Choosing Wisely: An idea worth sustaining.

OBJECTIVES: To evaluate the sustainability potential of Choosing Wisely (CW) to address unnecessary medical care at Ontario community hospitals. DATA SOURCES/STUDY SETTING: Ontario community hospitals and their affiliated family health teams (FHTs). STUDY DESIGN: A mixed-methods study involving the administration of a validated sustainability survey to CW implementation teams followed by their participation in focus groups. DATA COLLECTION/EXTRACTION METHODS: Survey data were collected using an Excel file with an embedded, automated scoring system. We collated individual survey scores and generated aggregate team scores. We also performed descriptive statistics for quantitative data (frequencies, means). Qualitative data were triangulated with quantitative assessments to support data interpretations using the meta-matrix method. PRINCIPAL FINDINGS: Fifteen CW implementation teams across four Ontario community hospitals and six affiliated primary care FHTs participated. CW priority areas investigated were de-prescribing of proton pump inhibitors (PPIs) and reducing Pre-Op testing and BUN/Urea lab testing. Survey results showed steady improvements in sustainability scores from baseline to final follow-up among most implementation teams: 10% increase for PPI de-prescribing (six FHTs) and 2% increase (three hospital teams); 18% increase in BUN/Urea lab testing (three hospital teams). Regardless of site or CW priority area, common facilitators were fit with existing processes and workflows, leadership support, and optimized team communication; common challenges were lack of awareness and buy-in, leadership engagement or a champion, and lack of fit with existing workflow and culture. All teams identified at least one challenge for which they co-designed and implemented a plan to maximize the sustainability potential of their CW initiative. CONCLUSIONS: Evaluating the sustainability potential of an innovation such as Choosing Wisely is critical to ensuring that they have the best potential for impact. Our work highlights that implementation teams can be empowered to influence implementation efforts and to realize positive outcomes for their health care services and patients.

Novel Coronavirus Infection in an Infant with Intussusception.

Background. Intussusception is the leading cause of intestinal obstruction in children under 4 years of age. Viral infections are the associated etiology in most cases, SARS-CoV-2 thereby being a plausible cause, although only 5 cases have been reported worldwide with both entities. We report a case of an infant with Intussusception and covid-19, its clinical approach, and surgical outcomes, throughout a retrospective review of electronic medical chart history with the authorization of the Ethics Committee on research of the "Hospital Universitario del Valle" and endorsement for publication. Case report. It is an 8-month-old male patient with 72 hours of fever 100.4°F; bloody diarrheal stools; episodic abdominal pain; signs of peritoneal irritation and sensation of a mass in the right quadrants of the abdomen. Intussusception confirmed by ultrasound led to surgery, with SARS-CoV-2 infection as a possible differential diagnosis. Surgical findings reported intussusception of the transverse colon, peritonitis, and intestinal ischemia of distal ileum and right colon, for which intestinal resection with consequent ileostomy and a mucous fistula was performed. Positive SARS-CoV-2 RT-PCR test result was confirmed. Pediatric intensive care unit support took place at the initial postoperative phase, and eventual intestinal, with enteral feeding tolerance at the fourth day, and respiratory improvement came off within the first week of medical and surgical treatment. Conclusion. Gastrointestinal symptoms are often the leading manifestations of COVID-19 in children, which can be isolated or as a common sign of a concomitant pathology such as intussusception, and they could also have a causal relationship.

Does Functionality Condition the Population Structure and Genetic Diversity of Endangered Dog Breeds under Island Territorial Isolation?

