Functional Copy Research Articles

Genome Sequencing of the Endangered Kingdonia uniflora (Circaeasteraceae, Ranunculales) Reveals Potential Mechanisms of Evolutionary Specialization.

SummaryKingdonia uniflora, an alpine herb, has an extremely narrow distribution and represents a model for studying evolutionary mechanisms of species that have adapted to undisturbed environments for evolutionarily long periods of time. We assembled a 1,004.7-Mb draft genome (encoding 43,301 genes) of K. uniflora and found significant overrepresentation in gene families associated with DNA repair, underrepresentation in gene families associated with stress response, and loss of most plastid ndh genes. During the evolutionary process, the overrepresentation of gene families involved in DNA repair could help asexual K. uniflora reduce the accumulation of deleterious mutations, while reducing genetic diversity, which is important in responding to environment fluctuations. The underrepresentation of gene families related to stress response and functional loss of ndh genes could be due to lack or loss of ability to respond to environmental changes caused by long-term adaptation to a relatively stable ecological environment.

Monomeric NADH-Oxidizing Methylenetetrahydrofolate Reductases from Mycobacterium smegmatis Lack Flavin Coenzyme.

5,10-Methylenetetrahydrofolate reductase (MetF/MTHFR) is an essential enzyme in one-carbon metabolism for de novo biosynthesis of methionine. Our in vivo and in vitro analyses of MSMEG_6664/MSMEI_6484, annotated as putative MTHFR in Mycobacterium smegmatis, failed to reveal their function as MTHFRs. However, we identified two hypothetical proteins, MSMEG_6596 and MSMEG_6649, as noncanonical MTHFRs in the bacterium. MTHFRs are known to be oligomeric flavoproteins. Both MSMEG_6596 and MSMEG_6649 are monomeric proteins and lack flavin coenzymes. In vitro, the catalytic efficiency (kcat/Km ) of MSMEG_6596 (MTHFR1) for 5,10-CH2-THF and NADH was ∼13.5- and 15.3-fold higher than that of MSMEG_6649 (MTHFR2). Thus, MSMEG_6596 is the major MTHFR. This interpretation was further supported by better rescue of the E. coli Δmthfr strain by MTHFR1 than by MTHFR2. As identified by liquid chromatography-tandem mass spectrometry, the product of MTHFR1- or MTHFR2-catalyzed reactions was 5-CH3-THF. The M. smegmatis Δmsmeg_6596 strain was partially auxotrophic for methionine and grew only poorly without methionine or without being complemented with a functional copy of MTHFR1 or MTHFR2. Furthermore, the Δmsmeg_6596 strain was more sensitive to folate pathway inhibitors (sulfachloropyridazine, p-aminosalicylic acid, sulfamethoxazole, and trimethoprim). The studies reveal that MTHFR1 and MTHFR2 are two noncanonical MTHFR proteins that are monomeric and lack flavin coenzyme. Both MTHFR1 and MTHFR2 are involved in de novo methionine biosynthesis and required for antifolate resistance in mycobacteria.IMPORTANCE MTHFR/MetF is an essential enzyme in a one-carbon metabolic pathway for de novo biosynthesis of methionine. MTHFRs are known to be oligomeric flavoproteins. Our in vivo and in vitro analyses of Mycobacterium smegmatis MSMEG_6664/MSMEI_6484, annotated as putative MTHFR, failed to reveal their function as MTHFRs. However, we identified two of the hypothetical proteins, MSMEG_6596 and MSMEG_6649, as MTHFR1 and MTHFR2, respectively. Interestingly, both MTHFRs are monomeric and lack flavin coenzymes. M. smegmatis deleted for the major mthfr (mthfr1) was partially auxotroph for methionine and more sensitive to folate pathway inhibitors (sulfachloropyridazine, para-aminosalicylic acid, sulfamethoxazole, and trimethoprim). The studies reveal that MTHFR1 and MTHFR2 are novel MTHFRs involved in de novo methionine biosynthesis and required for antifolate resistance in mycobacteria.

