flow sensor and attraction feature. In the present study, we profiled the specific expression of 20 neuron- and glial cell-marker genes in the adipose fin and seven other tissues (including dorsal and pectoral fin, brain, skin, muscle, head kidney, and liver) of the salmonid species rainbow trout Oncorhynchus mykiss and maraena whitefish Coregonus maraena. Moreover, we measured the transcript abundance of genes coding for 15 mechanoreceptive channel proteins from a variety of mechanoreceptors known in vertebrates. The overall expression patterns indicate the presence of the entire repertoire of neurons, glial cells and receptor proteins on the RNA level. This quantification suggests that the adipose fin contains considerable amounts of small nerve fibers with unmyelinated or slightly myelinated axons and most likely mechanoreceptive potential. The findings are consistent for both rainbow trout and maraena whitefish and support a previous hypothesis about the innervation and potential flow sensory function of the adipose fin. Moreover, our data suggest that the resection of the adipose fin has a stronger impact on the welfare of salmonid fish than previously assumed.

Description: Rotavirus remains a major cause of diarrhea in infants and young children worldwide. The permanent emergence of new genotypes puts the potential effectiveness of vaccines under serious question. The distribution of unusual genotypes subject to viral fitness is influenced by interactions among viral proteins. The present work aimed at analyzing the genetic constellation and the coevolution of rotavirus coding genes for the available rotavirus genotypes. Seventy-two full genome sequences of different genetic constellations were analyzed using a genetic algorithm. The results revealed an extensive genome-wide covariance network among the 12 viral proteins. Altogether, the emergence of new genotypes represents a challenge to the outcome and success of vaccination and the coevolutionary analysis of rotavirus proteins may boost efforts to better understand the interaction networks of proteins during viral replication/transcription.

Description: Maize lethal necrosis (MLN) occurs when maize chlorotic mottle virus (MCMV) and sugarcane mosaic virus (SCMV) co-infect maize plant. Yield loss of up to 100% can be experienced under severe infections. Identification and validation of genomic regions and their flanking markers can facilitate marker assisted breeding for resistance to MLN. To understand the status of previously identified quantitative trait loci (QTL)in diverse genetic background, F3 progenies derived from seven bi-parental populations were genotyped using 500 selected kompetitive allele specific PCR (KASP) SNPs. The F3 progenies were evaluated under artificial MLN inoculation for three seasons. Phenotypic analyses revealed significant variability (P ≤ 0.01) among genotypes for responses to MLN infections, with high heritability estimates (0.62 to 0.82) for MLN disease severity and AUDPC values. Linkage mapping and joint linkage association mapping revealed at least seven major QTL (qMLN3_130 and qMLN3_142, qMLN5_190 and qMLN5_202, qMLN6_85 and qMLN6_157 qMLN8_10 and qMLN9_142) spread across the 7-biparetal populations, for resistance to MLN infections and were consistent with those reported previously. The seven QTL appeared to be stable across genetic backgrounds and across environments. Therefore, these QTL could be useful for marker assisted breeding for resistance to MLN.

Description: The protease inhibitors (PIs) in plants are involved primarily in defense against pathogens and pests and in response to abiotic stresses. However, information about the PI gene families in tomato (Solanum lycopersicum), one of the most important model plant for crop species, is limited. In this study, in silico analysis identified 55 PI genes and their conserved domains, phylogenetic relationships, and chromosome locations were characterized. According to genetic structure and evolutionary relationships, the PI gene families were divided into seven families. Genome-wide microarray transcription analysis indicated that the expression of SlPI genes can be induced by abiotic (heat, drought, and salt) and biotic (Botrytis cinerea and tomato spotted wilt virus (TSWV)) stresses. In addition, expression analysis using RNA-seq in various tissues and developmental stages revealed that some SlPI genes were highly or preferentially expressed, showing tissue- and developmental stage-specific expression profiles. The expressions of four representative SlPI genes in response to abscisic acid (ABA), salicylic acid (SA), ethylene (Eth), gibberellic acid (GA). and methyl viologen (MV) were determined. Our findings indicated that PI genes may mediate the response of tomato plants to environmental stresses to balance hormone signals. The data obtained here will improve the understanding of the potential function of PI gene and lay a foundation for tomato breeding and transgenic resistance to stresses.

Description: Magnolia × soulangeana ‘Changchun’ are trees that bloom in spring and summer respectively after flower bud differentiation. Here, we use phenological and morphological observation and RNA-seq technology to study the molecular basis of flowering initiation in ‘Changchun’. During the process of flowering initiation in spring and summer, the growth of expanded flower buds increased significantly, and their shape was obviously enlarged, which indicated that flowering was initiated. A total of 168,120 expressed genes were identified in spring and summer dormant and expanded flower buds, of which 11,687 genes showed significantly differential expression between spring and summer dormant and expanded flower buds. These differentially expressed genes (DEGs) were mainly involved in plant hormone signal transduction, metabolic processes, cellular components, binding, and catalytic activity. Analysis of differential gene expression patterns revealed that gibberellin signaling, and some transcription factors were closely involved in the regulation of spring and summer flowering initiation in ‘Changchun’. A qRT-PCR (quantitative Real Time Polymerase Chain Reaction) analysis showed that BGISEQ-500 sequencing platform could truly reflect gene expression patterns. It also verified that GID1B (GIBBERELLIN INSENSITIVE DWARF1 B), GID1C, SPL8 (SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 8), and GASA (GIBBERELLIC ACID-STIMULATED ARABIDOPSIS) family genes were expressed at high levels, while the expression of SPY (SPINDLY) was low during spring and summer flowering initiation. Meanwhile, the up- and down-regulated expression of, respectively, AGL6 (AGAMOUS-LIKE 6) and DREB3 (DEHYDRATION-RESPONSIVE ELEMENT-BINDING PROTEIN 3), AG15, and CDF1 (CYCLIC DOF FACTOR 1) might also be involved in the specific regulation of spring and summer flowering initiation. Obviously, flowering initiation is an important stage of the flowering process in woody plants, involving the specific regulation of relevant genes and transcription factors. This study provides a new perspective for the regulation of the flowering process in perennial woody plants.

Description: Pancreatic ductal adenocarcinoma is an extremely aggressive disease with a high metastatic potential. Most patients are diagnosed with metastatic disease, at which the five-year survival rate is only 3%. A better understanding of the mechanisms that drive metastasis is imperative for the development of better therapeutic interventions. Here, we take the reader through our current knowledge of the parameters that support metastatic progression in pancreatic ductal adenocarcinoma, and the experimental models that are at our disposal to study this process. We also describe the advantages and limitations of these models to study the different aspects of metastatic dissemination.