Despite the undefinition of the origins of Ca de Rater (CR) and Ca de Bestiar (CB) dogs, references to these endangered autochthonous breeds highlighted their ratting/pet and shepherding/guard skills for centuries. Genealogical historical records were traced back to founders. Founder number in the reference population (146 and 53 for CR and CB, respectively), historical and reference maximum generations traced (eight and seven for CR and CB, respectively), and historical average number of complete generations (1.04 for both breeds) were determined. Structure assessment revealed the existence of subpopulations regarding criteria such as breeders (75 and 17), breeder location (32 and eight), owners (368 and 198), and owner location (73 and 51) for CR and CB, respectively. Average inbreeding (F) within breed subpopulations ranged from 0.27-1.20% for CB breeders and the rest of subpopulation criteria for both breeds, respectively, except for CB owners and owner location. F ranged from 0.27-1.41% for CB historical population and CR current population, respectively. The study of genetic diversity revealed a relatively similar genetic background between subpopulations. Average coancestry between and within breeds suggested a similar evolutionary process. However, Mann-Whitney U test determined significant differences for diversity parameters (F, ΔR, coancestry, nonrandom mating degree, maximum, complete, and equivalent generations, ΔF, and genetic conservation index) between breeds and their functionalities. Conclusively, functionality in dog breeds may determine the genetic diversity evolution of endangered breeds, even when these share the same geographic isolation conditions.

Increased expression of Phytophthora sojae genes encoding membrane-degrading enzymes appears to suggest an early onset of necrotrophy during Glycine max infection.

Phytophthora sojae is an oomycete pathogen that causes root, stem, and leaf rot in soybean plants, frequently leading to massive economic losses. Despite its importance, the mechanism by which P. sojae penetrates the host is not yet fully understood. Evidence indicates that P. sojae is not capable of penetrating the plant cell wall via mechanical force, suggesting that alternative factors facilitate breakdown of the host cell wall and membrane. Members of the carbohydrate esterase (CE) family 10 (carboxylesterases, arylesterases, sterol esterases and acetylcholine esterases, collectively known as CE10), are thought to be important for this penetration process. To gain insight into the potential role of CE10-coding genes in P. sojae pathogenesis, the newly revised version of the P. sojae genome was searched for putative CE10-coding genes, and various bioinformatic analyses were conducted using their amino acid and nucleotide sequences. In addition, in planta infection assays were conducted with P. sojae Race 4 and soybean cultivars Williams and Williams 82, and the transcriptional activity of P. sojae CE10-coding genes was evaluated at different time points during infection. Results suggest that these genes are important for both the biotrophic and necrotrophic stages of the P. sojae infection process and provide molecular evidence for stage distinction during infection progression. Furthermore, bioinformatic analyses have identified several conserved gene and protein sequence features that appear to have a significant impact on observed levels of expression during infection. Results agree with previous reports implicating other carbohydrate-active enzymes in P. sojae infection.

Risk factor meta-analysis and Bayesian estimation of genetic parameters and breeding values for hypersensibility to cutaneous habronematidosis in donkeys.

Cutaneous habronematidosis (CH) is a highly prevalent seasonally recurrent skin disease that affects donkeys as a result from the action of spirurid stomach worm larvae. Carrier flies mistakenly deposit these larvae on previous skin lesions or on the moisture of natural orifices, causing distress and inflicting relapsing wounds to the animals. First, we carried out a meta-analysis of the predisposing factors that could condition the development of CH in Andalusian donkeys. Second, basing on the empirical existence of an inter and intrafamilial variation previously addressed by owners, we isolated the genetic background behind the hypersensibility to this parasitological disease. To this aim, we designed a Bayesian linear model (BLM) to estimate the breeding values and genetic parameters for the hypersensibility to CH as a way to infer the potential selection suitability of this trait, seeking the improvement of donkey conservation programs. We studied the historical record of the cases of CH of 765 donkeys from 1984 to 2017. Fixed effects included birth year, birth season, sex, farm/owner, and husbandry system. Age was included as a linear and quadratic covariate. Although the effects of birth season and birth year were statistically non-significant (P > 0.05), their respective interactions with sex and farm/owner were statistically significant (P < 0.01), what translated into an increase of 40.5% in the specificity and of 0.6% of the sensibility of the model designed, when such interactions were included. Our BLM reported highly accurate genetic parameters as suggested by the low error of around 0.005, and the 95% credible interval for the heritability of ±0.0012. The CH hypersensibility heritability was 0.0346. The value of 0.1232 for additive genetic variance addresses a relatively low genetic variation in the Andalusian donkey breed. Our results suggest that farms managed under extensive husbandry conditions are the most protective ones against developing CH. Furthermore, these results provide evidence of the lack of repercussion of other factors such as age or sex. Potentially considering CH hypersensibility as a negative selection aimed goal in donkey breeding programs, may turn into a measure to improve animal welfare indirectly. However, the low heritability value makes it compulsory to control environmental factors to ensure the effectiveness of the breeding measures implemented to obtain individuals that may genetically be less prone to develop the condition.