Morphology and gene expression in mouse placentas lacking leptin receptors

In the pregnant mouse, the hormone leptin is primarily produced by adipose tissue and does not significantly cross the placenta into fetal circulation. Nonetheless, leptin treatment during gestation affects offspring phenotypes. Leptin treatment also affects placental trophoblast cells in vitro, by altering proliferation, invasion and nutrient transport. The goal of the present study was to determine whether the absence of placental leptin receptors alters placental development and gene expression. Leprdb−3j+ mice possessing only one functional copy of the leptin receptor were mated to obtain wildtype, Leprdb−3j+ and Leprdb−3j/db−3j conceptuses, which were then transferred to wildtype recipient dams. Placentas were collected at gestational d18.5 to examine placental morphology and gene expression. Placentas lacking functional leptin receptor had reduced weights, but were otherwise morphologically indistinguishable from control placentas. Relative mRNA levels, however, were altered in Leprdb−3j/db−3j placentas, particularly transcripts related to amino acid and lipid metabolism and transport. Consistent with a previous in vitro study, leptin was found to promote expression of stathmin, a positive regulator of trophoblast invasion, and of serotonin receptors, potential mediators of offspring neurological development. Overall placental leptin receptor was found not to play a significant role in morphological development of the placenta, but to regulate placental gene expression, including in metabolic pathways that affect fetal growth.

Loss of CLC‐5 reduces endocytic capacity and alters cholesterol distribution in the proximal tubule

Dent disease is a progressive X‐linked disorder affecting the kidney proximal tubule (PT) caused by loss of function of the Cl−/H+ exchanger, CLC‐5. Early symptoms include low molecular weight (LMW) proteinuria resulting from inefficient reabsorption of filtered proteins by the PT. However, the role of CLC‐5 in this process in unclear. Knockout of Clc‐5 in mice recapitulates the LMW proteinuria observed in human disease and causes decreased PT levels of megalin and cubilin, the receptors that bind to and recover filtered proteins. How loss of CLC‐5 leads to reduced receptor expression remains unknown. We used a previously developed opossum kidney (OK) cell culture model that recapitulates morphologic and functional features of the PT in vivo to study the role of Clc‐5 in the endocytic pathway. In Clc‐5 siRNA knockdown (KD) and CRISPR/Cas9 mediated Clc‐5 knockout (KO) cells, there was a significant decrease in endocytic capacity that was rescued by heterologous expression of wild‐type human CLC‐5. Additionally, we used CRISPR/Cas9 technology to generate Clc‐5 knockout mice and confirmed LMW proteinuria in the KO. Heterozygous females also have reduced PT albumin uptake and megalin expression. Previous gene expression studies in Clcn5 KO mice suggest there are alterations in cholesterol and lipid metabolic enzymes, and elevated cholesterol levels have been shown to alter recycling endosome organization and impair fast recycling. We hypothesize that loss of CLC‐5 disrupts cholesterol homeostasis, resulting in reduced recycling and concomitantly increased degradation of megalin. Consistent with this, we observed an accumulation and redistribution of cholesterol in Clc‐5 KD OK cells. We are currently working to quantify cholesterol in Clc‐5 KO OK cells. In female mice with only one functional copy of Clc‐5, we observed a dramatic a change in the distribution of cholesterol in the PT when compared to WT mice. Current studies are focused on quantifying the trafficking kinetics and distribution of megalin in the PT in the absence of CLC‐5 and determining whether altered cholesterol metabolism contributes to the reduced endocytic capacity of the PT and resulting LMW proteinuria in Dent disease.Support or Funding InformationNIH F31 DK121394, R01 DK101484, R01 DK100357, P30 DK079307, and ASN Pre‐Doctoral Fellowship

Registration of six partial waxy near‐isogenic hexaploid wheat genetic stock lines lacking one or two granule bound starch synthase I genes

AbstractStarch is the leading component of wheat (Triticum spp.) grain and flour. Six hexaploid wheat (T. aestivum L.) near‐isogenic lines (NILs), WQL19WX7A (Reg. no. GS‐192, PI 691847), WQL19WX4A (Reg. no. GS‐193, PI 691848), WQL19WX7D (Reg. no. GS‐194, PI 691849), WQL19WX7A.A4 (Reg. no. GS‐195, PI 691850), WQL19WX7A.7D (Reg. no. GS‐196, PI 691851), and WQL19WX4A.7D (Reg. no. GS‐197, PI 691852), were developed to facilitate studies examining the role of starch amylose and amylopectin on wheat processing and end‐product quality and as parents to develop reduced amylose germplasm. Each NIL lacks a functional copy of one or two of the Waxy granule bound starch synthase I (GBSSI) genes and conversely possesses one or two functional genes. These genetic stocks were produced in ‘Alpowa’ soft spring wheat. The NILs are BC7 sibs, selected among segregating BC7 progeny using allele‐specific primers. The GBSSI null alleles were derived from the waxy cultivar Waxy‐Pen (PI 637779). These NILs are complemented by wild‐type Alpowa and a full waxy (zero amylose) line approved for release, ‘USDA Lori’. These lines were developed at the USDA Western Wheat Quality Laboratory.

