Identification of Potential New Genes Related to the SREBP Pathway in Xanthophyllomyces dendrorhous.

The sterol regulatory element-binding protein (SREBP) pathway is an integral cellular mechanism that regulates lipid homeostasis, in which transcriptional activator SREBPs regulate the expression of various genes. In the carotenogenic yeast Xanthophyllomyces dendrorhous, Sre1 (the yeast SREBP homolog) regulates lipid biosynthesis and carotenogenesis, among other processes. Despite the characterization of several components of the SREBP pathway across various eukaryotes, the specific elements of this pathway in X. dendrorhous remain largely unknown. This study aimed to explore the potential regulatory mechanisms of the SREBP pathway in X. dendrorhous using the strain CBS.cyp61- as a model, which is known to have Sre1 in its active state under standard culture conditions, resulting in a carotenoid-overproducing phenotype. This strain was subjected to random mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine (NTG), followed by a screening methodology that focused on identifying mutants with altered Sre1 activation phenotypes. Single-nucleotide polymorphism (SNP) analysis of 20 selected mutants detected 5439 single-nucleotide variants (SNVs), narrowing them down to 1327 SNPs of interest after a series of filters. Classification based on SNP impact identified 116 candidate genes, including 49 genes with high impact and 68 genes with deleterious moderate-impact mutations. BLAST, InterProScan, and gene ontology enrichment analyses highlighted 25 genes as potential participants in regulating Sre1 in X. dendrorhous. The key findings of this study include the identification of genes potentially encoding proteins involved in protein import/export to the nucleus, sterol biosynthesis, the ubiquitin-proteasome system, protein regulatory activities such as deacetylases, a subset of kinases and proteases, as well as transcription factors that could be influential in SREBP regulation. These findings are expected to significantly contribute to the current understanding of the intricate regulation of the transcription factor Sre1 in X. dendrorhous, providing valuable groundwork for future research and potential biotechnological applications.

Effect of the Lys62Ala Mutation on the Thermal Stability of BstHPr Protein by Molecular Dynamics.

We analyzed the thermal stability of the BstHPr protein through the site-directed point mutation Lys62 replaced by Ala residue using molecular dynamics simulations at five different temperatures: 298, 333, 362, 400, and 450 K, for periods of 1 µs and in triplicate. The results from the mutant thermophilic BstHPrm protein were compared with those of the wild-type thermophilic BstHPr protein and the mesophilic BsHPr protein. Structural and molecular interaction analyses show that proteins lose stability as temperature increases. Mutant and wild-type proteins behave similarly up to 362 K. However, at 400 K the mutant protein shows greater structural instability, losing more buried hydrogen bonds and exposing more of its non-polar residues to the solvent. Therefore, in this study, we confirmed that the salt bridge network of the Glu3-Lys62-Glu36 triad, made up of the Glu3-Lys62 and Glu36-Lys62 ion pairs, provides thermal stability to the thermophilic BstHPr protein.

Almonertinib and alflutinib show novel inhibition on rare EGFR S768I mutant cells.

PURPOSE: EGFR classical mutations respond well to EGFR tyrosine kinase inhibitors. However, it is uncertain whether currently available EGFR-TKIs are effective against rare EGFR mutations and compound mutations. Herein, the effectiveness of almonertinib and alflutinib, the third-generation tyrosine kinase inhibitors developed in China, on rare EGFR S768I mutations and compound mutations is identified. METHODS: In this study, using CRISPR method, four EGFR S768I mutation cell lines were constructed, and the sensitivity of EGFR to almonertinib and alflutinib was tested, with positive controls being the 1st (gefitinib), 2nd (afatinib), and 3rd (osimertinib) generation drugs. RESULTS: The present results indicate that almonertinib and alflutinib can effectively inhibit cell viability and proliferation in rare EGFR S768I mutations through the ERK or AKT pathways in a time-dependent manner, by blocking the cell cycle and inhibiting apoptosis. CONCLUSIONS: These findings suggest that almonertinib and alflutinib may be potential therapeutic options for non-small cell lung cancer patients with the EGFR S768I mutation.