Structural characterization of a putative chitin synthase gene in Phytophthora spp. and analysis of its transcriptional activity during pathogenesis on potato and soybean plants.

Although chitin is a major component of the fungal cell wall, in oomycetes (fungal-like organisms), this compound has only been found in very little amounts, mostly in the cell wall of members of the genera Achlya and Saprolegnia. In the oomycetes Phytophthora infestans and P. sojae the presence of chitin has not been demonstrated; however, the gene putatively encoding chitin synthase (CHS), the enzyme that synthesizes chitin, is present in their genomes. The evolutionary significance of the CHS gene in P. infestans and P. sojae genomes is not fully understood and, therefore, further studies are warranted. We have cloned and characterized the putative CHS genes from two Phytophthora spp. and multiple isolates of P. infestans and P. sojae and analyzed their phylogenetic relationships. We also conducted CHS inhibition assays and measured CHS transcriptional activity in Phytophthora spp. during infection of susceptible plants. Results of our investigations suggest that CHS contains all the motifs that are typical in CHS genes of fungal origin and is expressed, at least at the mRNA level, during in vitro and in planta growth. In infected tissues, the highest levels of expression occurred in the first 12 h post inoculation. In addition, results from our inhibition experiments appear to suggest that CHS activity is important for P. infestans normal vegetative growth. Because of the considerable variation in expression during infection when compared to basal expression observed in in vitro cultures of non-sporulating mycelium, we hypothesize that CHS may have a meaningful role in Phytophthora pathogenicity.

The Pectin Methylesterase Gene Complement of Phytophthora sojae: Structural and Functional Analyses, and the Evolutionary Relationships with Its Oomycete Homologs.

Phytophthora sojae is an oomycete pathogen that causes the disease known as root and stem rot in soybean plants, frequently leading to massive economic damage. Additionally, P. sojae is increasingly being utilized as a model for phytopathogenic oomycete research. Despite the economic and scientific importance of P. sojae, the mechanism by which it penetrates the host roots is not yet fully understood. It has been found that oomycetes are not capable of penetrating the cell wall solely through mechanical force, suggesting that alternative factors facilitate breakdown of the host cell wall. Pectin methylesterases have been suggested to be important for Phytophthora pathogenicity, but no data exist on their role in the P. sojae infection process. We have scanned the newly revised version of the annotated P. sojae genome for the presence of putative pectin methylesterases genes and conducted a sequence analysis of all gene models found. We also searched for potential regulatory motifs in the promoter region of the proposed P. sojae models, and investigated the gene expression levels throughout the early course of infection on soybean plants. We found that P. sojae contains a large repertoire of pectin methylesterase-coding genes and that most of these genes display similar motifs in the promoter region, indicating the possibility of a shared regulatory mechanism. Phylogenetic analyses confirmed the evolutionary relatedness of the pectin methylesterase-coding genes within and across Phytophthora spp. In addition, the gene duplication events that led to the emergence of this gene family appear to have occurred prior to many speciation events in the genus Phytophthora. Our results also indicate that the highest levels of expression occurred in the first 24 hours post inoculation, with expression falling after this time. Our study provides evidence that pectin methylesterases may be important for the early action of the P. sojae infection process.

Comparative structural and functional analysis of genes encoding pectin methylesterases in Phytophthora spp.

We have scanned the Phytophthora infestans, P. ramorum, and P. sojae genomes for the presence of putative pectin methylesterase genes and conducted a sequence analysis of all gene models found. We also searched for potential regulatory motifs in the promoter region of the proposed P. infestans models, and investigated the gene expression levels throughout the course of P. infestans infection on potato plants, using in planta and detached leaf assays. We found that genes located on contiguous chromosomal regions contain similar motifs in the promoter region, indicating the possibility of a shared regulatory mechanism. Results of our investigations also suggest that, during the pathogenicity process, the expression levels of some of the analyzed genes vary considerably when compared to basal expression observed in in vitro cultures of non-sporulating mycelium. These results were observed both in planta and in detached leaf assays.

