CRISPR/Cas9-mediated knockout of a polyester synthase-like gene delays flowering time in alfalfa.

KEY MESSAGE: T-DNA and CRISPR/Cas9-mediated knockout of polyester synthase-like genes delays flowering time in Arabidopsis thaliana and Medicago sativa (alfalfa). Thus, we here present the first report of edited alfalfa with delayed flowering.

Inhibition of the 3-hydroxy-3-methyl-glutaryl-CoA reductase diminishes the survival and size of chondrocytes during orofacial morphogenesis in zebrafish.

OBJECTIVES: The 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR) is the enzyme controlling the rate-limiting step in the synthesis of cholesterol, sterols, and isoprenoids in the mevalonate biosynthetic pathway. Impaired function of HMGCR in zebrafish produces craniofacial malformations and orofacial cleft, mainly affecting the post-migratory neural crest cells with little earlier effect. Here we investigate morphogenetic and cellular mechanisms underlying the generation of these malformations. METHODS: The morphology of chondrocytes in the lower jaw and the proliferation/apoptosis in the ethmoid plate were analysed in hmgcr1b mutants and in embryos treated with atorvastatin. In the ceratohyal of treated embryos, we measured the number and dimensions of chondrocytes. In the ethmoid plate, we performed proliferation and apoptosis assays to quantify the number of cells undergoing each process in both hmgcr1b mutants and pharmacologically treated embryos. All embryos were imaged using confocal microscopy and processed to obtain maximum intensity z-projection. RESULTS: The shortening of the ceratohyal is produced by a moderate reduction in the number of cells combined with isometric shrinkage of the chondrocytes. At the same time, the shortening of the ethmoid plate is due to a combination of a slightly diminished proliferation with massive abnormal apoptosis at the proliferation front. CONCLUSION: HMGCR function is necessary for the normal survival and morphology of chondrocytes during condensation and chondrogenesis in the developing palate and jaw. Further studies are required to establish the pathways through which HMGCR acts on apoptosis, proliferation, and cell size during normal craniofacial development.

Metadherin Regulates Inflammatory Breast Cancer Invasion and Metastasis.

Inflammatory breast cancer (IBC) is one of the most lethal subtypes of breast cancer (BC), accounting for approximately 1-5% of all cases of BC. Challenges in IBC include accurate and early diagnosis and the development of effective targeted therapies. Our previous studies identified the overexpression of metadherin (MTDH) in the plasma membrane of IBC cells, further confirmed in patient tissues. MTDH has been found to play a role in signaling pathways related to cancer. However, its mechanism of action in the progression of IBC remains unknown. To evaluate the function of MTDH, SUM-149 and SUM-190 IBC cells were edited with CRISPR/Cas9 vectors for in vitro characterization studies and used in mouse IBC xenografts. Our results demonstrate that the absence of MTDH significantly reduces IBC cell migration, proliferation, tumor spheroid formation, and the expression of NF-κB and STAT3 signaling molecules, which are crucial oncogenic pathways in IBC. Furthermore, IBC xenografts showed significant differences in tumor growth patterns, and lung tissue revealed epithelial-like cells in 43% of wild-type (WT) compared to 29% of CRISPR xenografts. Our study emphasizes the role of MTDH as a potential therapeutic target for the progression of IBC.

Exploring the Role of the NO-Detoxifying Enzyme HmpA in the Evolution of Domesticated Alfalfa Rhizobia.

We have previously shown the extensive loss of genes during the domestication of alfalfa rhizobia and the high nitrous oxide emission associated with the extreme genomic instability of commercial inoculants. In the present note, we describe the molecular mechanism involved in the evolution of alfalfa rhizobia. Genomic analysis showed that most of the gene losses in inoculants are due to large genomic deletions rather than to small deletions or point mutations, a fact consistent with recurrent DNA double-strand breaks (DSBs) at numerous locations throughout the microbial genome. Genetic analysis showed that the loss of the NO-detoxifying enzyme HmpA in inoculants results in growth inhibition and high DSB levels under nitrosative stress, and large genomic deletions in planta but not in the soil. Therefore, besides its known function in the effective establishment of the symbiosis, HmpA can play a critical role in the preservation of the genomic integrity of alfalfa rhizobia under host-derived nitrosative stress.

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.

Generation of a multi-herbicide-tolerant alfalfa by using base editing.

