Polygenic risk impacts PDGFRA mutation penetrance in non-syndromic cleft lip and palate.

Non-syndromic cleft lip with or without cleft palate (NSCL/P) is a common, severe craniofacial malformation that imposes significant medical, psychosocial and financial burdens. NSCL/P is a multifactorial disorder with genetic and environmental factors playing etiologic roles. Currently, only 25% of the genetic variation underlying NSCL/P has been identified by linkage, candidate gene and genome-wide association studies. In this study, whole-genome sequencing and genome-wide genotyping followed by polygenic risk score (PRS) and linkage analyses were used to identify the genetic etiology of NSCL/P in a large three-generation family. We identified a rare missense variant in PDGFRA (c.C2740T; p.R914W) as potentially etiologic in a gene-based association test using pVAAST (P = 1.78 × 10-4) and showed decreased penetrance. PRS analysis suggested that variant penetrance was likely modified by common NSCL/P risk variants, with lower scores found among unaffected carriers. Linkage analysis provided additional support for PRS-modified penetrance, with a 7.4-fold increase in likelihood after conditioning on PRS. Functional characterization experiments showed that the putatively causal variant was null for signaling activity in vitro; further, perturbation of pdgfra in zebrafish embryos resulted in unilateral orofacial clefting. Our findings show that a rare PDGFRA variant, modified by additional common NSCL/P risk variants, have a profound effect on NSCL/P risk. These data provide compelling evidence for multifactorial inheritance long postulated to underlie NSCL/P and may explain some unusual familial patterns.

Multiple bHLH/MYB-based protein complexes regulate proanthocyanidin biosynthesis in the herbage of Lotus spp.

MAIN CONCLUSION: The complexes involving MYBPA2, TT2b, and TT8 proteins are the critical regulators of ANR and LAR genes to promote the biosynthesis of proanthocyanidins in the leaves of Lotus spp. The environmental impact and health of ruminants fed with forage legumes depend on the herbage's concentration and structure of proanthocyanidins (PAs). Unfortunately, the primary forage legumes (alfalfa and clover) do not contain substantial levels of PAs. No significant progress has been made to induce PAs to agronomically valuable levels in their edible organs by biotechnological approaches thus far. Building this trait requires a profound knowledge of PA regulators and their interplay in species naturally committed to accumulating these metabolites in the target organs. Against this background, we compared the shoot transcriptomes of two inter-fertile Lotus species, namely Lotus tenuis and Lotus corniculatus, polymorphic for this trait, to search for differentially expressed MYB and bHLH genes. We then tested the expression of the above-reported regulators in L. tenuis x L. corniculatus interspecific hybrids, several Lotus spp., and different L. corniculatus organs with contrasting PA levels. We identified a novel MYB activator and MYB-bHLH-based complexes that, when expressed in Nicotiana benthamiana, trans-activated the promoters of L. corniculatus anthocyanidin reductase and leucoanthocyanidin reductase 1 genes. The last are the two critical structural genes for the biosynthesis of PAs in Lotus spp. Competition between MYB activators for the transactivation of these promoters also emerged. Overall, by employing Lotus as a model genus, we refined the transcriptional network underlying PA biosynthesis in the herbage of legumes. These findings are crucial to engineering this trait in pasture legumes.

FishNET: An automated relational database for zebrafish colony management.

The zebrafish Danio rerio is a powerful model system to study the genetics of development and disease. However, maintenance of zebrafish husbandry records is both time intensive and laborious, and a standardized way to manage and track the large amount of unique lines in a given laboratory or centralized facility has not been embraced by the field. Here, we present FishNET, an intuitive, open-source, relational database for managing data and information related to zebrafish husbandry and maintenance. By creating a "virtual facility," FishNET enables users to remotely inspect the rooms, racks, tanks, and lines within a given facility. Importantly, FishNET scales from one laboratory to an entire facility with several laboratories to multiple facilities, generating a cohesive laboratory and community-based platform. Automated data entry eliminates confusion regarding line nomenclature and streamlines maintenance of individual lines, while flexible query forms allow researchers to retrieve database records based on user-defined criteria. FishNET also links associated embryonic and adult biological samples with data, such as genotyping results or confocal images, to enable robust and efficient colony management and storage of laboratory information. A shared calendar function with email notifications and automated reminders for line turnover, automated tank counts, and census reports promote communication with both end users and administrators. The expected benefits of FishNET are improved vivaria efficiency, increased quality control for experimental numbers, and flexible data reporting and retrieval. FishNET's easy, intuitive record management and open-source, end-user-modifiable architecture provides an efficient solution to real-time zebrafish colony management for users throughout a facility and institution and, in some cases, across entire research hubs.