Novel cellulose-binding-domain protein in Phytophthora is cell wall localized.

Cellulose binding domains (CBD) in the carbohydrate binding module family 1 (CBM1) are structurally conserved regions generally linked to catalytic regions of cellulolytic enzymes. While widespread amongst saprophytic fungi that subsist on plant cell wall polysaccharides, they are absent amongst most plant pathogenic fungal cellulases. A genome wide survey for CBM1 was performed on the highly destructive plant pathogen Phytophthora infestans, a fungal-like Stramenopile, to determine if it harbored cellulolytic enzymes with CBM1. Only five genes were found to encode CBM1, and none were associated with catalytic domains. Surveys of other genomes indicated that the CBM1-containing proteins, lacking other domains, represent a unique group of proteins largely confined to the Stramenopiles. Immunolocalization of one of these proteins, CBD1, indicated that it is embedded in the hyphal cell wall. Proteins with CBM1 domains can have plant host elicitor activity, but tests with Agrobacterium-mediated in planta expression and synthetic peptide infiltration failed to identify plant hypersensitive elicitation with CBD1. A structural basis for differential elicitor activity is proposed.

VdSNF1, the sucrose nonfermenting protein kinase gene of Verticillium dahliae, is required for virulence and expression of genes involved in cell-wall degradation.

Verticillium dahliae is a soilborne fungus causing vascular wilt in a diverse array of plant species. Its virulence has been attributed, among other factors, to the activity of hydrolytic cell wall-degrading enzymes (CWDE). The sucrose nonfermenting 1 gene (VdSNF1), which regulates catabolic repression, was disrupted in V. dahliae tomato race 1. Expression of CWDE in the resulting mutants was not induced in inductive medium and in simulated xylem fluid medium. Growth of the mutants was significantly reduced when grown with pectin or galactose as a carbon source whereas, with glucose, sucrose, and xylose, they grew similarly to wild-type and ectopic transformants. The mutants were severely impaired in virulence on tomato and eggplant (final disease severity reduced by an average of 87%). Microscopic observation of the infection behavior of a green fluorescent protein (gfp)-labeled VdSNF1 mutant (70ΔSF-gfp1) showed that it was defective in initial colonization of roots. Cross sections of tomato stem at the cotyledonary level showed that 70ΔSF-gfp1 colonized xylem vessels considerably less than the wild-type strain. The wild-type strain heavily colonized xylem vessels and adjacent parenchyma cells. Quantification of fungal biomass in plant tissues further confirmed reduced colonization of roots, stems, and cotyledons by 70ΔSF-gfp1 relative to that by the wild-type strain.

The CAZyome of Phytophthora spp.: a comprehensive analysis of the gene complement coding for carbohydrate-active enzymes in species of the genus Phytophthora.