KEYMESSAGE: We present the first report on base editing in alfalfa. Specifically, we showed edited alfalfa with tolerance to both sulfonylurea- and imidazolinone-type herbicides.

Immigrant assimilation and profiles of breast cancer screening behaviors among U.S. immigrant women.

Previous researchers have shown that despite increased prevention and cancer risk awareness, many immigrant women do not receive cancer screenings in a timely manner. We use data from the National Health Interview Survey and Latent Class Analysis to characterize immigrant women in the U.S. in accordance to their risk of engaging in breast cancer prevention. We identify three latent profiles based on use of cancer screenings, sociodemographic characteristics, family cancer history, and immigrant acculturation. Our results show that cancer screening behaviors of immigrants are consistent with patterns of both classic and segmented assimilation, as well as with greater immigrant diversity.

Spontaneous Mutations in the Nitrate Reductase Gene napC Drive the Emergence of Eco-friendly Low-N2O-Emitting Alfalfa Rhizobia in Regions with Different Climates.

We have recently shown that commercial alfalfa inoculants (e.g., Sinorhizobium meliloti B399), which are closely related to the denitrifier model strain Sinorhizobium meliloti 1021, have conserved nitrate, nitrite, and nitric oxide reductases associated with the production of the greenhouse gas nitrous oxide (N2O) from nitrate but lost the N2O reductase related to the degradation of N2O to gas nitrogen. Here, we screened a library of nitrogen-fixing alfalfa symbionts originating from different ecoregions and containing N2O reductase genes and identified novel rhizobia (Sinorhizobium meliloti INTA1-6) exhibiting exceptionally low N2O emissions. To understand the genetic basis of this novel eco-friendly phenotype, we sequenced and analyzed the genomes of these strains, focusing on their denitrification genes, and found mutations only in the nitrate reductase structural gene napC. The evolutionary analysis supported that, in these natural strains, the denitrification genes were inherited by vertical transfer and that their defective nitrate reductase napC alleles emerged by independent spontaneous mutations. In silico analyses showed that mutations in this gene occurred in ssDNA loop structures with high negative free energy (-ΔG) and that the resulting mutated stem-loop structures exhibited increased stability, suggesting the occurrence of transcription-associated mutation events. In vivo assays supported that at least one of these ssDNA sites is a mutational hot spot under denitrification conditions. Similar benefits from nitrogen fixation were observed when plants were inoculated with the commercial inoculant B399 and strains INTA4-6, suggesting that the low-N2O-emitting rhizobia can be an ecological alternative to the current inoculants without resigning economic profitability.

pBAR-H3.2, a native-optimized binary vector to bypass transgene silencing in alfalfa.

KEY MESSAGE: A novel genetic tool to bypass transgene silencing in alfalfa.

Pollen-Specific Aquaporins NIP4;1 and NIP4;2 Are Required for Pollen Development and Pollination in Arabidopsis thaliana.

In flowers with dry stigmas, pollen development, pollination, and pollen tube growth require spatial and temporal regulation of water and nutrient transport. To better understand the molecular mechanisms involved in reproductive processes, we characterized NIP4;1 and NIP4;2, two pollen-specific aquaporins of Arabidopsis thaliana. NIP4;1 and NIP4;2 are paralogs found exclusively in the angiosperm lineage. Although they have 84% amino acid identity, they displayed different expression patterns. NIP4;1 has low expression levels in mature pollen, while NIP4;2 expression peaks during pollen tube growth. Additionally, NIP4;1pro:GUS flowers showed GUS activity in mature pollen and pollen tubes, whereas NIP4;2pro:GUS flowers only in pollen tubes. Single T-DNA mutants and double artificial microRNA knockdowns had fewer seeds per silique and reduced pollen germination and pollen tube length. Transport assays in oocytes showed NIP4;1 and NIP4;2 function as water and nonionic channels. We also found that NIP4;1 and NIP4;2 C termini are phosphorylated by a pollen-specific CPK that modifies their water permeability. Survival assays in yeast indicated that NIP4;1 also transports ammonia, urea, boric acid, and H2O2 Thus, we propose that aquaporins NIP4;1 and NIP4;2 are exclusive components of the reproductive apparatus of angiosperms with partially redundant roles in pollen development and pollination.

[Development of a tool to conduct family studies in primary health care]. / Estudio de familia en Atención Primaria: construcción y validación de una herramienta para la valoración familiar.