SARS-CoV-2 infection in early first-trimester miscarriages: a prospective observational study.

RESEARCH QUESTION: Is there an association between SARS-CoV-2 infection and first-trimester miscarriage? DESIGN: This multicentre prospective study included a cohort of women with first-trimester miscarriages registered consecutively by seven Spanish hospitals where universal PCR screening for SARS-CoV-2 infection was implemented with both miscarriages and deliveries. The incidence of SARS-CoV-2 infection among women with first-trimester miscarriages was compared with the rate registered in women on admission to the delivery ward within the same time frame using a mixed-effects Poisson regression analysis, considering 'hospital' as random effect. The characteristics of SARS-CoV-2 positive and negative patients who miscarried were compared through two-sided univariable analyses. RESULTS: A total of 301 miscarriages were registered, 11 (3.7%) to SARS-CoV-2 infected and 290 to non-infected women. In the same time frame as the miscarriages, 1936 deliveries were registered, 44 [2.3%] of them were SARS-CoV-2 infected. No differences in terms of SARS-CoV-2 infection incidence were observed between infected miscarriages and infected deliveries (P = 0.233). Regarding the differences observed between miscarriages in SARS-CoV-2 positive and negative women, more inevitable miscarriages occurred in the group of infected women (36.4% versus 16.5% in non-infected women; P = 0.004), and there was greater surgical management of miscarriages (27.3% versus 8.2% in non-infected women; P = 0.036), probably in line with the greater number of inevitable miscarriages observed in this group. CONCLUSIONS: No association between SARS-CoV-2 infection and risk of first-trimester miscarriage was observed; however, the type of miscarriage seems to differ between SARS-CoV-2 positive and negative women, with inevitable miscarriage being more frequent among infected women.

Whole-Genome Resequencing of Spontaneous Oxidative Stress-Resistant Mutants Reveals an Antioxidant System of Bradyrhizobium japonicum Involved in Soybean Colonization.

Soybean is the most inoculant-consuming crop in the world, carrying strains belonging to the extremely related species Bradyrhizobium japonicum and Bradyrhizobium diazoefficiens. Currently, it is well known that B. japonicum has higher efficiency of soybean colonization than B. diazoefficiens, but the molecular mechanism underlying this differential symbiotic performance remains unclear. In the present study, genome resequencing of four spontaneous oxidative stress-resistant mutants derived from the commercial strain B. japonicum E109 combined with molecular and physiological studies allowed identifying an antioxidant cluster (BjAC) containing a transcriptional regulator (glxA) that controls the expression of a catalase (catA) and a phosphohydrolase (yfbR) related to the hydrolysis of hydrogen peroxide and oxidized nucleotides, respectively. Integrated synteny and phylogenetic analyses supported the fact that BjAC emergence in the B. japonicum lineage occurred after its divergence from the B. diazoefficiens lineage. The transformation of the model bacterium B. diazoefficiens USDA110 with BjAC from E109 significantly increased its ability to colonize soybean roots, experimentally recapitulating the beneficial effects of the occurrence of BjAC in B. japonicum. In addition, the glxA mutation significantly increased the nodulation competitiveness and plant growth-promoting efficiency of E109. Finally, the potential applications of these types of non-genetically modified mutant microbes in soybean production worldwide are discussed.

The association between SARS-CoV-2 infection and preterm delivery: a prospective study with a multivariable analysis.