The beta Subunit of Nascent Polypeptide Associated Complex Plays A Role in Flowers and Siliques Development of Arabidopsis thaliana.

The nascent polypeptide-associated (NAC) complex was described in yeast as a heterodimer composed of two subunits, α and β, and was shown to bind to the nascent polypeptides newly emerging from the ribosomes. NAC function was widely described in yeast and several information are also available about its role in plants. The knock down of individual NAC subunit(s) led usually to a higher sensitivity to stress. In Arabidopsis thaliana genome, there are five genes encoding NACα subunit, and two genes encoding NACβ. Double homozygous mutant in both genes coding for NACβ was acquired, which showed a delayed development compared to the wild type, had abnormal number of flower organs, shorter siliques and greatly reduced seed set. Both NACβ genes were characterized in more detail—the phenotype of the double homozygous mutant was complemented by a functional NACβ copy. Then, both NACβ genes were localized to nuclei and cytoplasm and their promoters were active in many organs (leaves, cauline leaves, flowers, pollen grains, and siliques together with seeds). Since flowers were the most affected organs by nacβ mutation, the flower buds’ transcriptome was identified by RNA sequencing, and their proteome by gel-free approach. The differential expression analyses of transcriptomic and proteomic datasets suggest the involvement of NACβ subunits in stress responses, male gametophyte development, and photosynthesis.

The Role of a Nascent Polypeptide-Associated Complex Subunit Alpha in Siderophore Biosynthesis, Oxidative Stress Response, and Virulence in Alternaria alternata.

The present study demonstrates that a nascent polypeptide-associated complex α subunit (Nac1) functions as a transcriptional regulator and plays both positive and negative roles in a vast array of functions in Alternaria alternata. Gain- and loss-of-function studies reveal that Nac1 is required for the formation and germination of conidia, likely via the regulation of Fus3 and Slt2 mitogen-activated protein kinase (MAPK)-coding genes, both implicated in conidiation. Nac1 negatively regulates hyphal branching and the production of cell wall-degrading enzymes. Importantly, Nac1 is required for the biosynthesis of siderophores, a novel phenotype that has not been reported to be associated with a Nac in fungi. The expression of Nac1 is positively regulated by iron, as well as by the Hog1 MAPK and the NADPH-dependent oxidase (Nox) complex. Nac1 confers cellular susceptibility to reactive oxygen species (ROS) likely via negatively regulating the expression of the genes encoding Yap1, Skn7, Hog1, and Nox, all involved in ROS resistance. The involvement of Nac1 in sensitivity to glucose-, mannitol-, or sorbitol-induced osmotic stress could be due to its ability to suppress the expression of Skn7. The requirement of Nac1 in resistance to salts is unlikely mediated through the transcriptional activation of Hog1. Although Nac1 plays no role in toxin production, Nac1 is required for fungal full virulence. All observed deficiencies can be restored by re-expressing a functional copy of Nac1, confirming that Nac1 contributes to the phenotypes. Thus, a dynamic regulation of gene expression via Nac1 is critical for developmental, physiological, and pathological processes of A. alternata.

Catabolism of aromaticβ-glucosides by bacteria can lead to antibiotics resistance.