High-throughput analysis reveals disturbances throughout the cell caused by Arabidopsis UCP1 and UCP3 double knockdown.

Three genes encoding mitochondrial uncoupling proteins (UCPs) have been described in Arabidopsis thaliana (UCP1 to UCP3). In plants, UCPs may act as an uncoupler or as an aspartate/glutamate exchanger. For instance, much of the data regarding UCP functionality were obtained for the UCP1 and UCP2 isoforms compared with UCP3. Here, to get a better understanding about the concerted action of UCP1 and UCP3 in planta, we investigated the transcriptome and metabolome profiles of ucp1 ucp3 double mutant plants during the vegetative phase. For that, 21-day-old mutant plants, which displayed the most evident phenotypic alterations compared to wild type (WT) plants, were employed. The double knockdown of UCP1 and UCP3, isoforms unequivocally present inside the mitochondria, promoted important transcriptional reprogramming with alterations in the expression of genes related to mitochondrial and chloroplast function as well as those responsive to abiotic stress, suggesting disturbances throughout the cell. The observed transcriptional changes were well integrated with the metabolomic data of ucp1 ucp3 plants. Alterations in metabolites related to primary and secondary metabolism, particularly enriched in the Alanine, Aspartate and Glutamate metabolism, were detected. These findings extend our knowledge of the underlying roles played by UCP3 in concert with UCP1 at the whole plant level.

Increased yield performance of mutation induced Soybean genotypes at varied agro-ecological conditions

Abstract In soybean breeding program, continuous selection pressure on traits response to yield created a genetic bottleneck for improvements of soybean through hybridization breeding technique. Therefore an initiative was taken to developed high yielding soybean variety applying mutation breeding techniques at Plant Breeding Division, Bangladesh Institute of Nuclear Agriculture (BINA), Bangladesh. Locally available popular cultivar BARI Soybean-5 was used as a parent material and subjected to five different doses of Gamma ray using Co60. In respect to seed yield and yield attributing characters, twelve true breed mutants were selected from M4 generation. High values of heritability and genetic advance with high genotypic coefficient of variance (GCV) for plant height, branch number and pod number were considered as favorable attributes for soybean improvement that ensure expected yield. The mutant SBM-18 obtained from 250Gy provided stable yield performance at diversified environments. It provided maximum seed yield of 3056 kg ha-1 with highest number of pods plant-1 (56). The National Seed Board of Bangladesh (NSB) eventually approved SBM-18 and registered it as a new soybean variety named Binasoybean-5 for large-scale planting because of its superior stability in various agro-ecological zones and consistent yield performance.

Resumo No programa de melhoramento da soja, a pressão pela seleção contínua para a resposta das características de rendimento criou um gargalo genético para melhorias da soja por meio da técnica de melhoramento por hibridação. Portanto, foi desenvolvida uma variedade de soja de alto rendimento, aplicando técnicas de reprodução por mutação, na Divisão de Melhoramento de Plantas, no Instituto de Agricultura Nuclear de Bangladesh (BINA), em Bangladesh. A cultivar popular BARI Soybean-5, disponível localmente, foi usada como material original e submetida a cinco doses diferentes de raios gama usando Co60. Em relação ao rendimento de sementes e às características de atribuição de rendimento, 12 mutantes genuínos foram selecionados a partir da geração M4. Altos valores de herdabilidade e avanço genético com alto coeficiente de variância genotípico (GCV) para altura da planta, número de ramos e número de vagens foram considerados atributos favoráveis ao melhoramento da soja, garantindo, assim, a produtividade esperada. O mutante SBM-18, obtido a partir de 250Gy, proporcionou desempenho de rendimento estável em ambientes diversificados e produtividade máxima de sementes de 3.056 kg ha-1 com o maior número de vagens planta-1 (56). O Conselho Nacional de Sementes de Bangladesh (NSB) finalmente aprovou o SBM-18 e o registrou como uma nova variedade de soja, chamada Binasoybean-5, para plantio em larga escala por causa de sua estabilidade superior em várias zonas agroecológicas e desempenho de rendimento consistente.