BACKGROUND: Enzymes involved in carbohydrate metabolism include Carbohydrate esterases (CE), Glycoside hydrolases (GH), Glycosyl transferases (GT), and Polysaccharide lyases (PL), commonly referred to as carbohydrate-active enzymes (CAZymes). The CE, GH, and PL superfamilies are also known as cell wall degrading enzymes (CWDE) due to their role in the disintegration of the plant cell wall by bacterial and fungal pathogens. In Phytophthora infestans, penetration of the plant cells occurs through a specialized hyphal structure called appressorium; however, it is likely that members of the genus Phytophthora also use CWDE for invasive growth because hyphal forces are below the level of tensile strength exhibited by the plant cell wall. Because information regarding the frequency and distribution of CAZyme coding genes in Phytophthora is currently unknown, we have scanned the genomes of P. infestans, P. sojae, and P. ramorum for the presence of CAZyme-coding genes using a homology-based approach and compared the gene collinearity in the three genomes. In addition, we have tested the expression of several genes coding for CE in cultures grown in vitro. RESULTS: We have found that P. infestans, P. sojae and P. ramorum contain a total of 435, 379, and 310 CAZy homologs; in each genome, most homologs belong to the GH superfamily. Most GH and PL homologs code for enzymes that hydrolyze substances present in the pectin layer forming the middle lamella of the plant cells. In addition, a significant number of CE homologs catalyzing the deacetylation of compounds characteristic of the plant cell cuticle were found. In general, a high degree of gene location conservation was observed, as indicated by the presence of sequential orthologous pairs in the three genomes. Such collinearity was frequently observed among members of the GH superfamily. On the other hand, the CE and PL superfamilies showed less collinearity for some of their putative members. Quantitative PCR experiments revealed that all genes are expressed in P. infestans when this pathogen grown in vitro. However, the levels of expression vary considerably and are lower than the expression levels observed for the constitutive control. CONCLUSIONS: In conclusion, we have identified a highly complex set of CAZy homologs in the genomes of P. infestans, P. sojae, and P. ramorum, a significant number of which could play roles critical for pathogenicity, by participating in the degradation of the plant cell wall.

Structural and functional profile of the carbohydrate esterase gene complement in Phytophthora infestans.

The plant cell cuticle is the first obstacle for penetration of the host by plant pathogens. To breach this barrier, most pathogenic fungi employ a complex assortment of cell wall-degrading enzymes including carbohydrate esterases, glycoside hydrolases, and polysaccharide lyases. We characterized the full complement of carbohydrate esterase-coding genes in three Phytophthora species and analyzed the expression of cutinase in vitro and in planta; we also determined the cutinase allele distribution in multiple isolates of P. infestans. Our investigations revealed that there are 49, 21, and 37 esterase homologs in the P. infestans, P. ramorum, and P. sojae genomes, respectively, with a considerable number predicted to be extracellular. Four cutinase gene copies were found in both the P. infestans and P. ramorum genomes, while 16 copies were found in P. sojae. Transcriptional analyses of cutinase in P. infestans revealed that its expression level during infection is significantly upregulated at all time points compared to that of the same gene in mycelium grown in vitro. Expression achieves maximum values at 15 hpi, declining at subsequent time points. These results may suggest, therefore, that cutinase most likely plays a role in P. infestans pathogenicity.

Vaccination with recombinant Plasmodium vivax MSP-10 formulated in different adjuvants induces strong immunogenicity but no protection.

Although largely considered benign, Plasmodium vivax causes disease in nearly 75 million people each year and the available strategies are not sufficient to reduce the burden of disease, therefore pointing to vaccine development as a cost-effective control measure. In this study, the P. vivax merozoite surface protein 10 (MSP-10) was expressed as a recombinant protein in Escherichia coli and purified by affinity chromatography. High antigenicity was observed since sera from P. vivax-infected patients strongly recognized rPvMSP10. The immunogenicity of rPvMSP10 was tested in Aotus monkeys, comparing responses induced by formulations with Freund's adjuvant, Montanide ISA720 or aluminum hydroxide. All formulations produced high antibody titers recognizing the native protein in late schizonts. Despite inducing strong antibody production, none of the formulations protected immunized Aotus monkeys upon experimental challenge.

Immunogenicity and protection-inducing ability of recombinant Plasmodium vivax rhoptry-associated protein 2 in Aotus monkeys: a potential vaccine candidate.

Rhoptry proteins have been extensively shown to be important in invasion and parasitophorous vacuole (PV) formation. This work evaluates the immunogenicity and protective efficacy of Plasmodium vivax RAP2 in the non-human Aotus primate model, when expressed as a recombinant molecule in E. coli and formulated in Freund and Alum hydroxide adjuvants. Our results show that rPvRAP2 is immunogenic in both formulations, finding a trend of higher cytokine levels in immunized monkeys, specially in IL-4 levels (using Freund's adjuvant) and IL-5 (using Alum hydroxide). RAP2 is suggested as a P. vivax-vaccine candidate since immunized monkeys exhibited lower parasitemias than control groups after being experimentally challenged with the P. vivax VCG-I strain.