Antimicrobial resistance is a serious public health threat worldwide today. Escherichia coli is known to resist low doses of antibiotics in the presence of sodium salicylate and related compounds by mounting non-heritable transient phenotypic antibiotic resistance (PAR). In the present study, we demonstrate that Bgl+ bacterial strains harboring a functional copy of the β-glucoside (bgl) operon and are actively hydrolyzing plant-derived aromatic β-glucosides such as salicin show PAR to low doses of antibiotics. The aglycone released during metabolism of aromatic β-glucosides is responsible for conferring this phenotype by de-repressing the multiple antibiotics resistance (mar) operon. We also show that prolonged exposure of Bgl+ bacteria to aromatic β-glucosides in the presence of sub-lethal doses of antibiotics can lead to a significant increase in the frequency of mutants that show heritable resistance to higher doses of antibiotics. Although heritable drug resistance in many cases is known to reduce the fitness of the carrier strain, we did not see a cost associated with resistance in the mutants, most of which carry clinically relevant mutations. These findings indicate that the presence of the activated form of the bgl operon in the genome facilitates the survival of bacteria in environments in which both aromatic β-glucosides and antibiotics are present.

Quantification of denitrifier genes population size and its relationship with environmental factors.

The objectives of this study were to use real-time PCR for culture-independent quantification of the copy numbers of 16S rRNA and denitrification functional genes, and also the relationships between gene copy numbers and soil physicochemical properties. In this study, qPCR analysis of the soil samples showed 16S rRNA, nirS, nirK, nosZI and nosZII average densities of 3.0 × 108, 2.25 × 107, 2.9 × 105, 4.0 × 106 and 1.75 × 107 copies per gram of dry soil, respectively. In addition, the abundances of (nirS + nirK), nosZI and nosZII relative to 16S rRNA genes were 1.4-34.1%, 0.06-3.95% and 1.3-39%, respectively, confirming the low proportion of denitrifiers to total bacteria in soil. This showed that the non-denitrifying nosZII-type bacteria may contribute significantly to N2O consumption in the soil. Furthermore, the shifts in abundance and diversity of the total bacteria and denitrification functional gene copy numbers correlated significantly with the various soil factors. It is the first study in Turkey about the population size of denitrification functional genes in different soil samples. It also aims to draw attention to nitrous oxide-associated global warming.

Verticillium dahliae VdTHI20, Involved in Pyrimidine Biosynthesis, Is Required for DNA Repair Functions and Pathogenicity

Verticillium dahliae (V. dahliae) infects roots and colonizes the vascular vessels of host plants, significantly reducing the economic yield of cotton and other crops. In this study, the protein VdTHI20, which is involved in the thiamine biosynthesis pathway, was characterized by knocking out the corresponding VdTHI20 gene in V. dahliae via Agrobacterium tumefaciens-mediated transformation (ATMT). The deletion of VdTHI20 resulted in several phenotypic defects in vegetative growth and conidiation and in impaired virulence in tobacco seedlings. We show that VdTHI20 increases the tolerance of V. dahliae to UV damage. The impaired vegetative growth of ΔVdTHI20 mutant strains was restored by complementation with a functional copy of the VdTHI20 gene or by supplementation with additional thiamine. Furthermore, the root infection and colonization of the ΔVdTHI20 mutant strains were suppressed, as indicated by green fluorescent protein (GFP)-labelling under microscope observation. When the RNAi constructs of VdTHI20 were used to transform Nicotiana benthamiana, the transgenic lines expressing dsVdTHI20 showed elevated resistance to V. dahliae. Together, these results suggest that VdTHI20 plays a significant role in the pathogenicity of V. dahliae. In addition, the pathogenesis-related gene VdTHI20 exhibits potential for controlling V. dahliae in important crops.

PRH1 mediates ARF7-LBD dependent auxin signaling to regulate lateral root development in Arabidopsis thaliana.

The development of lateral roots in Arabidopsis thaliana is strongly dependent on signaling directed by the AUXIN RESPONSE FACTOR7 (ARF7), which in turn activates LATERAL ORGAN BOUNDARIES DOMAIN (LBD) transcription factors (LBD16, LBD18 and LBD29). Here, the product of PRH1, a PR-1 homolog annotated previously as encoding a pathogen-responsive protein, was identified as a target of ARF7-mediated auxin signaling and also as participating in the development of lateral roots. PRH1 was shown to be strongly induced by auxin treatment, and plants lacking a functional copy of PRH1 formed fewer lateral roots. The transcription of PRH1 was controlled by the binding of both ARF7 and LBDs to its promoter region.

Longitudinal metabolomic analysis of plasma enables modeling disease progression in Duchenne muscular dystrophy mouse models.