Description: Rust caused by the fungus Puccinia helianthi and downy mildew (DM) caused by the obligate pathogen Plasmopara halstedii are two of the most globally important sunflower diseases. Resistance to rust and DM is controlled by race-specific single dominant genes. The present study aimed at pyramiding rust resistance genes combined with a DM resistance gene, using molecular markers. Four rust resistant lines, HA-R3 (carrying the R4 gene), HA-R2 (R5), HA-R8 (R15), and RHA 397 (R13b), were each crossed with a common line, RHA 464, carrying a rust gene R12 and a DM gene PlArg. An additional cross was made between HA-R8 and RHA 397. Co-dominant simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers linked to the target genes were used to discriminate between homozygotes and heterozygotes in F2 populations. Five pyramids with different combinations of rust resistance genes were selected in the homozygous condition through marker-assisted selection, and three of them were combined with a DM resistance gene PlArg: R4/R12/PlArg, R5/R12/PlArg, R13b/R12/PlArg, R15/R12, and R13b/R15. The pyramiding lines with the stacking of two rust and one DM genes were resistant to all known races of North American sunflower rust and all known races of the pathogen causing DM, potentially providing multiple and durable resistance to both rust and DM. A cluster of 12 SNP markers spanning a region of 34.5 Mb on chromosome 1, which co-segregate with PlArg, were tested in four populations. Use of those markers, located in a recombination suppressed region in marker selection, is discussed.

Description: Functional (meta) genomics allows the high-throughput identification of functional genes in a premise-free way. However, it is still difficult to perform Sanger sequencing for high GC DNA templates, which hinders the functional genomic exploration of a high GC genomic library. Here, we developed a procedure to resolve this problem by coupling the Sanger and PacBio sequencing strategies. Identification of cadmium (Cd) resistance genes from a small-insert high GC genomic library was performed to test the procedure. The library was generated from a high GC (75.35%) bacterial genome. Nineteen clones that conferred Cd resistance to Escherichia coli subject to Sanger sequencing directly. The positive clones were in parallel subject to in vivo amplification in host cells, from which recombinant plasmids were extracted and linearized by selected restriction endonucleases. PacBio sequencing was performed to obtain the full-length sequences. As the identities, partial sequences from Sanger sequencing were aligned to the full-length sequences from PacBio sequencing, which led to the identification of seven unique full-length sequences. The unique sequences were further aligned to the full genome sequence of the source strain. Functional screening showed that the identified positive clones were all able to improve Cd resistance of the host cells. The functional genomic procedure developed here couples the Sanger and PacBio sequencing methods and overcomes the difficulties in PCR approaches for high GC DNA. The procedure can be a promising option for the high-throughput sequencing of functional genomic libraries, and realize a cost-effective and time-efficient identification of the positive clones, particularly for high GC genetic materials.

Description: Ionising radiation (IR) is known to induce a wide variety of lesions in DNA. In this review, we compared three different techniques that examined the DNA sequence preference of IR-induced DNA damage at nucleotide resolution. These three techniques were: the linear amplification/polymerase stop assay, the end-labelling procedure, and Illumina next-generation genome-wide sequencing. The DNA sequence preference of IR-induced DNA damage was compared in purified DNA sequences including human genomic DNA. It was found that the DNA sequence preference of IR-induced DNA damage identified by the end-labelling procedure (that mainly detected single-strand breaks) and Illumina next-generation genome-wide sequencing (that mainly detected double-strand breaks) was at C nucleotides, while the linear amplification/polymerase stop assay (that mainly detected base damage) was at G nucleotides. A consensus sequence at the IR-induced DNA damage was found to be 5′-AGGC*C for the end-labelling technique, 5′-GGC*MH (where * is the cleavage site, M is A or C, H is any nucleotide except G) for the genome-wide technique, and 5′-GG* for the linear amplification/polymerase stop procedure. These three different approaches are important because they provide a deeper insight into the mechanism of action of IR-induced DNA damage.

Description: Strawberry (Fragaria) and raspberry (Rubus) are very popular crops, and improving their nutritional quality and disease resistance are important tasks in their breeding programs that are becoming increasingly based on use of functional DNA markers. We identified 118 microsatellite (simple sequence repeat—SSR) loci in the nucleotide sequences of flavonoid biosynthesis and pathogenesis-related genes and developed 24 SSR markers representing some of these structural and regulatory genes. These markers were used to assess the genetic diversity of 48 Fragaria and Rubus specimens, including wild species and rare cultivars, which differ in berry color, ploidy, and origin. We have demonstrated that a high proportion of the developed markers are transferable within and between Fragaria and Rubus genera and are polymorphic. Transferability and polymorphism of the SSR markers depended on location of their polymerase chain reaction (PCR) primer annealing sites and microsatellite loci in genes, respectively. High polymorphism of the SSR markers in regulatory flavonoid biosynthesis genes suggests their allelic variability that can be potentially associated with differences in flavonoid accumulation and composition. This set of SSR markers may be a useful molecular tool in strawberry and raspberry breeding programs for improvement anthocyanin related traits.

Description: Myostatin acts as a negative regulator of muscle growth; therefore, its role is important with regard to animal growth and meat production. This study was undertaken with the objective to detect polymorphisms in the first intron and c.*1232 position of the MSTN gene and to analyze effects of the detected alleles/genotypes on growth and carcass traits in Colored Polish Merino sheep. In total, 23 traits were analyzed, i.e., seven describing lamb growth and 16 carcass traits. Single nucleotide polymorphisms (SNPs) in the first intron and the c.*1232 position were identified using polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) and PCR-restriction fragment length polymorphism (PCR-RFLP) methods, respectively. The MIXED procedure of the SAS software package was used to analyze allelic and genotypic effects of the MSTN gene on growth and carcass traits. Polymorphisms were only detected in the first intron of the MSTN gene. All investigated sheep were monomorphic G in the c.*1232 position. The MSTN genotype was found to have significant effect on body weight at 2nd day of life (BW2) and loin and fore shank weights. Significant allelic effects were detected with respect to BW2, scrag, leg, fore, and hind shank weights. These results suggest that polymorphisms in the first intron of the MSTN gene are relevant with respect to several carcass traits and BW2 in Colored Polish Merino sheep.

Description: Pancreatic ductal adenocarcinoma (PDAC) is among the most lethal cancer types world-wide. Its high mortality is related to the difficulty in the diagnosis, which often occurs when the disease is already advanced. As of today, no early diagnostic tests are available, while only a limited number of prognostic tests have reached clinical practice. The main reason is the lack of reliable biomarkers that are able to capture the early development or the progression of the disease. Hence, the discovery of biomarkers for early diagnosis or prognosis of PDAC remains, de facto, an unmet need. An increasing number of studies has shown that cell-free DNA (cfDNA) methylation analysis represents a promising non-invasive approach for the discovery of biomarkers with diagnostic or prognostic potential. In particular, cfDNA methylation could be utilized for the identification of disease-specific signatures in pre-neoplastic lesions or chronic pancreatitis (CP), representing a sensitive and non-invasive method of early diagnosis of PDAC. In this review, we will discuss the advantages and pitfalls of cfDNA methylation studies. Further, we will present the current advances in the discovery of pancreatic cancer biomarkers with early diagnostic or prognostic potential, focusing on pancreas-specific (e.g., CUX2 or REG1A) or abnormal (e.g., ADAMTS1 or BNC1) cfDNA methylation signatures in high risk pre-neoplastic conditions and PDAC.

