Manejo de la pandemia de COVID-19 en hospitalización domiciliaria en dos hospitales comarcales españoles: qué significó, quiénes atendimos, quién falleció y como evolucionó la asistencia a lo largo del tiempo / Management of COVID-19 pandemic in hospital at home in two regional Spanish hospitals. what it meant, who we cared for, who died and how assistance has evolved over time

Introducción. La Hospitalización a Domicilio (HAD) es una modalidad de ingreso alternativa que puede resultar de gran utilidad en momentos de estrés sanitario como la pandemia de COVID-19. En el presente trabajo se recoge el manejo de los pacientes ingresados con COVID-19 en HAD en dos hospitales comarcales españoles durante dos años.Métodos. Se realizó un estudio descriptivo, observacional y retrospectivo de los pacientes ingresados en HAD con COVID-19. Posteriormente se realizó un análisis para caracterizar a los pacientes que fallecieron en HAD o a 30 días del alta y otro para comparar el manejo entre la primera fase del estudio (2020) y la segunda (2021 y parte de 2022).Resultados. Se reclutaron 167 pacientes. Un 52,1% se trasladaron para vigilar que continuaban mejorando frente a un 40,7% en los que se hizo para vigilar que no empeoraran. Los pacientes que fallecieron en HAD resultaron más ancianos (87,5 años de media), más comórbidos y con mayor probabilidad de ser no reanimables en caso de paro cardiaco (No RCP) (85%). En la segunda fase del estudio se ingresaron pacientes más ancianos, más comórbidos y en mayor grado No RCP que los ingresados en 2020.Conclusiones. La HAD es un recurso útil para aumentar la resiliencia de los sistemas sanitarios en casos de estrés como supuso la enfermedad por COVID-19. El desarrollo y crecimiento de las unidades ya existentes, así como la creación de otras nuevas allá donde no existan, puede ser una herramienta básica para la medicina del futuro (AU)

Introduction. Home Hospitalization (HH) is an alternative hospitalization modality that can be very useful in times of health stress such as the COVID-19 pandemic. This paper includes the management of patients admitted with COVID-19 in HH in two county spanish hospitals for two years.Methods. A descriptive, observational and retrospective study of all patients admitted at HH with a diagnosis of COVID-19 disease was carried out. Subsequently, further analysis was carried out to characterize the patients who died in HH or 30 days after discharge and another to compare the management between the first phase of the study (2020) and the second one (2021 and part of 2022).Results. A total of 167 patients were recruited. A 52.1% moved to watch that the recovery continued compared to 40.7% in which it was done to watch that they did not worsen. The patients who died in HAD were older (mean 87.5 years), more comorbid and more likely to have do-not resucitate orders (DNR) in case of cardiac arrest (85%). In the second phase of the study, older patients, more comorbid patients and with a greater degree of DNR orders were admitted than those admitted throughout 2020.Conclusions. HAD is a useful resource to increase the resilience of health systems in cases of stress such as the disease caused by COVID-19. The development and growth of existing units, as well as the creation of new ones where they do not exist, could be a basic tool for the medicine of the future (AU)

Suppression among alleles encoding nucleotide-binding-leucine-rich repeat resistance proteins interferes with resistance in F1 hybrid and allele-pyramided wheat plants.

The development of high-yielding varieties with broad-spectrum durable disease resistance is the ultimate goal of crop breeding. In plants, immune receptors of the nucleotide-binding-leucine-rich repeat (NB-LRR) class mediate race-specific resistance against pathogen attack. When employed in agriculture this type of resistance is often rapidly overcome by newly adapted pathogen races. The stacking of different resistance genes or alleles in F1 hybrids or in pyramided lines is a promising strategy for achieving more durable resistance. Here, we identify a molecular mechanism which can negatively interfere with the allele-pyramiding approach. We show that pairwise combinations of different alleles of the powdery mildew resistance gene Pm3 in F1 hybrids and stacked transgenic wheat lines can result in suppression of Pm3-based resistance. This effect is independent of the genetic background and solely dependent on the Pm3 alleles. Suppression occurs at the post-translational level, as levels of RNA and protein in the suppressed alleles are unaffected. Using a transient expression system in Nicotiana benthamiana, the LRR domain was identified as the domain conferring suppression. The results of this study suggest that the expression of closely related NB-LRR resistance genes or alleles in the same genotype can lead to dominant-negative interactions. These findings provide a molecular explanation for the frequently observed ineffectiveness of resistance genes introduced from the secondary gene pool into polyploid crop species and mark an important step in overcoming this limitation.

Three-dimensional modeling and diversity analysis reveals distinct AVR recognition sites and evolutionary pathways in wild and domesticated wheat Pm3 R genes.