Duchenne muscular dystrophy is a severe pediatric neuromuscular disorder caused by the lack of dystrophin. Identification of biomarkers is needed to support and accelerate drug development. Alterations of metabolites levels in muscle and plasma have been reported in pre-clinical and clinical cross-sectional comparisons. We present here a 7-month longitudinal study comparing plasma metabolomic data in wild-type and mdx mice. A mass spectrometry approach was used to study metabolites in up to five time points per mouse at 6, 12, 18, 24 and 30 weeks of age, providing an unprecedented in depth view of disease trajectories. A total of 106 metabolites were studied. We report a signature of 31 metabolites able to discriminate between healthy and disease at various stages of the disease, covering the acute phase of muscle degeneration and regeneration up to the deteriorating phase. We show how metabolites related to energy production and chachexia (e.g. glutamine) are affected in mdx mice plasma over time. We further show how the signature is connected to molecular targets of nutraceuticals and pharmaceutical compounds currently in development as well as to the nitric oxide synthase pathway (e.g. arginine and citrulline). Finally, we evaluate the signature in a second longitudinal study in three independent mouse models carrying 0, 1 or 2 functional copies of the dystrophin paralog utrophin. In conclusion, we report an in-depth metabolomic signature covering previously identified associations and new associations, which enables drug developers to peripherally assess the effect of drugs on the metabolic status of dystrophic mice.

Evaluation of theanti-apoptotic activity of bovine alphaherpesvirus type 5 US3 protein kinase in insect cells using a recombinant baculovirus.

Bovine alphaherpesvirus type 5 (BoHV-5) is one of the main agents responsible for meningoencephalitis in cattle in Brazil, causing significant economic losses. It is known that other viruses of the Herpesviridae family such as Bovine alphaherpesvirus type 1, Swine alphaherpesvirus type 1, and the Human alphaherpesvirus types 1 and 2 encode genes homologous to BoHV-5, with recognized action in the control of apoptosis. The objective of this work was to express the BoHV-5 US3 gene in a baculovirus-based expression system for the production of the serine/threonine kinase protein and to evaluate its activity in the control of apoptosis in vitro. A recombinant baculovirus derived from the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) containing the US3 gene and a deletion in the baculovirus anti-apoptotic p35 gene was constructed using the Bac-to-Bac™ system. This recombinant baculovirus was used to evaluate the anti-apoptotic activity of the recombinant US3 protein in insect cells comparing with two other AcMNPV recombinants, one containing a functional copy of the AcMNPV anti-apoptotic p35 gene and an AcMNPV p35 knockout virus with the anti-apoptotic iap-3 gene from Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV). We found that the caspase level was higher in insect cells infected with the US3-contanining recombinant virus than in cells infected with the AcMNPV recombinants containing the p35 and iap-3 genes. These results indicate that the BoHV-5 US3 protein kinase gene is not able to block apoptosis in insect cells induced by the infection of a p35 knockout AcMNPV.

A novel integrative performance evaluation of constructed wetland on removal of viable bacterial cells and related pathogenic, virulent and multi-drug resistant genes from wastewater systems

Integrative performance evaluation of constructed wetland in removal different aspects of bacteria under specific local environmental conditions needs to be explored in detail to ensure selection of appropriate and highly efficient macrophytes candidates. To achieve this, integrative purification performance evaluation approach that holistically considers all the aspects of pathogenic bacterial biology (colony numbers, functional gene, species, virulent, pathogenicity and resistant genes) needs to be adopted rather than the commonly known unidimensional approaches that take into account a single bacterial aspect. This study experimentally evaluated the individual performance of three native and one exotic winter tolerant submerged macrophytes combined with a single emergent macrophyte in eradicating faecal related bacterial species and pathotypes across horizontal surface flow constructed wetland. It involved the new multi-dimensional approach that integrated the faecal bacterial colony numbers, functional gene copies, species survival, virulent and pathogenicity as well as antimicrobial resistant in constructed wetland purification evaluation. The results showed Elodea nuttallii and Myriophyllum spicatum as the best candidate partners to Typha latifolia for the highest Purification Efficiency (P < 0.05), of above 97% for removal of faecal bacteria colonies and functional genes, and more than 75% for removal of faecal bacterial strains, pathotypes, virulent and well as resistant genes. However, M. spicatum being a Chinese native species should be much preferred to the invasive E. nuttallii. Therefore, the study recommends the application of local macrophytes such as M. spicatum as the best candidates and the decision should emanate from such a multidimensional/intergrative purification-based evaluation approach.