BACKGROUND: To determine whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, the cause of COVID-19 disease) exposure in pregnancy, compared to non-exposure, is associated with infection-related obstetric morbidity. METHODS: We conducted a multicentre prospective study in pregnancy based on a universal antenatal screening program for SARS-CoV-2 infection. Throughout Spain 45 hospitals tested all women at admission on delivery ward using polymerase-chain-reaction (PCR) for COVID-19 since late March 2020. The cohort of positive mothers and the concurrent sample of negative mothers was followed up until 6-weeks post-partum. Multivariable logistic regression analysis, adjusting for known confounding variables, determined the adjusted odds ratio (aOR) with 95% confidence intervals (95% CI) of the association of SARS-CoV-2 infection and obstetric outcomes. MAIN OUTCOME MEASURES: Preterm delivery (primary), premature rupture of membranes and neonatal intensive care unit admissions. RESULTS: Among 1009 screened pregnancies, 246 were SARS-CoV-2 positive. Compared to negative mothers (763 cases), SARS-CoV-2 infection increased the odds of preterm birth (34 vs 51, 13.8% vs 6.7%, aOR 2.12, 95% CI 1.32-3.36, p = 0.002); iatrogenic preterm delivery was more frequent in infected women (4.9% vs 1.3%, p = 0.001), while the occurrence of spontaneous preterm deliveries was statistically similar (6.1% vs 4.7%). An increased risk of premature rupture of membranes at term (39 vs 75, 15.8% vs 9.8%, aOR 1.70, 95% CI 1.11-2.57, p = 0.013) and neonatal intensive care unit admissions (23 vs 18, 9.3% vs 2.4%, aOR 4.62, 95% CI 2.43-8.94, p <  0.001) was also observed in positive mothers. CONCLUSION: This prospective multicentre study demonstrated that pregnant women infected with SARS-CoV-2 have more infection-related obstetric morbidity. This hypothesis merits evaluation of a causal association in further research.

Frontalis Suspension Using Autologous Fascia Lata in Children Under 3 Years Old.

PURPOSE: To analyze functional results and complications of the use of autologous fascia lata in frontalis suspension in children under 3 years old and to confirm its technical viability. METHODS: A retrospective review of 8 patients (12 eyes) who underwent frontalis suspension using autologous fascia lata sling. RESULTS: Twelve eyes of 8 patients were analyzed, with an average age of 1.8 ± 0.6 years. Preoperatively, the mean margin-to-reflex distance 1 was -0.17 ± 0.577 mm. Postoperatively the mean margin-to-reflex distance 1 was 2.66 ± 0.492 mm without any graft donor site or corneal complications. No recurrence was recorded in the follow-up period (mean follow-up period 28.5 ± 32.33 months). CONCLUSIONS: Autologous fascia lata is an eligible material in frontalis suspension in children under 3 years old, despite the traditional oculoplastic dogma that advises against.

Dynamic regulation of VEGF-inducible genes by an ERK/ERG/p300 transcriptional network.

The transcriptional pathways activated downstream of vascular endothelial growth factor (VEGF) signaling during angiogenesis remain incompletely characterized. By assessing the signals responsible for induction of the Notch ligand delta-like 4 (DLL4) in endothelial cells, we find that activation of the MAPK/ERK pathway mirrors the rapid and dynamic induction of DLL4 transcription and that this pathway is required for DLL4 expression. Furthermore, VEGF/ERK signaling induces phosphorylation and activation of the ETS transcription factor ERG, a prerequisite for DLL4 induction. Transcription of DLL4 coincides with dynamic ERG-dependent recruitment of the transcriptional co-activator p300. Genome-wide gene expression profiling identified a network of VEGF-responsive and ERG-dependent genes, and ERG chromatin immunoprecipitation (ChIP)-seq revealed the presence of conserved ERG-bound putative enhancer elements near these target genes. Functional experiments performed in vitro and in vivo confirm that this network of genes requires ERK, ERG and p300 activity. Finally, genome-editing and transgenic approaches demonstrate that a highly conserved ERG-bound enhancer located upstream of HLX (which encodes a transcription factor implicated in sprouting angiogenesis) is required for its VEGF-mediated induction. Collectively, these findings elucidate a novel transcriptional pathway contributing to VEGF-dependent angiogenesis.

Molecular characterization of a novel cytorhabdovirus with a unique genomic organization infecting yerba mate (Ilex paraguariensis) in Argentina.