Alternate intron processing of family 5 endoglucanase transcripts from the genus Phytophthora.

Twenty-one homologs of family 5 endo-(1-4)-beta-glucanase genes (EGLs) were identified and characterized in the oomycete plant pathogens Phytophthora infestans, P. sojae, and P. ramorum, providing the first comprehensive analysis of this family in Phytophthora. Phylogenetic analysis revealed that these genes constitute a unique eukaryotic group, with closest similarity to bacterial endoglucanases. Many of the identified EGL copies were clustered in a few genomic regions, and contained from zero to three introns. Using reverse transcription PCR to study in vitro and in planta gene expression levels of P. sojae, we detected partially processed RNA transcripts retaining one or more of their introns. In some cases, the positions of intron/exon splicing sites were also found to be variable. The relative proportions of these transcripts remain apparently unchanged under various growing conditions, but differ among orthologous copies of the three Phytophthora species. The alternate processing of introns in this group of EGLs generates both coding and non-coding RNA isoforms. This is the first report on Phytophthora family 5 endoglucanases, and the first record for alternative intron processing of oomycete transcripts.

Phytophthora genome sequences uncover evolutionary origins and mechanisms of pathogenesis.

Draft genome sequences have been determined for the soybean pathogen Phytophthora sojae and the sudden oak death pathogen Phytophthora ramorum. Oömycetes such as these Phytophthora species share the kingdom Stramenopila with photosynthetic algae such as diatoms, and the presence of many Phytophthora genes of probable phototroph origin supports a photosynthetic ancestry for the stramenopiles. Comparison of the two species' genomes reveals a rapid expansion and diversification of many protein families associated with plant infection such as hydrolases, ABC transporters, protein toxins, proteinase inhibitors, and, in particular, a superfamily of 700 proteins with similarity to known oömycete avirulence genes.

Mutations in VMK1, a mitogen-activated protein kinase gene, affect microsclerotia formation and pathogenicity in Verticillium dahliae.

Verticillium dahliae is an important soil-borne fungal pathogen that causes vascular wilt diseases in a large variety of important crop plants. Due to its persistence in the soil, control of Verticillium wilt relies heavily on soil fumigation. The global ban on methyl bromide, a highly effective soil fumigant, poses an urgent need to develop alternative control measures against Verticillium wilt; and these might be more forthcoming with a better understanding of the molecular and cellular mechanisms that underpin the pathogenicity of V. dahliae. In this study, we assessed the role in growth, development, and pathogenicity of VMK1, a gene encoding a mitogen-activated protein (MAP) kinase (hence, Verticillium MAP Kinase 1). Disruption of VMK1 via Agrobacterium tumefaciens-mediated transformation, in two V. dahliae isolates, one from lettuce and the other from tomato, resulted in severely reduced virulence in diverse host plants, suggesting that VMK1 is essential for pathogenicity and that the MAP kinase-mediated signaling pathway has a conserved role in fungal pathogenicity. The vmk1 mutants also exhibited reduced conidiation and microsclerotia formation, suggesting that the gene is important for multiple cellular processes.

Loss of function of the Fusarium oxysporum SNF1 gene reduces virulence on cabbage and Arabidopsis.

Fusarium oxysporum pathogenicity is believed to require the activity of cell wall-degrading enzymes. Production of these enzymes in fungi is subject to carbon catabolite repression, a process that in yeast is mostly controlled by the SNF1 (sucrose non-fermenting 1) gene. To elucidate the role of cell wall-degrading enzymes in F. oxysporum pathogenicity, we cloned and disrupted its SNF1 homologue ( FoSNF1). The fosnf1 mutants had a reduced expression of several genes encoding cell wall-degrading enzymes and grew poorly on certain carbon sources. Infection assays on Arabidopsis thaliana and Brassica oleracea revealed that progression of wilt symptoms in plants infected by fosnf1 mutants was considerably delayed, in comparison with those infected by a wild-type strain. In conclusion, mutations in FoSNF1 prevent F. oxysporum from properly derepressing the production of cell wall-degrading enzymes, compromise the utilization of certain carbon sources, and reduce its virulence on A. thaliana and B. oleracea.