BACKGROUND: The Family and Community Health Model (MAIS) establishes the continuity of care as an essential principle. The Family Study, as a clinical strategy, allows to have sufficient and timely information and knowledge about users of health care services, facilitates their accompaniment and is a source of information to improve the quality of care and the management of health centers. AIM: To develop a tool to conduct family studies, devised by experts in Primary Health Care. MATERIAL AND METHODS: Using a qualitative method, an electronic Delphi was conducted on 24 experts on primary health care. Afterwards, the content validation was carried out with the participation of judges. RESULTS: The resulting tool considers two levels of family assessment. It allows to distinguish those families that would benefit from interventions of greater complexity than those derived from the usual care of health centers. CONCLUSIONS: The tool to perform family studies responds to the informational and continuity component of Continuity of Patient Care principle. It may be a proposal for the continuous improvement of Chilean primary care.

The Ammonium Channel NOD26 is the Evolutionary Innovation that Drives the Emergence, Consolidation, and Dissemination of Nitrogen-Fixing Symbiosis in Angiosperms.

Increasing evidence indicates that N-fixing symbiosis has evolved several times in the N-fixing clade of angiosperms and that this evolution is driven by a single evolutionary innovation. However, the genetics of this ancestral predisposition to N-fixing symbiosis remains unclear. A natural candidate for such molecular innovation is the ammonium channel NOD26, the main protein component of the symbiosome membrane, which facilitates the plant uptake of the nitrogen fixed by symbiotic bacteria. Here, in concordance with the emergence of N-fixing symbiosis in angiosperms but not in ancestral plants, phylogenetic analysis showed that NOD26 belongs to an angiosperm-exclusive subgroup of aquaporins. Integrated genomic, phylogenetic, and gene expression analyses supported NOD26 occurrence in the N-fixing clade, the increase in the NOD26 copy number by block and tandem duplications in legumes, and the low-copy number or even the loss of NOD26 in non-legume species of the N-fixing clade, which correlated with the possibility to lose N-fixing symbiosis in legume and non-legume lineages. Metabolic reconstructions showed that retention of NOD26 in N-fixing precursor could represent an adaptive mechanism to bypass energy crisis during anaerobic stress by ammonium detoxification. Finally, we discuss the potential use of NOD26 to transfer N-fixation to non-N-fixing crops as cereals.

Maximizing the expression of transgenic traits into elite alfalfa germplasm using a supertransgene configuration in heterozygous conditions.

KEY MESSAGE: A novel process for the production of transgenic alfalfa varieties. Numerous species of legumes, including alfalfa, are critical factors for agroecosystems due to their ability to grow without nitrogen fertilizers derived from non-renewable fossil fuels, their contribution of organic nitrogen to the soil, and their increased nutritional value. Alfalfa is the main source of vegetable proteins in meat and milk production systems worldwide. Despite the economic and ecological importance of this autotetraploid and allogamous forage crop, little progress has been made in the incorporation of transgenic traits into commercial alfalfa. This is mainly due to the unusually strong transgene silencing and complex reproductive behavior of alfalfa, which limit the production of events with high transgene expression and the introgression of selected events within heterogeneous synthetic populations, respectively. In this report, we describe a novel procedure, called supertransgene process, where a glufosinate-tolerant alfalfa variety was developed using a single event containing the BAR transgene associated with an inversion. This approach can be used to maximize the expression of transgenic traits into elite alfalfa germplasm and to reduce the cost of production of transgenic alfalfa cultivars, contributing to the public improvement of this legume forage and other polyploid and outcrossing crop species.

Understanding the intracellular-to-extracellular localization switch of polyhydroxybutyrate polymerase in pseudomonas backgrounds as a microevolutionary process.

After gene duplication, paralogous genes evolve independently, and consequently, the new proteins encoded by these duplicated genes are exposed to changes in their subcellular location. Although there are increasing evidence that phylogenetically related proteins play different functions in different subcellular compartments, the number of evolutionary steps required for the emergence of a novel protein with a novel subcellular localization remains unclear. Regarding this intriguing topic, here we examine in depth our previous reports describing both intracellular and extracellular polyhydroxybutyrate polymerases (PhaC) in the Pseudomonadales group. The recapitulation of the intracellular-to-extracellular localization switch of PhaC in these strains shows a gradual evolution from a simple cytosolic PhaC form to a complex extracellular PhaC form specifically secreted via the type 1 secretion system. This gradual evolution includes several adaptive and pre-adaptive changes at the genomic, genetic and enzymatic levels, which are intimately related to the lifestyle of organisms during the evolution of protein localization. We conclude that the protein localization switch can be an extremely complex process in nature.