The genome of a novel rhabdovirus was detected in yerba mate (Ilex paraguariensis St. Hil.). The newly identified virus, tentatively named "yerba mate virus A" (YmVA), has a genome of 14,961 nucleotides. Notably, eight open reading frames were identified in the antigenomic orientation of the negative-sense, single-stranded viral RNA, including two novel accessory genes, in the order 3'-N-P-3-4-M-G-L-8-5'. Sequence comparisons of the encoded proteins as well as phylogenetic analysis suggest that YmVA is a new member of the genus Cytorhabdovirus, family Rhabdoviridae. YmVA's unique genomic organization and phylogenetic relationships indicate that this virus likely represents a distinct evolutionary lineage among the cytorhabdoviruses.

Innovación pedagógica para mejorar la calidad del trato en la atención de la salud de mujeres indígenas.

Objetivo. Presentar los resultados de una intervención pedagógica para mejorar la calidad en el trato que brindan los prestadores de servicios de salud en regiones indígenas. Material y métodos. Se diseñó una metodología didáctica con enfoque crítico-constructivista dirigida a personal de salud que atiende a población indígena en cinco entidades de México. Resultados. Entre 09/2016 a 01/2020 se capacitó a 1 825 trabajadores de la salud que deconstruyeron las creencias que determinan prácticas de discriminación y maltrato durante la atención a la salud de los usuarios indígenas. La intervención logró un aprendizaje significativo transformador de las valoraciones y prácticas sociales, con propuestas para evitar cualquier forma de maltrato y garantizar el trato digno. Conclusiones. Para la construcción de un sistema de salud universal y equitativo, es necesario incluir intervenciones que actúen sobre las creencias determinantes de las prácticas de discriminación y maltrato en los servicios de salud hacia grupos sociales vulnerables.

Impact of Self-Assembled Monolayer Design and Electrochemical Factors on Impedance-Based Biosensing.

Real-time sensing of proteins, especially in wearable devices, remains a substantial challenge due to the need to convert a binding event into a measurable signal that is compatible with the chosen analytical instrumentation. Impedance spectroscopy enables real-time detection via either measuring electrostatic interactions or electron transfer reactions while simultaneously being amenable to miniaturization for integration into wearable form-factors. To create a more robust methodology for optimizing impedance-based sensors, additional fundamental studies exploring components influencing the design and implementation of these sensors are needed. This investigation addresses a sub-set of these issues by combining optical and electrochemical characterization to validate impedance-based sensor performance as a function of (1) biorecognition element density, (2) self-assembled monolayer chain length, (3) self-assembled monolayer charge density, (4) the electrochemical sensing mechanism and (5) the redox reporter selection. Using a pre-existing lysozyme aptamer and lysozyme analyte combination, we demonstrate a number of design criteria to advance the state-of-the-art in protein sensing. For this model system we demonstrated the following: First, denser self-assembled monolayers yielded substantially improved sensing results. Second, self-assembled monolayer composition, including both thickness and charge density, changed the observed peak position and peak current. Third, single frequency measurements, while less informative, can be optimized to replace multi-frequency measurements and in some cases (such as that with zwitterionic self-assembled monolayers) are preferred. Finally, various redox reporters traditionally not used in impedance sensing should be further explored. Collectively, these results can help limit bottlenecks associated with device development, enabling realization of next-generation impedance-based biosensing with customize sensor design for the specific application.

A toolbox to study epidermal cell types in zebrafish.

Epithelia provide a crucial protective barrier for our organs and are also the sites where the majority of carcinomas form. Most studies on epithelia and carcinomas use cell culture or organisms where high-resolution live imaging is inaccessible without invasive techniques. Here, we introduce the developing zebrafish epidermis as an excellent in vivo model system for studying a living epithelium. We developed tools to fluorescently tag specific epithelial cell types and express genes in a mosaic fashion using five Gal4 lines identified from an enhancer trap screen. When crossed to a variety of UAS effector lines, we can now track, ablate or monitor single cells at sub-cellular resolution. Using photo-cleavable morpholino oligonucleotides that target gal4, we can also express genes in a mosaic fashion at specific times during development. Together, this system provides an excellent in vivo alternative to tissue culture cells, without the intrinsic concerns of culture conditions or transformation, and enables the investigation of distinct cell types within living epithelial tissues.