Description: Maize lethal necrosis (MLN), caused by co-infection of maize chlorotic mottle virus and sugarcane mosaic virus, can lead up to 100% yield loss. Identification and validation of genomic regions can facilitate marker assisted breeding for resistance to MLN. Our objectives were to identify marker-trait associations using genome wide association study and assess the potential of genomic prediction for MLN resistance in a large panel of diverse maize lines. A set of 1400 diverse maize tropical inbred lines were evaluated for their response to MLN under artificial inoculation by measuring disease severity or incidence and area under disease progress curve (AUDPC). All lines were genotyped with genotyping by sequencing (GBS) SNPs. The phenotypic variation was significant for all traits and the heritability estimates were moderate to high. GWAS revealed 32 significantly associated SNPs for MLN resistance (at p 〈 1.0 × 10−6). For disease severity, these significantly associated SNPs individually explained 3–5% of the total phenotypic variance, whereas for AUDPC they explained 3–12% of the total proportion of phenotypic variance. Most of significant SNPs were consistent with the previous studies and assists to validate and fine map the big quantitative trait locus (QTL) regions into few markers’ specific regions. A set of putative candidate genes associated with the significant markers were identified and their functions revealed to be directly or indirectly involved in plant defense responses. Genomic prediction revealed reasonable prediction accuracies. The prediction accuracies significantly increased with increasing marker densities and training population size. These results support that MLN is a complex trait controlled by few major and many minor effect genes.

Description: Transposable elements (TEs), activated as a response to unfavorable conditions, have been proposed to contribute to the generation of genetic and phenotypic diversity in diatoms. Here we explore the transcriptome of three warm water strains of the diatom Leptocylindrus aporus, and the possible involvement of TEs in their response to changing temperature conditions. At low temperature (13 °C) several stress response proteins were overexpressed, confirming low temperature to be unfavorable for L. aporus, while TE-related transcripts of the LTR retrotransposon superfamily were the most enriched transcripts. Their expression levels, as well as most of the stress-related proteins, were found to vary significantly among strains, and even within the same strains analysed at different times. The lack of overexpression after many months of culturing suggests a possible role of physiological plasticity in response to growth under controlled laboratory conditions. While further investigation on the possible central role of TEs in the diatom stress response is warranted, the strain-specific responses and possible role of in-culture evolution draw attention to the interplay between the high intraspecific variability and the physiological plasticity of diatoms, which can both contribute to the adaptation of a species to a wide range of conditions in the marine environment.

Description: The transcription factor PAX6 is essential in ocular development in vertebrates, being considered the master regulator of the eye. During eye development, it is essential for the correct patterning and formation of the multi-layered optic cup and it is involved in the developing lens and corneal epithelium. In adulthood, it is mostly expressed in cornea, iris, and lens. PAX6 is a dosage-sensitive gene and it is highly regulated by several elements located upstream, downstream, and within the gene. There are more than 500 different mutations described to affect PAX6 and its regulatory regions, the majority of which lead to PAX6 haploinsufficiency, causing several ocular and systemic abnormalities. Aniridia is an autosomal dominant disorder that is marked by the complete or partial absence of the iris, foveal hypoplasia, and nystagmus, and is caused by heterozygous PAX6 mutations. Other ocular abnormalities have also been associated with PAX6 changes, and genotype-phenotype correlations are emerging. This review will cover recent advancements in PAX6 regulation, particularly the role of several enhancers that are known to regulate PAX6 during eye development and disease. We will also present an updated overview of the mutation spectrum, where an increasing number of mutations in the non-coding regions have been reported. Novel genotype-phenotype correlations will also be discussed.

Description: Since ancient times, the importance of physical activity (PA) and of a wholesome diet for human health has been clearly recognized. However, only recently, it has been acknowledged that PA can reverse at least some of the unwanted effects of a sedentary lifestyle, contributing to the treatment of pathologies such as hypertension and diabetes, to the delay of aging and neurodegeneration, and even to the improvement of immunity and cognitive processes. At the same time, the cellular and molecular bases of these effects are beginning to be uncovered. The original research articles and reviews published in this Special Issue on “Genetic and Epigenetic Modulation of Cell Functions by Physical Exercise” focus on different aspects of the genetics and molecular biology of PA effects on health and, in addition, on the effects of different genotypes on the ability to perform PA. All authors have read and agreed to the published version of the manuscript.

Description: Laccases belong to ligninolytic enzymes and play important roles in various biological processes of filamentous fungi, including fruiting-body formation and lignin degradation. The process of fruiting-body development in Lentinula edodes is complex and is greatly affected by environmental conditions. In this paper, 14 multicopper oxidase-encoding (laccase) genes were analyzed in the draft genome sequence of L. edodes strain W1-26, followed by a search of multiple stress-related Cis-elements in the promoter region of these laccase genes, and then a transcription profile analysis of 14 laccase genes (Lelcc) under the conditions of different carbon sources, temperatures, and photoperiods. All laccase genes were significantly regulated by varying carbon source materials. The expression of only two laccase genes (Lelcc5 and Lelcc6) was induced by sodium-lignosulphonate and the expression of most laccase genes was specifically upregulated in glucose medium. Under different temperature conditions, the expression levels of most laccase genes decreased at 39 °C and transcription was significantly increased for Lelcc1, Lelcc4, Lelcc5, Lelcc9, Lelcc12, Lelcc13, and Lelcc14 after induction for 24 h at 10 °C, indicating their involvement in primordium differentiation. Tyrosinase, which is involved in melanin synthesis, was clustered with the same group as Lelcc4 and Lelcc7 in all the different photoperiod treatments. Meanwhile, five laccase genes (Lelcc8, Lelcc9, Lelcc12, Lelcc13, and Lelcc14) showed similar expression profiles to that of two blue light receptor genes (LephrA and LephrB) in the 12 h light/12 h dark treatment, suggesting the involvement of laccase genes in the adaptation process of L. edodes to the changing environment and fruiting-body formation. This study contributes to our understanding of the function of the different Lelcc genes and facilitates the screening of key genes from the laccase gene family for further functional research.