Plant Growth-Promoting Genes can Switch to be Virulence Factors via Horizontal Gene Transfer.

There are increasing evidences that horizontal gene transfer (HGT) is a critical mechanism of bacterial evolution, while its complete impact remains unclear. A main constraint of HGT effects on microbial evolution seems to be the conservation of the function of the horizontally transferred genes. From this perspective, inflexible nomenclature and functionality criteria have been established for some mobile genetic elements such as pathogenic and symbiotic islands. Adhesion is a universal prerequisite for both beneficial and pathogenic plant-microbe interactions, and thus, adhesion systems (e.g., the Lap cluster) are candidates to have a dual function depending on the genomic background. In this study, we showed that the virulent factor Lap of the phytopathogen Erwinia carotovora SCRI1043, which is located within a genomic island, was acquired by HGT and probably derived from Pseudomonas. The transformation of the phytopathogen Erwinia pyrifoliae Ep1/96 with the beneficial factor Lap from the plant growth-promoting bacterium Pseudomonas fluorescens Pf-5 significantly increased its natural virulence, experimentally recapitulating the beneficial-to-virulence functional switch of the Lap cluster via HGT. To our knowledge, this is the first report of a functional switch of an individual gene or a cluster of genes mediated by HGT.

Absence of the Nitrous Oxide Reductase Gene Cluster in Commercial Alfalfa Inoculants Is Probably Due to the Extensive Loss of Genes During Rhizobial Domestication.

As other legume crops, alfalfa cultivation increases the emission of the greenhouse gas nitrous oxide (N2O). Since legume-symbiotic nitrogen-fixing bacteria play a crucial role in this emission, it is important to understand the possible impacts of rhizobial domestication on the evolution of denitrification genes. In comparison with the genomes of non-commercial strains, those of commercial alfalfa inoculants exhibit low total genome size, low number of ORFs and high numbers of both frameshifted genes and pseudogenes, suggesting a dramatic loss of genes during bacterial domestication. Genomic analysis focused on denitrification genes revealed that commercial strains have perfectly conserved the nitrate (NAP), nitrite (NIR) and nitric (NOR) reductase clusters related to the production of N2O from nitrate but completely lost the nitrous oxide (NOS) reductase cluster (nosRZDFYLX genes) associated with the reduction of N2O to gas nitrogen. Based on these results, we propose future screenings for alfalfa-nodulating isolates containing both nitrogen fixation and N2O reductase genes for environmental sustainability of alfalfa production.

Plant growth-promoting bacterium Pseudomonas fluorescens FR1 secrets a novel type of extracellular polyhydroxybutyrate polymerase involved in abiotic stress response in plants.

OBJECTIVES: Identification of novel microbial factors contributing to plant protection against abiotic stress. RESULTS: The genome of plant growth-promoting bacterium Pseudomonas fluorescens FR1 contains a short mobile element encoding a novel type of extracellular polyhydroxybutyrate (PHB) polymerase (PhbC) associated with a type I secretion system. Genetic analysis using a phbC mutant strain and plants showed that this novel extracellular enzyme is related to the PHB production in planta and suggests that PHB could be a beneficial microbial compound synthesized during plant adaptation to cold stress. CONCLUSION: Extracellular PhbC can be used as a new tool for improve crop production under abiotic stress.

[User Participation in Clinical Decision-making in Primary Health Care in ChileParticipação dos usuários nas decisões clínicas na atenção primária à saúde no Chile]. / Participación de los usuarios en las decisiones clínicas en la atención primaria de salud en Chile.

OBJECTIVE: Describe users' experience with participation in clinical decision-making at Family Health Centers (CESFAM) in the Primary Health Care (PHC) system. METHODS: Qualitative descriptive study. Focus groups made up of CESFAM users were held in southeastern Santiago, Chile, and the information was thematically analyzed using Atlas.ti version 6® software. RESULTS: Five focus groups were held (n = 41). The main themes that emerged from the discussions were the passive role of users in decisions and delegated decision-making, based on their trust in the health professional. Users' passive role limits their opportunities for participation in clinical decision-making, ceding power to the health professional. However, establishing a therapeutic partnership allows users to feel that they are being treated as unique individuals whom the professional listens to and respects, ensuring that the decisions of the health team can be trusted, since they look out for the users' real interests. CONCLUSION: Users' participation in clinical encounters is still limited in Chile. However, bolstering that participation is essential for increasing user satisfaction and promoting people-centered care.