Transcript and metabolic adjustments triggered by drought in Ilex paraguariensis leaves.

MAIN CONCLUSION: Abscisic acid is involved in the drought response of Ilex paraguariensis. Acclimation includes root growth stimulation, stomatal closure, osmotic adjustment, photoprotection, and regulation of nonstructural carbohydrates and amino acid metabolisms. Ilex paraguariensis (yerba mate) is cultivated in the subtropical region of South America, where the occurrence of drought episodes limit yield. To explore the mechanisms that allow I. paraguariensis to overcome dehydration, we investigated (1) how gene expression varied between water-stressed and non-stressed plants and (2) in what way the modulation of gene expression was linked to physiological status and metabolite composition. A total of 4920 differentially expressed transcripts were obtained through RNA-Seq after water deprivation. Drought induced the expression of several transcripts involved in the ABA-signalling pathway. Stomatal closure and leaf osmotic adjustments were promoted to minimize water loss, and these responses were accompanied by a high transcriptional remodeling of stress perception, signalling and transcriptional regulation, the photoprotective and antioxidant systems, and other stress-responsive genes. Simultaneously, significant changes in metabolite contents were detected. Glutamine, phenylalanine, isomaltose, fucose, and malate levels were shown to be positively correlated with dehydration. Principal component analysis showed differences in the metabolic profiles of control and stressed leaves. These results provide a comprehensive overview of how I. paraguariensis responds to dehydration at transcriptional and metabolomic levels and provide further characterization of the molecular mechanisms associated with drought response in perennial subtropical species.

A Bacterial Endophyte from Apoplast Fluids Protects Canola Plants from Different Phytopathogens via Antibiosis and Induction of Host Resistance.

Endophytic bacteria colonize inner plant tissues and thrive at the apoplast, which is considered its main reservoir. Because this niche is the place where the main molecular events take place between beneficial and pathogenic microorganisms, the aim of this work was to characterize culturable endophytic bacteria from apoplastic fluids obtained from field-grown canola leaves and analyze their potential for biological control of diseases caused by Xanthomonas campestris, Sclerotinia sclerotiorum, and Leptosphaeria maculans. Dual-culture analysis indicated that three isolates (Apo8, Apo11, and Apo12) were able to inhibit the growth of all three phytopathogens. Sequencing of the 16S ribosomal RNA and rpoD genes of these isolates revealed that they are closely related to Pseudomonas viridiflava. One of the isolates, Apo11, was able to diminish the propagation of X. campestris in whole-plant assays. At the same time, Apo11 inoculation reduced the necrotic lesions provoked by S. sclerotiorum on canola leaves. This protective effect might be due to the induction of resistance in the host mediated by salicylic and jasmonic acid signaling pathways or the production of compounds with antimicrobial activity. At the same time, Apo11 inoculation promoted canola plant growth. Thus, the isolate characterized in this work has several desirable characteristics, which make it a potential candidate for the formulation of biotechnological products to control plant diseases or promote plant growth.

Characterization of the Copper Transporters from Lotus spp. and Their Involvement under Flooding Conditions.

Forage legumes are an important livestock nutritional resource, which includes essential metals, such as copper. Particularly, the high prevalence of hypocuprosis causes important economic losses to Argentinian cattle agrosystems. Copper deficiency in cattle is partially due to its low content in forage produced by natural grassland, and is exacerbated by flooding conditions. Previous results indicated that incorporation of Lotus spp. into natural grassland increases forage nutritional quality, including higher copper levels. However, the biological processes and molecular mechanisms involved in copper uptake by Lotus spp. remain poorly understood. Here, we identify four genes that encode putative members of the Lotus copper transporter family, denoted COPT in higher plants. A heterologous functional complementation assay of the Saccharomyces cerevisiae ctr1∆ctr3∆ strain, which lacks the corresponding yeast copper transporters, with the putative Lotus COPT proteins shows a partial rescue of the yeast phenotypes in restrictive media. Under partial submergence conditions, the copper content of L. japonicus plants decreases and the expression of two Lotus COPT genes is induced. These results strongly suggest that the Lotus COPT proteins identified in this work function in copper uptake. In addition, the fact that environmental conditions affect the expression of certain COPT genes supports their involvement in adaptive mechanisms and envisages putative biotechnological strategies to improve cattle copper nutrition.