Description: Chameleons are well-known, highly distinctive lizards characterized by unique morphological and physiological traits, but their karyotypes and sex determination system have remained poorly studied. We studied karyotypes in six species of Madagascan chameleons of the genus Furcifer by classical (conventional stain, C-banding) and molecular (comparative genomic hybridization, in situ hybridization with rDNA, microsatellite, and telomeric sequences) cytogenetic approaches. In contrast to most sauropsid lineages, the chameleons of the genus Furcifer show chromosomal variability even among closely related species, with diploid chromosome numbers varying from 2n = 22 to 2n = 28. We identified female heterogamety with cytogenetically distinct Z and W sex chromosomes in all studied species. Notably, multiple neo-sex chromosomes in the form Z1Z1Z2Z2/Z1Z2W were uncovered in four species of the genus (F. bifidus, F. verrucosus, F. willsii, and previously studied F. pardalis). Phylogenetic distribution and morphology of sex chromosomes suggest that multiple sex chromosomes, which are generally very rare among vertebrates with female heterogamety, possibly evolved several times within the genus Furcifer. Although acrodontan lizards (chameleons and dragon lizards) demonstrate otherwise notable variability in sex determination, it seems that female heterogamety with differentiated sex chromosomes remained stable in the chameleons of the genus Furcifer for about 30 million years.

Description: ADORA2A has been shown to be responsible for the wakefulness-promoting effect of caffeine and the 1976T〉C genotype (SNP rs5751876, formerly 1083T〉C) to contribute to individual sensitivity to caffeine effects on sleep. We investigate the association between six single nucleotide polymorphisms (SNP) from ADORA2A and self-reported sleep characteristics and caffeine consumption in 1023 active workers of European ancestry aged 18–60 years. Three groups of caffeine consumers were delineated: low (0–50 mg/day, less than one expresso per day), moderate (51–300 mg/day), and high (〉300 mg/day). We found that at caffeine levels higher than 300 mg/day, total sleep time (TST) decreased (F = 13.9, p 〈 0.01), with an increase of insomnia (ORa [95%CI] = 1.5 [1.1–1.9]) and sleep complaints (ORa [95%CI] = 1.9 [1.1–3.3]), whatever the ADORA2A polymorphism. Odds ratios were adjusted (ORa) for sex, age, and tobacco. However, in low caffeine consumers, lower TST was observed in the T allele compared to homozygote rs5751876 and rs3761422 C carriers. Conversely, higher TST was observed in rs2298383 T allele compared to C and in rs4822492G allele compared to the homozygote C (p 〈 0.05). These 4 SNPs are in strong linkage disequilibrium. Haplotype analysis confirmed the influence of multiple ADORA2a SNPs on TST. In addition, the rs2298383 T and rs4822492 G alleles were associated with higher risk of sleep complaints (Ora = 1.9 [1.2–3.1] and Ora = 1.5 [1.1–2.1]) and insomnia (Ora = 1.5 [1.3–2.5] and Ora = 1.9 [1.3–3.2). The rs5751876 T allele was associated with a decreased risk of sleep complaints (Ora = 0.7 [0.3–0.9]) and insomnia (Ora = 0.5 [0.3–0.9]). Our results identified ADORA2A polymorphism influences in the less-than-300-mg-per-day caffeine consumers. This opens perspectives on the diagnosis and pharmacology of sleep complaints and caffeine chronic consumption.

Description: A telomere consists of repeated DNA sequences (TTAGGG)n as part of a nucleoprotein structure at the end of the linear chromosome, and their progressive shortening induces DNA damage response (DDR) that triggers cellular senescence. The telomere can be maintained by telomerase activity (TA) in the majority of cancer cells (particularly cancer stem cells) and pluripotent stem cells (PSCs), which exhibit unlimited self-proliferation. However, some cells, such as telomerase-deficient cancer cells, can add telomeric repeats by an alternative lengthening of the telomeres (ALT) pathway, showing telomere length heterogeneity. In this review, we focus on the mechanisms of the ALT pathway and potential clinical implications. We also discuss the characteristics of telomeres in PSCs, thereby shedding light on the therapeutic significance of telomere length regulation in age-related diseases and regenerative medicine.

Description: Genetic information of all organisms is coded in double-stranded DNA. DNA helicases are essential for unwinding this double strand when it comes to replication, repair or transcription of genetic information. In this review, we will focus on what is known about a variety of DNA helicases that are required to ensure genome stability in plants. Due to their sessile lifestyle, plants are especially exposed to harmful environmental factors. Moreover, many crop plants have large and highly repetitive genomes, making them absolutely dependent on the correct interplay of DNA helicases for safeguarding their stability. Although basic features of a number of these enzymes are conserved between plants and other eukaryotes, a more detailed analysis shows surprising peculiarities, partly also between different plant species. This is additionally of high relevance for plant breeding as a number of these helicases are also involved in crossover control during meiosis and influence the outcome of different approaches of CRISPR/Cas based plant genome engineering. Thus, gaining knowledge about plant helicases, their interplay, as well as the manipulation of their pathways, possesses the potential for improving agriculture. In the long run, this might even help us cope with the increasing obstacles of climate change threatening food security in completely new ways.

Description: Rapid radiation associated with phenotypic divergence and convergence provides an opportunity to study the genetic mechanisms of evolution. Here we investigate the genus Takifugu that has undergone explosive radiation relatively recently and contains a subset of closely-related species with a scale-loss phenotype. By using observations during development and genetic mapping approaches, we show that the scale-loss phenotype of two Takifugu species, T. pardalis Temminck & Schlegel and T. snyderi Abe, is largely controlled by an overlapping genomic segment (QTL). A search for candidate genes underlying the scale-loss phenotype revealed that the QTL region contains no known genes responsible for the evolution of scale-loss phenotype in other fishes. These results suggest that the genes used for the scale-loss phenotypes in the two Takifugu are likely the same, but the genes used for the similar phenotype in Takifugu and distantly related fishes are not the same. Meanwhile, Fgfrl1, a gene predicted to function in a pathway known to regulate bone/scale development was identified in the QTL region. Since Fgfr1a1, another memebr of the Fgf signaling pathway, has been implicated in scale loss/scale shape in fish distantly related to Takifugu, our results suggest that the convergence of the scale-loss phenotype may be constrained by signaling modules with conserved roles in scale development.

Description: Non-syndromic cleft lip with or without cleft palate (nsCL/P) ranks among the most common human congenital malformations, and has a multifactorial background in which both exogenous and genetic risk factors act in concert. The present report describes a genome-wide association study (GWAS) involving a total of 285 nsCL/P patients and 1212 controls from the Netherlands and Belgium. Twenty of the 40 previously reported nsC/LP susceptibility loci were replicated, which underlined the validity of this sample. SNV-based analysis of the data identified an as yet unreported suggestive locus at chromosome 16p12.1 (p-value of the lead SNV: 4.17 × 10−7). This association was replicated in two of three patient/control replication series (Central European and Yemeni). Gene analysis of the GWAS data prioritized SH3PXD2A at chromosome 10q24.33 as a candidate gene for nsCL/P. To date, support for this gene as a cleft gene has been restricted to data from zebrafish and a knockout mouse model. The present GWAS was the first to implicate SH3PXD2A in non-syndromic cleft formation in humans. In summary, although performed in a relatively small sample, the present GWAS generated novel insights into nsCL/P etiology.