OBJETIVO: Descrever a experiência de participação nas decisões clínicas dos usuários dos Centros de Saúde Familiar (CESFAM) de atenção primária à saúde. MÉTODOS: Estudo qualitativo descritivo conduzido em grupos de discussão formados por usuários dos CESFAM da região sudeste da cidade de Santiago, no Chile. Foi realizada uma análise temática das informações com o uso do programa de software ATLAS.ti® versão 6. RESULTADOS: Foram formados cinco grupos de discussão (n = 41). Os principais tópicos abordados foram o papel passivo dos usuários nas decisões e a tomada de decisão delegada aos profissionais na base da confiança. O papel passivo restringe as oportunidades de participação dos usuários nas decisões clínicas e mantém o poder nas mãos dos profissionais da saúde. Apesar disso, ao ser criada uma aliança terapêutica, os usuários se sentem tratados como indivíduos únicos que são ouvidos e respeitados pelos profissionais, o que assegura que as decisões tomadas pela equipe de saúde sejam confiáveis porque protegem os reais interesses dos usuários. CONCLUSÃO: Os usuários ainda têm pouca participação nas interações clínicas no Chile. Porém, é fundamental reforçar esta participação para melhorar a satisfação do usuário e promover uma atenção mais centrada na pessoa.

[Model of comprehensive care in family and community health in primary care in ChileModelo de atenção integral na saúde da família e da comunidade na atenção básica chilena]. / Modelo de atención integral en salud familiar y comunitaria en la atención primaria chilena.

OBJECTIVE: Describe the current status of the implementation of the Model of Comprehensive Care in Family and Community Health (MAIS, by its acronym in Spanish) in primary care in Chile. METHODS: Cross-sectional study that evaluated the implementation of MAIS in a total of 1 263 primary care facilities. Through correlations, the relationship between internal self-evaluation and external evaluation of health services for each center was studied. The factors of facilities, communes and regions associated with the level of implementation of the MAIS were evaluated with multilevel analyses. RESULTS: The correlation between internal self-evaluation and the external evaluation of the total implementation of the MAIS was very high (0.819, p <0.001). The technology axis presented the highest implementation (83.0% compliance), and family focus the lowest (37.8% compliance). The facilities with the highest implementation were family health centers, the urban communes, those with the highest number of enrollees and those with the lowest poverty index. A statistically significant association was not identified between the implementation of the MAIS and the total community expenses (p = 0.122) nor specific health expenditures (p = 0.244). CONCLUSIONS: Most of the primary care health facilities have evaluated the level of implementation of the MAIS. The accompanying strategies for its implementation are priorities for primary care facilities located in rural areas and with a low number of registered users. Improving the family focus and the quality of care -key aspects of health care- are still a challenge.

OBJETIVO: Descrever o estado atual da implementação do Modelo de Assistência Integral em Saúde da Família e da Comunidade (MAIS) na atenção primária no Chile. MÉTODOS: Estudo transversal que avaliou a implementação do MAIS em um total de 1 263 estabelecimentos de atenção primária. Por meio de correlações, foi estudada a relação entre a autoavaliação (interna) e a avaliação dos serviços de saúde (externa) para cada centro. Os fatores dos estabelecimentos, municípios e regiões associados ao nível de implementação do MAIS foram avaliados com análises multiníveis. RESULTADOS: A correlação entre a autoavaliação interna e a avaliação externa da implementação total do MAIS foi muito alta (0,819, p <0,001). O eixo tecnológico apresentou maior implementação (83,0% de cumprimento) e foco familiar o menor (37,8% de cumprimento). Os centros de saúde da família, as comunas urbanas, aqueles com maior número de inscritos e com o menor índice de pobreza, foram os estabelecimentos que apresentaram a maior implementação. Não foi identificada associação estatisticamente significativa entre a implementação do MAIS e as despesas totais da comunidade (p = 0,122) nem gastos específicos com saúde (p = 0,244). CONCLUSÕES: A maioria dos estabelecimentos de atenção primária avaliaram o nível de implementação do MAIS. As estratégias de acompanhamento para sua implementação são prioritárias para estabelecimentos de atenção primária em áreas rurais e com baixo número de usuários cadastrados. Enfatiza-se o desafio de avançar na instalação do enfoque familiar e na qualidade de atenção.