The Tolerance of Salinity in Rice Requires the Presence of a Functional Copy of FLN2.

A panel of ethane-methyl-sulfonate-mutagenized japonica rice lines was grown in the presence of salinity in order to identify genes required for the expression of salinity tolerance. A highly nontolerant selection proved to harbor a mutation in FLN2, a gene which encodes fructokinase-like protein2. Exposure of wild-type rice to salinity up-regulated FLN2, while a CRISPR/Cas9-generated FLN2 knockout line was hypersensitive to the stress. Both ribulose 1,5-bisphosphate carboxylase/oxygenase activity and the abundance of the transcript generated by a number of genes encoding components of sucrose synthesis were lower in the knockout line than in wild-type plants’ leaves, while the sucrose contents of the leaf and root were, respectively, markedly increased and decreased. That sugar partitioning to the roots was impaired in FLN2 knockout plants was confirmed by the observation that several genes involved in carbon transport were down-regulated in both the leaf and in the leaf sheath. The levels of sucrose synthase, acid invertase, and neutral invertase activity were distinctly lower in the knockout plants’ roots than in those of wild-type plants, particularly when the plants were exposed to salinity stress. The compromised salinity tolerance exhibited by the FLN2 knockout plants was likely a consequence of an inadequate supply of the assimilate required to support growth, a problem which was rectifiable by providing an exogenous supply of sucrose. The conclusion was that FLN2, on account of its influence over sugar metabolism, is important in the context of seedling growth and the rice plant’s response to salinity stress.

Ammonium release via dissolution and biological mineralization of food pellets used in salmon farming

We studied the input of ammonium coming from the dissolution of food pellets commonly used in salmon farming as well as its potential for mineralization via ammonification and ammonium oxidation by natural bacterioplankton communities. Through experiments in central and southern Chile, we evaluated the potential for photo‐ and bio‐ammonification of food pellets in surface and subsurface waters (30 m depth) using irradiated (PAR + UV; IPS) and non‐irradiated dissolved‐pellet solutions (NIPS). Ammonium release was observed in both IPS and NIPS treatments and occurred at short time scales (<2 hr). Experiments in southern Chile during late winter showed high rates of pellet ammonium release in surface waters (up to 3.8 µmol L⁻¹ min⁻¹) compared with deeper samples (30 m depth). In contrast, in spring higher rates were observed at 30 m depth and reached up to 45 µmol L⁻¹ min⁻¹ while in surface waters they accounted for 9 µmol L⁻¹ min⁻¹. Ammonium oxidizing bacteria and archaea showed seasonal variability, but archaea were detected in all experiments reaching high potential activity, based in RT‐qPCR of functional genes expression copy numbers, in IPS treatments during the winter season in Chiloe. Overall, our results indicate that food pellets can be a source of ammonium, which can exceed by two orders of magnitude the average natural concentrations commonly found in the euphotic layer in coastal environments. The ammonium enrichment from dissolved pellet solutions was rapidly consumed by microbial communities including nitrifying assemblages. This has implications for the evaluation of aquaculture nutrient input and its recycling in the water column in areas highly impacted by climate change and aquaculture pressure.

Loss of IL-10 signaling in macrophages limits bacterial killing driven by prostaglandin E2.

Loss of IL-10 signaling in macrophages (Mφs) leads to inflammatory bowel disease (IBD). Induced pluripotent stem cells (iPSCs) were generated from an infantile-onset IBD patient lacking a functional IL10RB gene. Mφs differentiated from IL-10RB-/- iPSCs lacked IL-10RB mRNA expression, were unable to phosphorylate STAT3, and failed to reduce LPS induced inflammatory cytokines in the presence of exogenous IL-10. IL-10RB-/- Mφs exhibited a striking defect in their ability to kill Salmonella enterica serovar Typhimurium, which was rescuable after experimentally introducing functional copies of the IL10RB gene. Genes involved in synthesis and receptor pathways for eicosanoid prostaglandin E2 (PGE2) were more highly induced in IL-10RB-/- Mφs, and these Mφs produced higher amounts of PGE2 after LPS stimulation compared with controls. Furthermore, pharmacological inhibition of PGE2 synthesis and PGE2 receptor blockade enhanced bacterial killing in Mφs. These results identify a regulatory interaction between IL-10 and PGE2, dysregulation of which may drive aberrant Mφ activation and impaired host defense contributing to IBD pathogenesis.