Description: Soybean seeds produce valuable protein that is a major component of livestock feed. However, soybean seeds also contain the anti-nutritional raffinose family oligosaccharides (RFOs) raffinose and stachyose, which are not digestible by non-ruminant animals. This requires the proportion of soybean meal in the feed to be limited, or risk affecting animal growth rate or overall health. While reducing RFOs in soybean seed has been a goal of soybean breeding, efforts are constrained by low genetic variability for carbohydrate traits and the difficulty in identifying these within the soybean germplasm. We used reverse genetics Targeting Induced Local Lesions in Genomes (TILLING)-by-sequencing approach to identify a damaging polymorphism that results in a missense mutation in a conserved region of the RAFFINOSE SYNTHASE3 gene. We demonstrate that this mutation, when combined as a double mutant with a previously characterized mutation in the RAFFINOSE SYNTHASE2 gene, eliminates nearly 90% of the RFOs in soybean seed as a proportion of the total seeds carbohydrates, and results in increased levels of sucrose. This represents a proof of concept for TILLING by sequencing in soybean.

Description: Sepsis is a life-threatening disease induced by a systemic inflammatory response, which leads to organ dysfunction and mortality. In sepsis, the host immune response is depressed and unable to cope with infection; no drug is currently available to treat this. The lungs are frequently the starting point for sepsis. This study aimed to identify potential genes for diagnostics and therapeutic purposes in sepsis by a comprehensive bioinformatics analysis. Our criteria are to unravel sepsis-associated signature genes from gene expression datasets. Differentially expressed genes (DEGs) were identified from samples of sepsis patients using a meta-analysis and then further subjected to functional enrichment and protein‒protein interaction (PPI) network analysis for examining their potential functions. Finally, the expression of the topmost upregulated genes (ARG1, IL1R2, ELANE, MMP9) was quantified by reverse transcriptase-PCR (RT-PCR), and myeloperoxidase (MPO) expression was confirmed by immunohistochemistry (IHC) staining in the lungs of a well-established sepsis mouse model. We found that all the four genes were upregulated in semiquantitative RT-PCR studies; however, MMP9 showed a nonsignificant increase in expression. MPO staining showed strong immunoreactivity in sepsis as compared to the control. This study demonstrates the role of significant and widespread immune activation (IL1R2, MMP9), along with oxidative stress (ARG1) and the recruitment of neutrophils, in sepsis (ELANE, MPO).

Description: The association of candidate genes and psychological symptoms of depression, anxiety, and stress among women with gestational diabetes mellitus (GDM) in Malaysia was determined in this study, followed by the determination of their odds of getting psychological symptoms, adjusted for socio-demographical background, maternal, and clinical characteristics. Single nucleotide polymorphisms (SNPs) recorded a significant association between SNP of EPHX2 (rs17466684) and depression symptoms (AOR = 7.854, 95% CI = 1.330–46.360) and stress symptoms (AOR = 7.664, 95% CI = 1.579–37.197). Associations were also observed between stress symptoms and SNP of OXTR (rs53576) and (AOR = 2.981, 95% CI = 1.058–8.402) and SNP of NRG1 (rs2919375) (AOR = 9.894, 95% CI = 1.159–84.427). The SNP of EPHX2 (rs17466684) gene polymorphism is associated with depression symptoms among Malaysian women with GDM. SNP of EPHX2 (rs17466684), OXTR (rs53576) and NRG1 (rs2919375) are also associated with stress symptoms.

Description: Eukaryotic genomes are rich in repetitive DNA sequences grouped in two classes regarding their genomic organization: tandem repeats and dispersed repeats. In tandem repeats, copies of a short DNA sequence are positioned one after another within the genome, while in dispersed repeats, these copies are randomly distributed. In this review we provide evidence that both tandem and dispersed repeats can have a similar organization, which leads us to suggest an update to their classification based on the sequence features, concretely regarding the presence or absence of retrotransposons/transposon specific domains. In addition, we analyze several studies that show that a repetitive element can be remodeled into repetitive non-coding or coding sequences, suggesting (1) an evolutionary relationship among DNA sequences, and (2) that the evolution of the genomes involved frequent repetitive sequence reshuffling, a process that we have designated as a “DNA remodeling mechanism”. The alternative classification of the repetitive DNA sequences here proposed will provide a novel theoretical framework that recognizes the importance of DNA remodeling for the evolution and plasticity of eukaryotic genomes.

Description: Moths and butterflies (Lepidoptera) are the largest group with heterogametic females. Although the ancestral sex chromosome system is probably Z0/ZZ, most lepidopteran species have the W chromosome. When and how the W chromosome arose remains elusive. Existing hypotheses place the W origin either at the common ancestor of Ditrysia and Tischeriidae, or prefer independent origins of W chromosomes in these two groups. Due to their phylogenetic position at the base of Ditrysia, bagworms (Psychidae) play an important role in investigating the W chromosome origin. Therefore, we examined the W chromosome status in three Psychidae species, namely Proutia betulina, Taleporia tubulosa, and Diplodoma laichartingella, using both classical and molecular cytogenetic methods such as sex chromatin assay, comparative genomic hybridization (CGH), and male vs. female genome size comparison by flow cytometry. In females of all three species, no sex chromatin was found, no female-specific chromosome regions were revealed by CGH, and a Z-chromosome univalent was observed in pachytene oocytes. In addition, the genome size of females was significantly smaller than males. Overall, our study provides strong evidence for the absence of the W chromosome in Psychidae, thus supporting the hypothesis of two independent W chromosome origins in Tischeriidae and in advanced Ditrysia.

Description: Plants constantly fight with stressful factors as high or low temperature, drought, soil salinity and flooding. Plants have evolved a set of stress response mechanisms, which involve physiological and biochemical changes that result in adaptive or morphological changes. At a molecular level, stress response in plants is performed by genetic networks, which also undergo changes in the process of evolution. The study of the network structure and evolution may highlight mechanisms of plants adaptation to adverse conditions, as well as their response to stresses and help in discovery and functional characterization of the stress-related genes. We performed an analysis of Arabidopsis thaliana genes associated with several types of abiotic stresses (heat, cold, water-related, light, osmotic, salt, and oxidative) at the network level using a phylostratigraphic approach. Our results show that a substantial fraction of genes associated with various types of abiotic stress is of ancient origin and evolves under strong purifying selection. The interaction networks of genes associated with stress response have a modular structure with a regulatory component being one of the largest for five of seven stress types. We demonstrated a positive relationship between the number of interactions of gene in the stress gene network and its age. Moreover, genes of the same age tend to be connected in stress gene networks. We also demonstrated that old stress-related genes usually participate in the response for various types of stress and are involved in numerous biological processes unrelated to stress. Our results demonstrate that the stress response genes represent the ancient and one of the fundamental molecular systems in plants.