Increased yield performance of mutation induced Soybean genotypes at varied agro-ecological conditions / Aumento no desempenho de rendimento de genótipos de soja induzidos por mutação em condições agroecológicas variadas

Abstract In soybean breeding program, continuous selection pressure on traits response to yield created a genetic bottleneck for improvements of soybean through hybridization breeding technique. Therefore an initiative was taken to developed high yielding soybean variety applying mutation breeding techniques at Plant Breeding Division, Bangladesh Institute of Nuclear Agriculture (BINA), Bangladesh. Locally available popular cultivar BARI Soybean-5 was used as a parent material and subjected to five different doses of Gamma ray using Co60. In respect to seed yield and yield attributing characters, twelve true breed mutants were selected from M4 generation. High values of heritability and genetic advance with high genotypic coefficient of variance (GCV) for plant height, branch number and pod number were considered as favorable attributes for soybean improvement that ensure expected yield. The mutant SBM-18 obtained from 250Gy provided stable yield performance at diversified environments. It provided maximum seed yield of 3056 kg ha-1 with highest number of pods plant-1 (56). The National Seed Board of Bangladesh (NSB) eventually approved SBM-18 and registered it as a new soybean variety named 'Binasoybean-5' for large-scale planting because of its superior stability in various agro-ecological zones and consistent yield performance.

Resumo No programa de melhoramento da soja, a pressão pela seleção contínua para a resposta das características de rendimento criou um gargalo genético para melhorias da soja por meio da técnica de melhoramento por hibridação. Portanto, foi desenvolvida uma variedade de soja de alto rendimento, aplicando técnicas de reprodução por mutação, na Divisão de Melhoramento de Plantas, no Instituto de Agricultura Nuclear de Bangladesh (BINA), em Bangladesh. A cultivar popular BARI Soybean-5, disponível localmente, foi usada como material original e submetida a cinco doses diferentes de raios gama usando Co60. Em relação ao rendimento de sementes e às características de atribuição de rendimento, 12 mutantes genuínos foram selecionados a partir da geração M4. Altos valores de herdabilidade e avanço genético com alto coeficiente de variância genotípico (GCV) para altura da planta, número de ramos e número de vagens foram considerados atributos favoráveis ​​ao melhoramento da soja, garantindo, assim, a produtividade esperada. O mutante SBM-18, obtido a partir de 250Gy, proporcionou desempenho de rendimento estável em ambientes diversificados e produtividade máxima de sementes de 3.056 kg ha-1 com o maior número de vagens planta-1 (56). O Conselho Nacional de Sementes de Bangladesh (NSB) finalmente aprovou o SBM-18 e o registrou como uma nova variedade de soja, chamada 'Binasoybean-5', para plantio em larga escala por causa de sua estabilidade superior em várias zonas agroecológicas e desempenho de rendimento consistente.

PEGylation Strategy for Improving the Pharmacokinetic and Antitumoral Activity of the IL-2 No-alpha Mutein.

BACKGROUND: In a previous work, an IL-2Rßγ biased mutant derived from human IL-2 and called IL-2noα, was designed and developed. Greater antitumor effects and lower toxicity were observed compared to native IL-2. Nevertheless, mutein has some disadvantages, such as a very short half-life of about 9-12 min, propensity for aggregation, and solubility problems. OBJECTIVE: In this study, PEGylation was employed to improve the pharmacokinetic and antitumoral properties of the novel protein. METHODS: Pegylated IL-2noα was characterized by polyacrylamide gel electrophoresis, size exclusion chromatography, in vitro cell proliferation and in vivo cell expansion bioassays, and pharmacokinetic and antitumor studies. RESULTS: IL-2noα-conjugates with polyethylene glycol (PEG) of 1.2 kDa, 20 kDa, and 40 kDa were obtained by classical acylation. No significant changes in the secondary and tertiary structures of the modified protein were detected. A decrease in biological activity in vitro and a significant improvement in half-life were observed, especially for IL-2noα-PEG20K. PEGylation of IL-2noα with PEG20K did not affect the capacity of the mutant to induce preferential expansion of T effector cells over Treg cells. This pegylated IL-2noα exhibited a higher antimetastatic effect compared to unmodified IL-2noα in the B16F0 experimental metastases model, even when administered at lower doses and less frequently. CONCLUSION: PEG20K was selected as the best modification strategy, to improve the blood circulation time of the IL-2noα with a superior antimetastatic effect achieved with lower doses.