Electrostatically Tunable Adhesion in a High Speed Sliding Interface.

Contact hysteresis between sliding interfaces is a widely observed phenomenon from macro- to nanoscale sliding interfaces. Most such studies are done using an atomic force microscope (AFM) where the sliding speed is a few µm/s. Here, we present a unique study on stiction between the head-disk interface of commercially available hard disk drives, wherein the vertical clearance between the head and the disk is of the same order as in various AFM-based fundamental studies but with a sliding speed that is nearly 6 orders of magnitude higher. We demonstrate that, although the electrostatic force (dc or ac voltage) is an attractive force, the ac-voltage-induced out-of-plane oscillation of the head with respect to the disk is able to completely suppress the contact hysteresis.

Application of a glass fertilizer in sustainable tomato plant crops.

BACKGROUND: Glass frits with a P2 O5 /K2 O ratio equal to 0.65 and different concentrations of SiO2 (6.5-56.0 wt%), CaO (0-5.1 wt%) and Al2 O3 (0-14.6 wt%) have been prepared and analysed as potential glass fertilizers (GF). RESULTS: P2 O5 and K2 O were leached primarily within the first 5 days but the total amount was dependent on the SiO2 /Al2 O3 ratio of the glass frit. Sustained P2 O5 and K2 O leaching for more than 40 days could be obtained if the glass frit contained more than 7% Al2 O3 . A final glass frit composition (29% SiO2 , 5% CaO, 7% Al2 O3 , 30% P2 O5 , 25% K2 O) was selected and used in an experimental tomato crop of 1 ha area, where one half was fertilized with an NPK conventional fertilizer and the other half with the GF fertilizer. CONCLUSION: After harvesting the tomato crops, the GF fertilizer was found to result in a higher yield than the NPK conventional fertilizer: 59980 kg and 74 360 kg for NKP and GF, respectively. The general characteristics of the harvested tomato fruits and leaves were similar in the two cases and the differences fell within statistical error. No differences in any of the soil characteristics were detected as a result of the use of GF. © 2018 Society of Chemical Industry.

A comprehensive multi-omics approach uncovers adaptations for growth and survival of Pseudomonas aeruginosa on n-alkanes.

BACKGROUND: Examination of complex biological systems has long been achieved through methodical investigation of the system's individual components. While informative, this strategy often leads to inappropriate conclusions about the system as a whole. With the advent of high-throughput "omic" technologies, however, researchers can now simultaneously analyze an entire system at the level of molecule (DNA, RNA, protein, metabolite) and process (transcription, translation, enzyme catalysis). This strategy reduces the likelihood of improper conclusions, provides a framework for elucidation of genotype-phenotype relationships, and brings finer resolution to comparative genomic experiments. Here, we apply a multi-omic approach to analyze the gene expression profiles of two closely related Pseudomonas aeruginosa strains grown in n-alkanes or glycerol. RESULTS: The environmental P. aeruginosa isolate ATCC 33988 consumed medium-length (C10-C16) n-alkanes more rapidly than the laboratory strain PAO1, despite high genome sequence identity (average nucleotide identity >99%). Our data shows that ATCC 33988 induces a characteristic set of genes at the transcriptional, translational and post-translational levels during growth on alkanes, many of which differ from those expressed by PAO1. Of particular interest was the lack of expression from the rhl operon of the quorum sensing (QS) system, resulting in no measurable rhamnolipid production by ATCC 33988. Further examination showed that ATCC 33988 lacked the entire lasI/lasR arm of the QS response. Instead of promoting expression of QS genes, ATCC 33988 up-regulates a small subset of its genome, including operons responsible for specific alkaline proteases and sphingosine metabolism. CONCLUSION: This work represents the first time results from RNA-seq, microarray, ribosome footprinting, proteomics, and small molecule LC-MS experiments have been integrated to compare gene expression in bacteria. Together, these data provide insights as to why strain ATCC 33988 is better adapted for growth and survival on n-alkanes.