Description: Signalling molecules and their cognate receptors are central components of the Metazoa endocrine system. Defining their presence or absence in extant animal lineages is critical to accurately devise evolutionary patterns, physiological shifts and the impact of endocrine disrupting chemicals. Here, we address the evolution of retinoic acid (RA) signalling in the Priapulida worm, Priapulus caudatus Lamarck, 1816, an Ecdysozoa. RA signalling has been shown to be central to chordate endocrine homeostasis, participating in multiple developmental and physiological processes. Priapulids, with their slow rate of molecular evolution and phylogenetic position, represent a key taxon to investigate the early phases of Ecdysozoa evolution. By exploring a draft genome assembly, we show, by means of phylogenetics and functional assays, that an orthologue of the nuclear receptor retinoic acid receptor (RAR) subfamily, a central mediator of RA signalling, is present in Ecdysozoa, contrary to previous perception. We further demonstrate that the Priapulida RAR displays low-affinity for retinoids (similar to annelids), and is not responsive to common endocrine disruptors acting via RAR. Our findings provide a timeline for RA signalling evolution in the Bilateria and give support to the hypothesis that the increase in RA affinity towards RAR is a late acquisition in the evolution of the Metazoa.

Description: Using the RNA-dependent RNA polymerase (RdRp) from poliovirus (PV) as our model system, we have shown that Lys-359 in motif-D functions as a general acid in the mechanism of nucleotidyl transfer. A K359H (KH) RdRp derivative is slow and faithful relative to wild-type enzyme. In the context of the KH virus, RdRp-coding sequence evolves, selecting for the following substitutions: I331F (IF, motif-C) and P356S (PS, motif-D). We have evaluated IF-KH, PS-KH, and IF-PS-KH viruses and enzymes. The speed and fidelity of each double mutant are equivalent. Each exhibits a unique recombination phenotype, with IF-KH being competent for copy-choice recombination and PS-KH being competent for forced-copy-choice recombination. Although the IF-PS-KH RdRp exhibits biochemical properties within twofold of wild type, the virus is impaired substantially for recombination in cells. We conclude that there are biochemical properties of the RdRp in addition to speed and fidelity that determine the mechanism and efficiency of recombination. The interwoven nature of speed, fidelity, the undefined property suggested here, and recombination makes it impossible to attribute a single property of the RdRp to fitness. However, the derivatives described here may permit elucidation of the importance of recombination on the fitness of the viral population in a background of constant polymerase speed and fidelity.

Description: Non-alcoholic fatty liver disease (NAFLD) is a progressive condition of the liver encompassing a range of pathologies including steatosis, non-alcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular carcinoma. Research into this disease is imperative due to its rapid growth in prevalence, economic burden, and current lack of FDA approved therapies. NAFLD involves a highly complex etiology that calls for multi-tissue multi-omics network approaches to uncover the pathogenic genes and processes, diagnostic biomarkers, and potential therapeutic strategies. In this review, we first present a basic overview of disease pathogenesis, risk factors, and remaining knowledge gaps, followed by discussions of the need and concepts of multi-tissue multi-omics approaches, various network methodologies and application examples in NAFLD research. We highlight the findings that have been uncovered thus far including novel biomarkers, genes, and biological pathways involved in different stages of NAFLD, molecular connections between NAFLD and its comorbidities, mechanisms underpinning sex differences, and druggable targets. Lastly, we outline the future directions of implementing network approaches to further improve our understanding of NAFLD in order to guide diagnosis and therapeutics.

Description: Seed-flooding stress is one of the major abiotic constraints severely affecting soybean yield and quality. Understanding the molecular mechanism and genetic basis underlying seed-flooding tolerance will be of greatly importance in soybean breeding. However, very limited information is available about the genetic basis of seed-flooding tolerance in soybean. The present study performed Genome-Wide Association Study (GWAS) to identify the quantitative trait nucleotides (QTNs) associated with three seed-flooding tolerance related traits, viz., germination rate (GR), normal seedling rate (NSR) and electric conductivity (EC), using a panel of 347 soybean lines and the genotypic data of 60,109 SNPs with MAF 〉 0.05. A total of 25 and 21 QTNs associated with all three traits were identified via mixed linear model (MLM) and multi-locus random-SNP-effect mixed linear model (mrMLM) in three different environments (JP14, HY15, and Combined). Among these QTNs, three major QTNs, viz., QTN13, qNSR-10 and qEC-7-2, were identified through both methods MLM and mrMLM. Interestingly, QTN13 located on Chr.13 has been consistently identified to be associated with all three studied traits in both methods and multiple environments. Within the 1.0 Mb physical interval surrounding the QTN13, nine candidate genes were screened for their involvement in seed-flooding tolerance based on gene annotation information and available literature. Based on the qRT-PCR and sequence analysis, only one gene designated as GmSFT (Glyma.13g248000) displayed significantly higher expression level in all tolerant genotypes compared to sensitive ones under flooding treatment, as well as revealed nonsynonymous mutation in tolerant genotypes, leading to amino acid change in the protein. Additionally, subcellular localization showed that GmSFT was localized in the nucleus and cell membrane. Hence, GmSFT was considered as the most likely candidate gene for seed-flooding tolerance in soybean. In conclusion, the findings of the present study not only increase our knowledge of the genetic control of seed-flooding tolerance in soybean, but will also be of great utility in marker-assisted selection and gene cloning to elucidate the mechanisms of seed-flooding tolerance.

Description: Notch signaling is a primitive signaling pathway having various roles in the normal origin and development of each multicellular organisms. Therefore, any aberration in the pathway will inevitably lead to deadly outcomes such as cancer. It has now been more than two decades since Notch was acknowledged as an oncogene in mouse mammary tumor virus-infected mice. Since that discovery, activated Notch signaling and consequent up-regulation of tumor-promoting Notch target genes have been observed in human breast cancer. Moreover, consistent over-expression of Notch ligands and receptors has been shown to correlate with poor prognosis in human breast cancer. Notch regulates a number of key processes during breast carcinogenesis, of which, one key phenomenon is epithelial–mesenchymal transition (EMT). EMT is a key process for large-scale cell movement during morphogenesis at the time of embryonic development. Cancer cells aided by transcription factors usurp this developmental program to execute the multi-step process of tumorigenesis and metastasis. In this review, we recapitulate recent progress in breast cancer research that has provided new perceptions into the molecular mechanisms behind Notch-mediated EMT regulation during breast tumorigenesis.