AKT1 induces Nanog promoter in a SUMOylation-dependent manner in different pluripotent contexts.

AKT/PKB is a kinase crucial for pluripotency maintenance in pluripotent stem cells. Multiple post-translational modifications modulate its activity. We have previously demonstrated that AKT1 induces the expression of the pluripotency transcription factor Nanog in a SUMOylation-dependent manner in mouse embryonic stem cells. Here, we studied different cellular contexts and main candidates that could mediate this induction. Our results strongly suggest the pluripotency transcription factors OCT4 and SOX2 are not essential mediators. Additionally, we concluded that this induction takes place in different pluripotent contexts but not in terminally differentiated cells. Finally, the cross-matching analysis of ESCs, iPSCs and MEFs transcriptomes and AKT1 phosphorylation targets provided new clues about possible factors that could be involved in the SUMOylation-dependent Nanog induction by AKT.

Mutant p53 gain-of-function stimulates canonical Wnt signaling via PI3K/AKT pathway in colon cancer.

Aberrant canonical Wnt signaling is a hallmark of colon cancer. The TP53 tumor suppressor gene is altered in many solid tumors, including colorectal cancer, resulting in mutant versions of p53 (mut-p53) that lose their tumor suppressor capacities and acquire new-oncogenic functions (GOFs) critical for disease progression. Although the mechanisms related to mut-p53 GOF have been explored extensively, the relevance of mut-p53 in the canonical Wnt pathway is not well defined. This work investigated the influence of mut-p53 compared to wt-p53 in ß-catenin-dependent Wnt signaling. Using the TCGA public data from Pan-Cancer and the GEPIA2 platform, an in silico analysis of wt-p53 versus mut-p53 genotyped colorectal cancer patients showed that TP53 (p53) and CTNNB1 (ß-catenin) are significantly overexpressed in colorectal cancer, compared with normal tissue. Using p53 overexpression or p53 knockdown assays of wt-p53 or mut-p53, we found that while wt-p53 antagonizes canonical Wnt signaling, mut-p53 induces the opposite effect, improving the ß-catenin-dependent transcriptional activity and colony formation ability of colon cancer cells, which were both decreased by mut-p53 knockdown expression. The mechanism involved in mut-p53-induced activation of canonical Wnt appears to be via AKT-mediated phosphorylation of Ser 552 of ß-catenin, which is known to stabilize and enhance its transcriptional activity. We also found that while wt-p53 expression contributes to 5-FU sensitivity in colon cancer cells, the RITA p53 reactivating molecule counteracted the resistance against 5-FU in cells expressing mut-p53. Our results indicate that mut-p53 GOF acts as a positive regulator of canonical Wnt signaling and participates in the induction of resistance to 5-FU in colon cancer cells.

Mutant p53 Gain-of-Function Induces Migration and Invasion through Overexpression of miR-182-5p in Cancer Cells.

The master-key TP53 gene is a tumor suppressor that is mutated in more than 50% of human cancers. Some p53 mutants lose their tumor suppressor activity and acquire new oncogenic functions, known as a gain of function (GOF). Recent studies have shown that p53 mutants can exert oncogenic effects through specific miRNAs. We identified the differentially expressed miRNA profiles of the three most frequent p53 mutants (p53R273C, p53R248Q, and p53R175H) after their transfection into the Saos-2 cell line (null p53) as compared with p53WT transfected cells. The associations between these miRNAs and the signaling pathways in which they might participate were identified with miRPath Software V3.0. QRT-PCR was employed to validate the miRNA profiles. We observed that p53 mutants have an overall negative effect on miRNA expression. In the global expression profile of the human miRNome regulated by the p53R273C mutant, 72 miRNAs were underexpressed and 35 overexpressed; in the p53R175H miRNAs profile, our results showed the downregulation of 93 and upregulation of 10 miRNAs; and in the miRNAs expression profile regulated by the p53R248Q mutant, we found 167 decreased and 6 increased miRNAs compared with p53WT. However, we found overexpression of some miRNAs, like miR-182-5p, in association with processes such as cell migration and invasion. In addition, we explored whether the induction of cell migration and invasion by the p53R48Q mutant was dependent on miR-182-5p because we found overexpression of miR-182-5p, which is associated with processes such as cell migration and invasion. Inhibition of mutant p53R248Q and miR-182-5p increased FOXF2-MTSS1 levels and decreased cell migration and invasion. In summary, our results suggest that p53 mutants increase the expression of miR-182-5p, and this miRNA is necessary for the p53R248Q mutant to induce cell migration and invasion in a cancer cell model.