The Pm3 gene confers resistance against wheat powdery mildew. Studies of Pm3 diversity have shown that Pm3 alleles isolated from southern populations of wild emmer wheat located in Lebanon, Jordan, Israel, and Syria are more diverse and more distant from bread wheat alleles than alleles from the northern wild wheat populations located in Turkey, Iran, and Iraq. Therefore, southern populations from Israel were studied extensively to reveal novel Pm3 alleles that are absent from the cultivated gene pool. Candidate Pm3 genes were isolated via a polymerase chain reaction cloning approach. Known and newly identified Pm3 genes were subjected to variation analysis and polymorphic amino acid residues were superimposed on a three-dimensional (3D) model of PM3. The region of highest interspecies diversity between Triticum aestivum and T. dicoccoides lies in leucine-rich repeats (LRR) 19 to 24, whereas most intraspecies diversity in T. aestivum is located in LRR 25 to 28. Interestingly, these two regions are separated by one large LRR whose propensity for flexibility facilitates the conformation of the PM3 LRR domain into two differently structured models. The combination of evolutionary and protein 3D structure analysis revealed that Pm3 genes in wild and domesticated wheat show different evolutionary histories which might have been triggered through different interactions with the powdery mildew pathogen.

Substitutions of two amino acids in the nucleotide-binding site domain of a resistance protein enhance the hypersensitive response and enlarge the PM3F resistance spectrum in wheat.

Proteins with nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domains are major components of the plant immune system. They usually mediate resistance against a subgroup of races of a specific pathogen. For the allelic series of the wheat powdery mildew resistance gene Pm3, alleles with a broad and a narrow resistance spectrum have been described. Here, we show that a broad Pm3 spectrum range correlates with a fast and intense hypersensitive response (HR) in a Nicotiana transient-expression system and this activity can be attributed to two particular amino acids in the ARC2 subdomain of the NBS. The combined substitution of these amino acids in narrow-spectrum PM3 proteins enhances their capacity to induce an HR in Nicotiana benthamiana, and we demonstrate that these substitutions also enlarge the resistance spectrum of the Pm3f allele in wheat. Finally, using Bph14, we show that the region carrying the relevant amino acids also plays a role in the HR regulation of another coiled-coil NBS-LRR resistance protein. These results highlight the importance of an optimized NBS-'molecular switch' for the conversion of initial pathogen perception by the LRR into resistance-protein activation, and we describe a possible approach to extend the effectiveness of resistance genes via minimal targeted modifications in the NBS domain.

Rye Pm8 and wheat Pm3 are orthologous genes and show evolutionary conservation of resistance function against powdery mildew.

The improvement of wheat through breeding has relied strongly on the use of genetic material from related wild and domesticated grass species. The 1RS chromosome arm from rye was introgressed into wheat and crossed into many wheat lines, as it improves yield and fungal disease resistance. Pm8 is a powdery mildew resistance gene on 1RS which, after widespread agricultural cultivation, is now widely overcome by adapted mildew races. Here we show by homology-based cloning and subsequent physical and genetic mapping that Pm8 is the rye orthologue of the Pm3 allelic series of mildew resistance genes in wheat. The cloned gene was functionally validated as Pm8 by transient, single-cell expression analysis and stable transformation. Sequence analysis revealed a complex mosaic of ancient haplotypes among Pm3- and Pm8-like genes from different members of the Triticeae. These results show that the two genes have evolved independently after the divergence of the species 7.5 million years ago and kept their function in mildew resistance. During this long time span the co-evolving pathogens have not overcome these genes, which is in strong contrast to the breakdown of Pm8 resistance since its introduction into commercial wheat 70 years ago. Sequence comparison revealed that evolutionary pressure acted on the same subdomains and sequence features of the two orthologous genes. This suggests that they recognize directly or indirectly the same pathogen effectors that have been conserved in the powdery mildews of wheat and rye.

The wheat Mla homologue TmMla1 exhibits an evolutionarily conserved function against powdery mildew in both wheat and barley.

The race-specific barley powdery mildew (Blumeria graminis f. sp. hordei) resistance gene Mla occurs as an allelic series and encodes CC-NB-LRR type resistance proteins. Inter-generic allele mining resulted in the isolation and characterisation of an Mla homologue from diploid wheat, designated TmMla1, which shares 78% identity with barley HvMLA1 at the protein level. TmMla1 was found to be a functional resistance gene against Blumeria graminis f. sp. tritici in wheat, hereby providing an example of R gene orthologs controlling the same disease in two different species. TmMLA1 exhibits race-specific resistance activity and its N-terminal coiled-coil domain interacts with the barley transcription factor HvWRKY1. Interestingly, TmMLA1 was not functional in barley transient assays. Replacement of the TmMLA1 LRR domain with that of HvMLA1 revealed that this fusion protein conferred resistance against B. graminis f. sp. hordei isolate K1 in barley. Thus, TmMLA1 not only confers resistance in wheat but possibly also in barley against an as yet unknown barley powdery mildew race. The conservation of functional R gene orthologs over at least 12 million years is surprising given the observed rapid breakdown of Mla-based resistance against barley mildew in agricultural ecosystems. This suggests a high stability of Mla resistance in the natural environment before domestication.

Diversity at the Mla powdery mildew resistance locus from cultivated barley reveals sites of positive selection.

