Metabolic engineering of the shikimate pathway in Amycolatopsis strains for optimized glycopeptide antibiotic production.

Glycopeptide antibiotics (GPA) consist of a glycosylated heptapeptide backbone enriched in aromatic residues originating from the shikimate pathway. Since the enzymatic reactions within the shikimate pathway are highly feedback-regulated, this raises the question as to how GPA producers control the delivery of precursors for GPA assembly. We chose Amycolatopsis balhimycina, the producer of balhimycin, as a model strain for analyzing the key enzymes of the shikimate pathway. A. balhimycina contains two copies each of the key enzymes of the shikimate pathway, deoxy-d-arabino-heptulosonate-7-phosphate synthase (Dahp) and prephenate dehydrogenase (Pdh), with one pair (Dahpsec and Pdhsec) encoded within the balhimycin biosynthetic gene cluster and one pair (Dahpprim and Pdhprim) in the core genome. While overexpression of the dahpsec gene resulted in a significant (>4-fold) increase in balhimycin yield, no positive effects were observed after overexpression of the pdhprim or pdhsec genes. Investigation of allosteric enzyme inhibition revealed that cross-regulation between the tyrosine and phenylalanine pathways plays an important role. Tyrosine, a key precursor of GPAs, was found to be a putative activator of prephenate dehydratase (Pdt), which catalyzes the first step reaction from prephenate to phenylalanine in the shikimate pathway. Surprisingly, overexpression of pdt in A. balhimycina led to an increase in antibiotic production in this modified strain. In order to demonstrate that this metabolic engineering approach is generally applicable to GPA producers, we subsequently applied this strategy to Amycolatopsis japonicum and improved the production of ristomycin A, which is used in diagnosis of genetic disorders. Comparison of "cluster-specific" enzymes with the isoenzymes from the primary metabolism's pathway provided insights into the adaptive mechanisms used by producers to ensure adequate precursor supply and GPA yields. These insights further demonstrate the importance of a holistic approach in bioengineering efforts that takes into account not only peptide assembly but also adequate precursor supply.

Bioinformatic Analysis Reveals both Oversampled and Underexplored Biosynthetic Diversity in Nonribosomal Peptides.

The traditional natural product discovery approach has accessed only a fraction of the chemical diversity in nature. The use of bioinformatic tools to interpret the instructions encoded in microbial biosynthetic genes has the potential to circumvent the existing methodological bottlenecks and greatly expand the scope of discovery. Structural prediction algorithms for nonribosomal peptides (NRPs), the largest family of microbial natural products, lie at the heart of this new approach. To understand the scope and limitation of the existing prediction algorithms, we evaluated their performances on NRP synthetase biosynthetic gene clusters. Our systematic analysis shows that the NRP biosynthetic landscape is uneven. Phenylglycine and its derivatives as a group of NRP building blocks (BBs), for example, have been oversampled, reflecting an extensive historical interest in the glycopeptide antibiotics family. In contrast, the benzoyl BB, including 2,3-dihydroxybenzoate (DHB), has been the most underexplored, hinting at the possibility of a reservoir of as yet unknown DHB containing NRPs with functional roles other than a siderophore. Our results also suggest that there is still vast unexplored biosynthetic diversity in nature, and the analysis presented herein shall help guide and strategize future natural product discovery campaigns. We also discuss possible ways bioinformaticians and biochemists could work together to improve the existing prediction algorithms.

Widespread presence of a highly virulent Edwardsiella ictaluri strain in farmed tilapia, Oreochromis spp.

Edwardsiella ictaluri is an emerging bacterial pathogen that affects farmed tilapia (Oreochromis spp.). This study reports the widespread presence of E. ictaluri in farmed tilapia in Vietnam. Among 26 disease outbreaks from nine provinces in Northern Vietnam during 2019-2021, 19 outbreaks originated from imported seeds, while outbreaks in seven farms were from domestic sources. Clinically sick fish showed the appearance of numerous white spots in visceral organs, and accumulative mortality reached 30%-65%. A total of 26 representative bacterial isolates recovered from 26 disease outbreaks were identified as E. ictaluri based on a combination of phenotypic tests, genus- and species-specific polymerase chain reaction assays, 16S rRNA and gyrB sequencing, and phylogenetic analysis. All isolates harboured the same virulence gene profiles esrC+ , evpC+ , ureA-C+ , eseI- , escD- and virD4- . Antimicrobial susceptibility tests revealed that 80.8%-100% of isolates were multidrug resistant, with resistance to 4-8 antimicrobials in the groups of penicillin, macrolides, sulfonamides, amphenicols and glycopeptides. The experimental challenge successfully induced disease that mimicked natural infection. The median lethal doses (LD50 ) of the tested isolates (n = 4) were 42-61 colony forming units/fish, indicating their extremely high virulence. This emerging pathogen is established and has spread to various geographical locations, causing serious impacts on farmed tilapia in northern Vietnam. It is likely that this pathogen will continue to spread through contaminated stocks (both imported and domestic sources) and persist. Thus, increased awareness, combined with biosecurity measures and emergent vaccination programs is essential to mitigate the negative impact of this emerging disease on the tilapia farming industry.