Tyrosine-Mutant AAV8 Vector Mediated Efficient and Safe Gene Transfer of Pigment Epithelium-Derived Factor to Mouse Lungs.

BACKGROUND/AIMS: Recombinant adeno-associated viruses (rAAV) are an important tool for lung targeted gene therapy. Substitution of tyrosine with phenylalanine residues (Y-F) in the capsid have been shown to protect the AAV vector from ubiquitin/proteasome degradation, increasing transduction efficiency. We tested the mutant Y733F-AAV8 vector for mucus diffusion, as well as the safety and efficacy of pigment epithelium-derived factor (PEDF) gene transfer to the lung. METHODS: For this purpose, Y733F-AAV8-PEDF (1010 viral genome) was administered intratracheally to C57BL/6 mice. Lung mechanics, morphometry, and inflammation were evaluated 7, 14, 21, and 28 days after injection. RESULTS: The tyrosine-mutant AAV8 vector was efficient at penetrating mucus in ex vivo assays and at transferring the gene to lung cells after in vivo instillation. Increased levels of transgene mRNA were observed 28 days after vector administration. Overexpression of PEDF did not affect in vivo lung parameters. CONCLUSION: These findings provide a basis for further development of Y733F-AAV8-based gene therapies for safe and effective delivery of PEDF, which has anti-angiogenic, anti-inflammatory and anti-fibrotic activities and might be a promising therapy for lung inflammatory disorders.

PSEN1 E280A Cholinergic-like Neurons and Cerebral Spheroids Derived from Mesenchymal Stromal Cells and from Induced Pluripotent Stem Cells Are Neuropathologically Equivalent.

Alzheimer's disease (AD) is a chronic neurological condition characterized by the severe loss of cholinergic neurons. Currently, the incomplete understanding of the loss of neurons has prevented curative treatments for familial AD (FAD). Therefore, modeling FAD in vitro is essential for studying cholinergic vulnerability. Moreover, to expedite the discovery of disease-modifying therapies that delay the onset and slow the progression of AD, we depend on trustworthy disease models. Although highly informative, induced pluripotent stem cell (iPSCs)-derived cholinergic neurons (ChNs) are time-consuming, not cost-effective, and labor-intensive. Other sources for AD modeling are urgently needed. Wild-type and presenilin (PSEN)1 p.E280A fibroblast-derived iPSCs, menstrual blood-derived menstrual stromal cells (MenSCs), and umbilical cord-derived Wharton Jelly's mesenchymal stromal cells (WJ-MSCs) were cultured in Cholinergic-N-Run and Fast-N-Spheres V2 medium to obtain WT and PSEN 1 E280A cholinergic-like neurons (ChLNs, 2D) and cerebroid spheroids (CSs, 3D), respectively, and to evaluate whether ChLNs/CSs can reproduce FAD pathology. We found that irrespective of tissue source, ChLNs/CSs successfully recapitulated the AD phenotype. PSEN 1 E280A ChLNs/CSs show accumulation of iAPPß fragments, produce eAß42, present TAU phosphorylation, display OS markers (e.g., oxDJ-1, p-JUN), show loss of ΔΨm, exhibit cell death markers (e.g., TP53, PUMA, CASP3), and demonstrate dysfunctional Ca2+ influx response to ACh stimuli. However, PSEN 1 E280A 2D and 3D cells derived from MenSCs and WJ-MSCs can reproduce FAD neuropathology more efficiently and faster (11 days) than ChLNs derived from mutant iPSCs (35 days). Mechanistically, MenSCs and WJ-MSCs are equivalent cell types to iPSCs for reproducing FAD in vitro.