Description: Aphids cause serious losses to the production of wheat. The grain aphid, Sitobion avenae, which is the dominant species of aphid in all wheat regions of China, is resistant to a variety of insecticides, including imidacloprid and chlorpyrifos. However, the resistance and mechanism of insecticide tolerance of S. avenae are still unclear. Therefore, this study employed transcriptome analysis to compare the expression patterns of stress response genes under imidacloprid and chlorpyrifos treatment for 15 min, 3 h, and 36 h of exposure. S. avenae adult transcriptome was assembled and characterized first, after which samples treated with insecticides for different lengths of time were compared with control samples, which revealed 602267 differentially expressed unigenes (DEUs). Among these DEUs, 31–790 unigenes were classified into 66–786 categories of gene ontology (GO) functional groups, and 24–760 DEUs could be mapped into 54–268 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Finally, 11 insecticide-tolerance-related unigenes were chosen to confirm the relative expression by quantitative real-time polymerase chain reaction (qRT-PCR) in each treatment. Most of the results between qRT-PCR and RNA sequencing (RNA-Seq) are well-established. The results presented herein will facilitate molecular research investigating insecticide resistance in S. avenae, as well as in other wheat aphids.

Description: Duchenne muscular dystrophy (DMD) is a progressive hereditary muscular disease with X-linked recessive inheritance, that leads patients to premature death. The loss of dystrophin determines membrane instability, causing cell damage and inflammatory response. Macrophage migration inhibitory factor (MIF) is a cytokine that exerts pleiotropic properties and is implicated in the pathogenesis of a variety of diseases. Recently, converging data from independent studies have pointed to a possible role of MIF in dystrophic muscle disorders, including DMD. In the present study, we have investigated the modulation of MIF and MIF-related genes in degenerative muscle disorders, by making use of publicly available whole-genome expression datasets. We show here a significant enrichment of MIF and related genes in muscle samples from DMD patients, as well as from patients suffering from Becker’s disease and limb-girdle muscular dystrophy type 2B. On the other hand, transcriptomic analysis of in vitro differentiated myotubes from healthy controls and DMD patients revealed no significant alteration in the expression levels of MIF-related genes. Finally, by analyzing DMD samples as a time series, we show that the modulation of the genes belonging to the MIF network is an early event in the DMD muscle and does not change with the increasing age of the patients, Overall, our analysis suggests that MIF may play a role in vivo during muscle degeneration, likely promoting inflammation and local microenvironment reaction.

Description: Background: In a previous survey, an elevated fasting glucose level (FG) and/or known type 2 diabetes mellitus (T2DM) were significantly more frequent in the Roma population than in the Hungarian general population. We assessed whether the distribution of 16 single nucleotide polymorphisms (SNPs) with unequivocal effects on the development of T2DM contributes to this higher prevalence. Methods: Genetic risk scores, unweighted (GRS) and weighted (wGRS), were computed and compared between the study populations. Associations between GRSs and FG levels and T2DM status were investigated in separate and combined study populations. Results: The Hungarian general population carried a greater genetic risk for the development of T2DM (GRSGeneral = 15.38 ± 2.70 vs. GRSRoma = 14.80 ± 2.68, p 〈 0.001; wGRSGeneral = 1.41 ± 0.32 vs. wGRSRoma = 1.36 ± 0.31, p 〈 0.001). In the combined population models, GRSs and wGRSs showed significant associations with elevated FG (p 〈 0.001) and T2DM (p 〈 0.001) after adjusting for ethnicity, age, sex, body mass index (BMI), high-density Lipoprotein Cholesterol (HDL-C), and triglyceride (TG). In these models, the effect of ethnicity was relatively strong on both outcomes (FG levels: βethnicity = 0.918, p 〈 0.001; T2DM status: ORethnicity = 2.484, p 〈 0.001). Conclusions: The higher prevalence of elevated FG and/or T2DM among Roma does not seem to be directly linked to their increased genetic load but rather to their environmental/cultural attributes. Interventions targeting T2DM prevention among Roma should focus on harmful environmental exposures related to their unhealthy lifestyle.

Description: Background: Radiation induces DNA double-strand breaks (DSBs), and chromosome aberrations (CA) form during the DSBs repair process. Several methods have been used to model the repair kinetics of DSBs including the bi-exponential model, i.e., N(t) = N1exp(−t/τ1) + N2exp(−t/τ2), where N(t) is the number of breaks at time t, and N1, N2, τ1 and τ2 are parameters. This bi-exponential fit for DSB decay suggests that some breaks are repaired rapidly and other, more complex breaks, take longer to repair. Methods: The bi-exponential repair kinetics model is implemented into a recent simulation code called RITCARD (Radiation Induced Tracks, Chromosome Aberrations, Repair, and Damage). RITCARD simulates the geometric configuration of human chromosomes, radiation-induced breaks, their repair, and the creation of various categories of CAs. The bi-exponential repair relies on a computational algorithm that is shown to be mathematically exact. To categorize breaks as complex or simple, a threshold for the local (voxel) dose was used. Results: The main findings are: i) the curves for the kinetics of restitution of DSBs are mostly independent of dose; ii) the fraction of unrepaired breaks increases with the linear energy transfer (LET) of the incident radiation; iii) the simulated dose–response curves for simple reciprocal chromosome exchanges that are linear-quadratic; iv) the alpha coefficient of the dose–response curve peaks at about 100 keV/µm.

Description: The root-knot nematode (RKN) is one of the most dangerous and widespread types of nematodes affecting tomatoes. There are few methods for controlling nematodes in tomatoes. Nature resistance genes (R-genes) are important in conferring resistance against nematodes. These genes that confer resistance to the RKN have already been identified as Mi-1, Mi-2, Mi-3, Mi-4, Mi-5, Mi-6, Mi-7, Mi-8, Mi-9, and Mi-HT. Only five of these genes have been mapped. The major problem is that their resistance breaks down at high temperatures. Some of these genes still work at high temperatures. In this paper, the mechanism and characteristics of these natural resistance genes are summarized. Other difficulties in using these genes in the resistance and how to improve them are also mentioned.

Description: Peanut (Arachis hypogaea L.) is one of the most important oil crops worldwide, and its yet increasing market demand may be met by genetic improvement of yield related traits, which may be facilitated by a good understanding of the underlying genetic base of these traits. Here, we have carried out a genome-wide association study (GWAS) with the aim to identify genomic regions and the candidate genes within these regions that may be involved in determining the phenotypic variation at seven yield-related traits in peanut. For the GWAS analyses, 195 peanut accessions were phenotyped and/or genotyped; the latter was done using a genotyping-by-sequencing approach, which produced a total of 13,435 high-quality single nucleotide polymorphisms (SNPs). Analyses of these SNPs show that the analyzed peanut accessions can be approximately grouped into two big groups that, to some extent, agree with the botanical classification of peanut at the subspecies level. By taking this genetic structure as well as the relationships between the analyzed accessions into consideration, our GWAS analyses have identified 93 non-overlapping peak SNPs that are significantly associated with four of the studied traits. Gene annotation of the genome regions surrounding these peak SNPs have found a total of 311 unique candidate genes. Among the 93 yield-related-trait-associated SNP peaks, 12 are found to be co-localized with the quantitative trait loci (QTLs) that were identified by earlier related QTL mapping studies, and these 12 SNP peaks are only related to three traits and are almost all located on chromosomes Arahy.05 and Arahy.16. Gene annotation of these 12 co-localized SNP peaks have found 36 candidates genes, and a close examination of these candidate genes found one very interesting gene (arahy.RI9HIF), the rice homolog of which produces a protein that has been shown to improve rice yield when over-expressed. Further tests of the arahy.RI9HIF gene, as well as other candidate genes especially those within the more confident co-localized genomic regions, may hold the potential for significantly improving peanut yield.