Pediatric retina imaging in aniridia

AbstractAniridia is a congenital complex ocular malformation characterized by complete or partial iris hypoplasia and foveal hypoplasia, resulting in nystagmus and low visual acuity from early infancy. In the majority of cases, aniridia is caused by the loss of function of one functional copy (haploinsufficiency) of the PAX6 protein resulting from mutations in PAX6 gene or chromosomal rearrangement at the 11p13 locus. High‐resolution foveal imaging by spectral‐domain optical coherence tomography (SD‐OCT) has improved over recent years the evaluation of foveal architecture analyze. We have analyzed foveal anomalies on SD‐OCT in a large cohort (89 patients) of aniridia patients. We have shown that the grade of foveal hypoplasia is correlated to visual acuity. We have also shown that foveal hypoplasia is a constant finding in PAX6 aniridia patients, although iris defects could vary from complete aniridia to slight iris defects, even in the same family. Foveal imaging and grading by SD‐OCT could offer a non‐invasive tool to determine the visual potential of aniridia eyes and indicate further investigations or treatments. Additionally, SD‐OCT could contribute in the diagnosis of congenital aniridia by detecting foveal hypoplasia as the first sign of the disease.

Targeted mutagenesis of BnTT8 homologs controls yellow seed coat development for effective oil production in Brassica napus L.

Yellow seed is a desirable trait with great potential for improving seed quality in Brassica crops. Unfortunately, no natural or induced yellow seed germplasms have been found in Brassica napus, an important oil crop, which likely reflects its genome complexity and the difficulty of the simultaneous random mutagenesis of multiple gene copies with functional redundancy. Here, we demonstrate the first application of CRISPR/Cas9 for creating yellow‐seeded mutants in rapeseed. The targeted mutations of the BnTT8 gene were stably transmitted to successive generations, and a range of homozygous mutants with loss‐of‐function alleles of the target genes were obtained for phenotyping. The yellow‐seeded phenotype could be recovered only in targeted mutants of both BnTT8 functional copies, indicating that the redundant roles of BnA09.TT8 and BnC09.TT8b are vital for seed colour. The BnTT8 double mutants produced seeds with elevated seed oil and protein content and altered fatty acid (FA) composition without any serious defects in the yield‐related traits, making it a valuable resource for rapeseed breeding programmes. Chemical staining and histological analysis showed that the targeted mutations of BnTT8 completely blocked the proanthocyanidin (PA)‐specific deposition in the seed coat. Further, transcriptomic profiling revealed that the targeted mutations of BnTT8 resulted in the broad suppression of phenylpropanoid/flavonoid biosynthesis genes, which indicated a much more complex molecular mechanism underlying seed colour formation in rapeseed than in Arabidopsis and other Brassica species. In addition, gene expression analysis revealed the possible mechanism through which BnTT8 altered the oil content and fatty acid composition in seeds.

Fermentation innovation through complex hybridization of wild and domesticated yeasts.

The most common fermented beverage, lager beer, is produced by interspecies hybrids of the brewing yeast Saccharomyces cerevisiae and its wild relative S. eubayanus. Lager-brewing yeasts are not the only example of hybrid vigour or heterosis in yeasts, but the full breadth of interspecies hybrids associated with human fermentations has received less attention. Here we present a comprehensive genomic analysis of 122 Saccharomyces hybrids and introgressed strains. These strains arose from hybridization events between two to four species. Hybrids with S. cerevisiae contributions originated from three lineages of domesticated S. cerevisiae, including the major wine-making lineage and two distinct brewing lineages. In contrast, the undomesticated parents of these interspecies hybrids were all from wild Holarctic or European lineages. Most hybrids have inherited a mitochondrial genome from a parent other than S. cerevisiae, which recent functional studies suggest could confer adaptation to colder temperatures. A subset of hybrids associated with crisp flavour profiles, including both lineages of lager-brewing yeasts, have inherited inactivated S. cerevisiae alleles of critical phenolic off-flavour genes and/or lost functional copies from the wild parent through multiple genetic mechanisms. These complex hybrids shed light on the convergent and divergent evolutionary trajectories of interspecies hybrids and their impact on innovation in lager brewing and other diverse fermentation industries.