Sumoylation and the oncogenic E17K mutation affect AKT1 subcellular distribution and impact on Nanog-binding dynamics to chromatin in embryonic stem cells.

AKT/PKB is a kinase involved in the regulation of a plethora of cell processes. Particularly, in embryonic stem cells (ESCs), AKT is crucial for the maintenance of pluripotency. Although the activation of this kinase relies on its recruitment to the cellular membrane and subsequent phosphorylation, multiple other post-translational modifications (PTMs), including SUMOylation, fine-tune its activity and target specificity. Since this PTM can also modify the localization and availability of different proteins, in this work we explored if SUMOylation impacts on the subcellular compartmentalization and distribution of AKT1 in ESCs. We found that this PTM does not affect AKT1 membrane recruitment, but it modifies the AKT1 nucleus/cytoplasm distribution, increasing its nuclear presence. Additionally, within this compartment, we found that AKT1 SUMOylation also impacts on the chromatin-binding dynamics of NANOG, a central pluripotency transcription factor. Remarkably, the oncogenic E17K AKT1 mutant produces major changes in all these parameters increasing the binding of NANOG to its targets, also in a SUMOylation dependent manner. These findings demonstrate that SUMOylation modulates AKT1 subcellular distribution, thus adding an extra layer of regulation of its function, possibly by affecting the specificity and interaction with its downstream targets.

Expanding the Toolbox for Functional Genomics in Fonsecaea pedrosoi: The Use of Split-Marker and Biolistic Transformation for Inactivation of Tryptophan Synthase (trpB) Gene.

Chromoblastomycosis (CBM) is a disease caused by several dematiaceous fungi from different genera, and Fonsecaea is the most common which has been clinically isolated. Genetic transformation methods have recently been described; however, molecular tools for the functional study of genes have been scarcely reported for those fungi. In this work, we demonstrated that gene deletion and generation of the null mutant by homologous recombination are achievable for Fonsecaea pedrosoi by the use of two approaches: use of double-joint PCR for cassette construction, followed by delivery of the split-marker by biolistic transformation. Through in silico analyses, we identified that F. pedrosoi presents the complete enzymatic apparatus required for tryptophan (trp) biosynthesis. The gene encoding a tryptophan synthase trpB -which converts chorismate to trp-was disrupted. The ΔtrpB auxotrophic mutant can grow with external trp supply, but germination, viability of conidia, and radial growth are defective compared to the wild-type and reconstituted strains. The use of 5-FAA for selection of trp- phenotypes and for counter-selection of strains carrying the trp gene was also demonstrated. The molecular tools for the functional study of genes, allied to the genetic information from genomic databases, significantly boost our understanding of the biology and pathogenicity of CBM causative agents.

Bioinformatics Insights on the Physicochemical Properties of SCN5A Mutant Proteins Associated with the Brugada Syndrome.

BACKGROUND: The Brugada syndrome (BrS) is a heart rhythm condition that is commonly associated with a strong predisposition for sudden cardiac death. Malignant ventricular arrhythmias could occur secondary to the dysfunction of the cardiac sodium voltage-gated Na(v)1.5 channel (SCN5A). OBJECTIVE: This study aimed to perform a multiparametric computational analysis of the physicochemical properties of SCN5A mutants associated with BrS using a set of bioinformatics tools. METHODS: In-house algorithms were calibrated to calculate, in a double-blind test, the Polarity Index Method (PIM) profile and protein intrinsic disorder predisposition (PIDP) profile of each sequence, and computer programs specialized in the genomic analysis were used. RESULTS: Specific regularities in the charge/polarity and PIDP profile of the SCN5A mutant proteins enabled the re-creation of the taxonomy, allowing us to propose a bioinformatics method that takes advantage of the PIM profile to identify this group of proteins from their sequence. CONCLUSION: Bioinformatics programs could reproduce characteristic PIM and PIDP profiles of the BrS-related SCN5A mutant proteins. This information can contribute to a better understanding of these altered proteins.