The R2R3-MYB TT2b and the bHLH TT8 genes are the major regulators of proanthocyanidin biosynthesis in the leaves of Lotus species.

MAIN CONCLUSION: By exploiting interspecific hybrids and their progeny, we identified key regulatory and transporter genes intimately related to proanthocyanidin biosynthesis in leaves of Lotus spp. Proanthocyanidins (PAs), known as condensed tannins, are polymeric flavonoids enriching forage legumes of key nutritional value to prevent bloating in ruminant animals. Unfortunately, major forage legumes such as alfalfa and clovers lack PAs in edible tissues. Therefore, engineering the PA trait in herbage of forage legumes is paramount to improve both ecological and economical sustainability of cattle production system. Progresses on the understanding of genetic determinants controlling PA biosynthesis and accumulation have been mainly made studying mutants of Arabidopsis, Medicago truncatula and Lotus japonicus, model species unable to synthesize PAs in the leaves. Here, we exploited interspecific hybrids between Lotus corniculatus, with high levels of PAs in the leaves, and Lotus tenuis, with no PAs in these organs, and relative F2 progeny, to identify among candidate PA regulators and transporters the genes mainly affecting this trait. We found that the levels of leaf PAs significantly correlate with the expression of MATE1, the putative transporter of glycosylated PA monomers, and, among the candidate regulatory genes, with the expression of the MYB genes TT2a, TT2b and MYB14 and the bHLH gene TT8. The expression levels of TT2b and TT8 also correlated with those of all key structural genes of the PA pathways investigated, MATE1 included. Our study unveils a different involvement of the three Lotus TT2 paralogs to the PA trait and highlights differences in the regulation of this trait in our Lotus genotypes with respect to model species. This information opens new avenues for breeding bloat safe forage legumes.

Transcriptomic Analyses Elucidate Adaptive Differences of Closely Related Strains of Pseudomonas aeruginosa in Fuel.

Pseudomonas aeruginosa can utilize hydrocarbons, but different strains have various degrees of adaptation despite their highly conserved genome. P. aeruginosa ATCC 33988 is highly adapted to hydrocarbons, while P. aeruginosa strain PAO1, a human pathogen, is less adapted and degrades jet fuel at a lower rate than does ATCC 33988. We investigated fuel-specific transcriptomic differences between these strains in order to ascertain the underlying mechanisms utilized by the adapted strain to proliferate in fuel. During growth in fuel, the genes related to alkane degradation, heat shock response, membrane proteins, efflux pumps, and several novel genes were upregulated in ATCC 33988. Overexpression of alk genes in PAO1 provided some improvement in growth, but it was not as robust as that of ATCC 33988, suggesting the role of other genes in adaptation. Expression of the function unknown gene PA5359 from ATCC 33988 in PAO1 increased the growth in fuel. Bioinformatic analysis revealed that PA5359 is a predicted lipoprotein with a conserved Yx(FWY)xxD motif, which is shared among bacterial adhesins. Overexpression of the putative resistance-nodulation-division (RND) efflux pump PA3521 to PA3523 increased the growth of the ATCC 33988 strain, suggesting a possible role in fuel tolerance. Interestingly, the PAO1 strain cannot utilize n-C8 and n-C10 The expression of green fluorescent protein (GFP) under the control of alkB promoters confirmed that alk gene promoter polymorphism affects the expression of alk genes. Promoter fusion assays further confirmed that the regulation of alk genes was different in the two strains. Protein sequence analysis showed low amino acid differences for many of the upregulated genes, further supporting transcriptional control as the main mechanism for enhanced adaptation.IMPORTANCE These results support that specific signal transduction, gene regulation, and coordination of multiple biological responses are required to improve the survival, growth, and metabolism of fuel in adapted strains. This study provides new insight into the mechanistic differences between strains and helpful information that may be applied in the improvement of bacterial strains for resistance to biotic and abiotic factors encountered during bioremediation and industrial biotechnological processes.