The Mla locus in barley (Hordeum vulgare) conditions isolate-specific immunity to the powdery mildew fungus (Blumeria graminis f. sp. hordei) and encodes intracellular coiled-coil (CC) domain, nucleotide-binding (NB) site, and leucine-rich repeat (LRR)-containing receptor proteins. Over the last decades, genetic studies in breeding material have identified a large number of functional resistance genes at the Mla locus. To study the structural and functional diversity of this locus at the molecular level, we isolated 23 candidate MLA cDNAs from barley accessions that were previously shown by genetic studies to harbor different Mla resistance specificities. Resistance activity was detected for 13 candidate MLA cDNAs in a transient gene-expression assay. Sequence alignment of the deduced MLA proteins improved secondary structure predictions, revealing four additional, previously overlooked LRR. Analysis of nucleotide diversity of the candidate and validated MLA cDNAs revealed 34 sites of positive selection. Recombination or gene conversion events were frequent in the first half of the gene but positive selection was also found when this region was excluded. The positively selected sites are all, except two, located in the LRR domain and cluster in predicted solvent-exposed residues of the repeats 7 to 15 and adjacent turns on the concave side of the predicted solenoid protein structure. This domain-restricted pattern of positively selected sites, together with the length conservation of individual LRR, suggests direct binding of effectors to MLA receptors.

Plant pathogen recognition mediated by promoter activation of the pepper Bs3 resistance gene.

Plant disease resistance (R) proteins recognize matching pathogen avirulence proteins. Alleles of the pepper R gene Bs3 mediate recognition of the Xanthomonas campestris pv. vesicatoria (Xcv) type III effector protein AvrBs3 and its deletion derivative AvrBs3Deltarep16. Pepper Bs3 and its allelic variant Bs3-E encode flavin monooxygenases with a previously unknown structure and are transcriptionally activated by the Xcv effector proteins AvrBs3 and AvrBs3Deltarep16, respectively. We found that recognition specificity resides in the Bs3 and Bs3-E promoters and is determined by binding of AvrBs3 or AvrBs3Deltarep16 to a defined promoter region. Our data suggest a recognition mechanism in which the Avr protein binds and activates the promoter of the cognate R gene.

Physical delimitation of the pepper Bs3 resistance gene specifying recognition of the AvrBs3 protein from Xanthomonas campestris pv. vesicatoria.

The pepper (Capsicum annuum) Bs3 gene confers resistance to avrBs3-expressing strains of the bacterial spot pathogen Xanthomonas campestris pv. vesicatoria. To physically delimit Bs3, a pepper YAC library was screened with two flanking DNA markers that are separated from Bs3 by 1.0 and 1.2 cM, respectively resulting in the identification of three YAC clones. Genetic mapping of the corresponding YACends revealed however, that these YACs do not cover Bs3 and subsequent screens with newly developed YACend markers failed to identify new YAC clones. Marker saturation at the Bs3 locus was carried out by amplified fragment length polymorphism (AFLP). The analysis of 1,024 primer combinations resulted in the identification of 47 new Bs3-linked AFLPs. High-resolution linkage mapping of Bs3 was accomplished by inspecting more than 4,000 F(2) segregants resulting in a genetic resolution of 0.01 cM. Using tightly Bs3-linked YACend- and AFLP-derived markers we established a Bs3-spanning BAC contig and physically delimited the target gene within one BAC clone. The analysis of the Bs3-containing genomic region revealed substantial local variation in the correlation of genetic and physical distances.

Gene-for-gene-mediated recognition of nuclear-targeted AvrBs3-like bacterial effector proteins.

Plant disease resistance (R) genes mediate specific recognition of pathogens via perception of cognate avirulence (avr) gene products. The numerous highly similar AvrBs3-like proteins from the bacterial genus Xanthomonas provide together with their corresponding R proteins a unique biological resource to dissect the molecular basis of recognition specificity. A central question in this context is if R proteins that mediate recognition of structurally similar Avr proteins are themselves functionally similar or rather dissimilar. The recent isolation of rice xa5, rice Xa27 and tomato Bs4, R genes that collectively mediate recognition of avrBs3-like genes, provides a first clue to the molecular mechanisms that plants employ to detect AvrBs3-like proteins. Their initial characterization suggests that these R proteins are structurally and functionally surprisingly diverge. This review summarizes the current knowledge on R-protein-mediated recognition of AvrBs3-like proteins and provides working models on how recognition is achieved at the molecular level.

Alternative splicing of transcripts encoding Toll-like plant resistance proteins - what's the functional relevance to innate immunity?

Innate immunity in plants and animals shares many structural and functional homologies, which suggests an ancient origin of cellular defense mechanisms in both kingdoms. Pathogen sensing in animal innate immunity is mediated by Toll-like receptors (TLRs). These receptors have TIR (Toll/interleukin-1 receptor) domains and leucine-rich repeats, which are modules also present in many plant resistance (R) proteins. Molecular analysis of transcripts encoding animal TLRs and Toll-like plant R proteins revealed many cases of alternative splicing. Recent studies of the tobacco N and the Arabidopsis RPS4 genes, both encoding Toll-like plant R proteins, showed that intron-deprived genes have reduced or no activity, suggesting that alternative splicing is a crucial component in these signaling pathways.