A collection of novel Lotus japonicus LORE1 mutants perturbed in the nodulation program induced by the Agrobacterium pusense strain IRBG74.

The Lotus japonicus population carrying new Lotus retrotransposon 1 (LORE1) insertions represents a valuable biological resource for genetic research. New insertions were generated by activation of the endogenous retroelement LORE1a in the germline of the G329-3 plant line and arranged in a 2-D system for reverse genetics. LORE1 mutants identified in this collection contributes substantially to characterize candidate genes involved in symbiotic association of L. japonicus with its cognate symbiont, the nitrogen-fixing bacteria Mesorhizobium loti that infects root nodules intracellularly. In this study we aimed to identify novel players in the poorly explored intercellular infection induced by Agrobacterium pusense IRBG74 sp. For this purpose, a forward screen of > 200,000 LORE1 seedlings, obtained from bulk propagation of G329-3 plants, inoculated with IRBG74 was performed. Plants with perturbed nodulation were scored and the offspring were further tested on plates to confirm the symbiotic phenotype. A total of 110 Lotus mutants with impaired nodulation after inoculation with IRBG74 were obtained. A comparative analysis of nodulation kinetics in a subset of 20 mutants showed that most of the lines were predominantly affected in nodulation by IRBG74. Interestingly, additional defects in the main root growth were observed in some mutant lines. Sequencing of LORE1 flanking regions in 47 mutants revealed that 92 Lotus genes were disrupted by novel LORE1 insertions in these lines. In the IM-S34 mutant, one of the insertions was located in the 5´UTR of the LotjaGi5g1v0179800 gene, which encodes the AUTOPHAGY9 protein. Additional mutant alleles, named atg9-2 and atg9-3, were obtained in the reverse genetic collection. Nodule formation was significantly reduced in these mutant alleles after M. loti and IRBG74 inoculation, confirming the effectiveness of the mutant screening. This study describes an effective forward genetic approach to obtain novel mutants in Lotus with a phenotype of interest and to identify the causative gene(s).

Functional characterization of the sugarcane (Saccharum spp.) ammonium transporter AMT2;1 suggests a role in ammonium root-to-shoot translocation.

AMMONIUM TRANSPORTER/METHYLAMMONIUM PERMEASE/RHESUS (AMT) family members transport ammonium across membranes in all life domains. Plant AMTs can be categorized into AMT1 and AMT2 subfamilies. Functional studies of AMTs, particularly AMT1-type, have been conducted using model plants but little is known about the function of AMTs from crops. Sugarcane (Saccharum spp.) is a major bioenergy crop that requires heavy nitrogen fertilization but depends on a low carbon-footprint for competitive sustainability. Here, we identified and functionally characterized sugarcane ScAMT2;1 by complementing ammonium uptake-defective mutants of Saccharomyces cerevisiae and Arabidopsis thaliana. Reporter gene driven by the ScAMT2;1 promoter in A. thaliana revealed preferential expression in the shoot vasculature and root endodermis/pericycle according to nitrogen availability and source. Arabidopsis quadruple mutant plants expressing ScAMT2;1 driven by the CaMV35S promoter or by a sugarcane endogenous promoter produced significantly more biomass than mutant plants when grown in NH4 + and showed more 15N-ammonium uptake by roots and nitrogen translocation to shoots. In A. thaliana, ScAMT2;1 displayed a Km of 90.17 µM and Vmax of 338.99 µmoles h-1 g-1 root DW. Altogether, our results suggest that ScAMT2;1 is a functional high-affinity ammonium transporter that might contribute to ammonium uptake and presumably to root-to-shoot translocation under high NH4 + conditions.

Outbreak of Klebsiella pneumoniae ST11 Resistant To Ceftazidime-Avibactam Producing KPC-31 and the Novel Variant KPC-115 during COVID-19 Pandemic in Argentina.