Description: Growing resistance is reported to carbamate insecticides in malaria vectors in Cameroon. However, the contribution of acetylcholinesterase (Ace-1) to this resistance remains uncharacterised. Here, we established that the G119S mutation is driving resistance to carbamates in Anopheles gambiae populations from Cameroon. Insecticide bioassay on field-collected mosquitoes from Bankeng, a locality in southern Cameroon, showed high resistance to the carbamates bendiocarb (64.8% ± 3.5% mortality) and propoxur (55.71% ± 2.9%) but a full susceptibility to the organophosphate fenitrothion. The TaqMan genotyping of the G119S mutation in field-collected adults revealed the presence of this resistance allele (39%). A significant correlation was observed between the Ace-1R and carbamate resistance at allelic ((bendiocarb; odds ratio (OR) = 75.9; p 〈 0.0001) and (propoxur; OR = 1514; p 〈 0.0001)) and genotypic (homozygote resistant vs. homozygote susceptible (bendiocarb; OR = 120.8; p 〈 0.0001) and (propoxur; OR = 3277; p 〈 0.0001)) levels. Furthermore, the presence of the mutation was confirmed by sequencing an Ace-1 portion flanking codon 119. The cloning of this fragment revealed a likely duplication of Ace-1 in Cameroon as mosquitoes exhibited at least three distinct haplotypes. Phylogenetic analyses showed that the predominant Ace-1R allele is identical to that from West Africa suggesting a recent introduction of this allele in Central Africa from the West. The spread of this Ace-1R represents a serious challenge to future implementation of indoor residual spraying (IRS)-based interventions using carbamates or organophosphates in Cameroon.

Description: Microglia, the main immune cells of the central nervous system, are increasingly implicated in Alzheimer’s disease (AD). Manifold transcriptomic studies in the brain have not only highlighted microglia’s role in AD pathogenesis, but also mapped crucial pathological processes and identified new therapeutic targets. An important component of many of these transcriptomic studies is the investigation of gene expression networks in AD brain, which has provided important new insights into how coordinated gene regulatory programs in microglia (and other cell types) underlie AD pathogenesis. Given the rapid technological advancements in transcriptional profiling, spanning from microarrays to single-cell RNA sequencing (scRNA-seq), tools used for mapping gene expression networks have evolved to keep pace with the unique features of each transcriptomic platform. In this article, we review the trajectory of transcriptomic network analyses in AD from brain to microglia, highlighting the corresponding methodological developments. Lastly, we discuss examples of how transcriptional network analysis provides new insights into AD mechanisms and pathogenesis.

Description: Colorectal cancer (CRC) is a heterogeneous disease that includes both hereditary and sporadic types of tumors. Tumor initiation and growth is driven by mutational or epigenetic changes that alter the function or expression of multiple genes. The genes predominantly encode components of various intracellular signaling cascades. In this review, we present mouse intestinal cancer models that include alterations in the Wnt, Hippo, p53, epidermal growth factor (EGF), and transforming growth factor β (TGFβ) pathways; models of impaired DNA mismatch repair and chemically induced tumorigenesis are included. Based on their molecular biology characteristics and mutational and epigenetic status, human colorectal carcinomas were divided into four so-called consensus molecular subtype (CMS) groups. It was shown subsequently that the CMS classification system could be applied to various cell lines derived from intestinal tumors and tumor-derived organoids. Although the CMS system facilitates characterization of human CRC, individual mouse models were not assigned to some of the CMS groups. Thus, we also indicate the possible assignment of described animal models to the CMS group. This might be helpful for selection of a suitable mouse strain to study a particular type of CRC.

Description: Cholesteryl ester transfer protein (CETP) plays an important role in lipid metabolism. Low levels of high-density lipoprotein cholesterol (HDL-C) increase the risk of type 2 diabetes (T2D). This study investigated CETP gene variants to assess the risk of T2D and specific complications of diabetic kidney disease (DKD) and diabetic retinopathy. Towards this, a total of 3023 Taiwanese individuals (1383 without T2D, 1640 with T2D) were enrolled in this study. T2D mice (+Leprdb/+Leprdb, db/db) were used to determine CETP expression in tissues. The A-alleles of rs3764261, rs4783961, and rs1800775 variants were found to be independently associated with 2.86, 1.71, and 0.91 mg/dL increase in HDL-C per allele, respectively. In addition, the A-allele of rs4783961 was significantly associated with a reduced T2D risk (odds ratio (OR), 0.82; 95% confidence interval (CI), 0.71‒0.96)), and the A-allele of rs1800775 was significantly related to a lowered DKD risk (OR, 0.78; 95% CI, 0.64‒0.96). CETP expression was significantly decreased in the T2D mice kidney compared to that in the control mice (T2D mice, 0.16 0.01 vs. control mice, 0.21 0.02; p = 0.02). These collective findings indicate that CETP variants in the promoter region may affect HDL-C levels. Taiwanese individuals possessing an allele associated with higher HDL-C levels had a lower risk of T2D and DKD.

Description: Aneuploidy, a deviation from a balanced genome by either gain or loss of chromosomes, is generally associated with impaired fitness and developmental defects in eukaryotic organisms. While the general physiological impact of aneuploidy remains largely elusive, many phenotypes associated with aneuploidy link to a common theme of stress adaptation. Here, we review previously identified mechanisms and observations related to aneuploidy, focusing on the highly diverse eukaryotes, fungi. Fungi, which have conquered virtually all environments, including several hostile ecological niches, exhibit widespread aneuploidy and employ it as an adaptive strategy under severe stress. Gambling with the balance between genome plasticity and stability has its cost and in fact, most aneuploidies have fitness defects. How can this fitness defect be reconciled with the prevalence of aneuploidy in fungi? It is likely that the fitness cost of the extra chromosomes is outweighed by the advantage they confer under life-threatening stresses. In fact, once the selective pressures are withdrawn, aneuploidy is often lost and replaced by less drastic mutations that possibly incur a lower fitness cost. We discuss representative examples across hostile environments, including medically and industrially relevant cases, to highlight potential adaptive mechanisms in aneuploid yeast.