We describe an outbreak of Klebsiella pneumoniae sequence type 11 (ST11) producing KPC variants resistant to ceftazidime-avibactam. Six patients hospitalized in the intensive care unit (mostly due to critical COVID pneumonia) presented infection or colonization by this bacterium. They had several comorbidities and required mechanical ventilation, central venous catheters, and urinary catheters. All 6 patients had a history of fecal colonization with KPC-producing Enterobacterales (KPC-E). Three of them had previous episodes of infection with ceftazidime-avibactam-susceptible KPC-producing K. pneumoniae, which were treated with ceftazidime-avibactam. Several phenotypic methods failed to detect carbapenemase production in these 6 ceftazidime-avibactam-resistant isolates, and they showed in vitro susceptibility to imipenem and meropenem. All of them rendered positive results for blaKPC by PCR, and amplicon sequencing identified blaKPC-31 variant in 5 isolates and a novel variant, named blaKPC-115, in the other. Moreover, matrix-assisted laser desorption ionization-time of flight mass spectrometry was able to detect KPC in all isolates. Ceftazidime-avibactam-resistant isolates, as well as those recovered from previous infection episodes (KPC-3-producing K. pneumoniae, ceftazidime-avibactam susceptible), displayed a unique pulse type and belonged to ST11. Based on whole-genome sequencing results of selected isolates, less than 7 single-nucleotide polymorphisms were identified among them, which was indicative of the presence of a unique clone. Both in vivo selection and horizontal transmission seemed to have occurred in our hospital. Detection of these strains is challenging for the laboratory. History of previous KPC-E infections or colonization and systematic testing for resistance to ceftazidime-avibactam might help raise awareness of this possibility. IMPORTANCE Klebsiella pneumoniae is one of the main bacteria that cause infections in health care settings. This pathogen has developed a high level of resistance to many antibiotics. Some K. pneumoniae isolates can produce an enzyme known as carbapenemase KPC, making carbapenems (considered the last line for therapy) not effective to treat their infections. The combination ceftazidime-avibactam, approved by FDA in 2015, is useful to treat infections caused by KPC-producing K. pneumoniae. This study describes the emergence, in one hospital in Argentina, of K. pneumoniae isolates that produce KPC variants (KPC-31 and KPC-115) resistant to ceftazidime-avibactam. The ceftazidime-avibactam-resistant bacteria were isolated in inpatients, including some that previously received this combination as treatment. Transmission of this strain to other patients also occurred in the studied period. Detection of these bacteria is challenging for the laboratory. The knowledge and awareness of the emergence of this pathogen in our region are highly valuable.

A putative siderophore receptor of Gallibacterium anatis 12656-12 under Fur control also binds hemoglobin.

Pasteurellaceae family members obtain iron directly from host proteins or through siderophore-dependent mechanisms. Although Gallibacterum anatis expresses different virulence factors, its response to growth under iron restriction is unknown. G. anatis cultured in the presence of 2,2'-dipyridyl, up-expressed an approximately 65 kDa protein and repressed the expression of a 70 kDa protein. MALDI-TOF analysis of those proteins indicated homology with CirA (65 kDa), a protein involved in iron-siderophore acquisition in Mannheimia succinoproducens and a TonB-dependent receptor (70 kDa protein), a protein that binds chicken hemoglobin; however, G. anatis siderophore production was not detected by chromo azurol S (CAS)-BHI agar determination. This putative G. anatis siderophore receptor is under Fur control, but not the hemoglobin binding protein, as observed in G. anatis 12656-12 fur mutant (Ω fur 126.13) grown in the presence or not of 2,2'-dipyridyl. The addition of FeCl3 to the culture medium diminished the growth and biofilm production in approximately 30% and 35%, respectively, in the wild-type strain, but the growth of Ω fur 126.13 strain was not affected and biofilm production increased in 35%. G. anatis Ω fur 126.13 presented lower virulence when it was inoculated to 35-day-old chickens in comparison to the wild-type strain. The induction of more than one iron uptake mechanism could benefit pathogenic microorganisms such as Gallibacterium.