Glycopeptide antibiotic discovery in the genomic era.

Glycopeptide antibiotics are essential drugs used to treat infections caused by multi-drug resistant Gram-positive pathogens. There is a continuous need for new antibiotics, including GPAs, to address emerging resistance and offer desirable pharmacological profiles for improved efficacy. Microbial natural products are proven sources of antibiotics, and this source has dominated drug discovery over the past century. Bacteria from the phylum Actinobacteria are particularly renowned for producing a diverse range of bioactive natural products including glycopeptide antibiotics. The traditional approach to mining this resource is through the culture and extraction of natural products followed by assay for cell-killing activity. Unfortunately, this method no longer efficiently yields new antibiotic leads, delivering instead known compounds. Whole-genome sequencing programs on the other hand are revealing thousands of unexplored natural product biosynthetic gene clusters in the chromosomes of Actinobacteria. These gene clusters encode the necessary enzymes, transport and resistance mechanisms, along with regulatory elements for the biosynthesis of a variety of antibiotics. Identification of uncharacterized or cryptic biosynthetic gene clusters to unlock the chemical "dark matter" represents a new direction for the discovery of new drug candidates. This chapter discusses the identification of glycopeptide antibiotic biosynthetic gene clusters in microbial genomes, the improved production of these antibiotics using the GPAHex synthetic biology platform, and methods for their purification.

Fucosylated AGP glycopeptides as biomarkers of HNF1A-Maturity onset diabetes of the young.

AIMS: We previously demonstrated that antennary fucosylated N-glycans on plasma proteins are regulated by HNF1A and can identify cases of Maturity-Onset Diabetes of the Young caused by HNF1A variants (HNF1A-MODY). Based on literature data, we further postulated that N-glycans with best diagnostic value mostly originate from alpha-1-acid glycoprotein (AGP). In this study we analyzed fucosylation of AGP in subjects with HNF1A-MODY and other types of diabetes aiming to evaluate its diagnostic potential. METHODS: A recently developed LC-MS method for AGP N-glycopeptide analysis was utilized in two independent cohorts: a) 466 subjects with different diabetes subtypes to test the fucosylation differences, b) 98 selected individuals to test the discriminative potential for pathogenic HNF1A variants. RESULTS: Our results showed significant reduction in AGP fucosylation associated to HNF1A-MODY when compared to other diabetes subtypes. Additionally, ROC curve analysis confirmed significant discriminatory potential of individual fucosylated AGP glycopeptides, where the best performing glycopeptide had an AUC of 0.94 (95% CI 0.90-0.99). CONCLUSIONS: A glycopeptide based diagnostic tool would be beneficial for patient stratification by providing information about the functionality of HNF1A. It could assist the interpretation of DNA sequencing results and be a useful addition to the differential diagnostic process.

Distinct shifts in site-specific glycosylation pattern of SARS-CoV-2 spike proteins associated with arising mutations in the D614G and Alpha variants.

Extensive glycosylation of the spike protein of severe acute respiratory syndrome coronavirus 2 virus not only shields the major part of it from host immune responses, but glycans at specific sites also act on its conformation dynamics and contribute to efficient host receptor binding, and hence infectivity. As variants of concern arise during the course of the coronavirus disease of 2019 pandemic, it is unclear if mutations accumulated within the spike protein would affect its site-specific glycosylation pattern. The Alpha variant derived from the D614G lineage is distinguished from others by having deletion mutations located right within an immunogenic supersite of the spike N-terminal domain (NTD) that make it refractory to most neutralizing antibodies directed against this domain. Despite maintaining an overall similar structural conformation, our mass spectrometry-based site-specific glycosylation analyses of similarly produced spike proteins with and without the D614G and Alpha variant mutations reveal a significant shift in the processing state of N-glycans on one specific NTD site. Its conversion to a higher proportion of complex type structures is indicative of altered spatial accessibility attributable to mutations specific to the Alpha variant that may impact its transmissibility. This and other more subtle changes in glycosylation features detected at other sites provide crucial missing information otherwise not apparent in the available cryogenic electron microscopy-derived structures of the spike protein variants.

Production enhancement of the glycopeptide antibiotic A40926 by an engineered Nonomuraea gerenzanensis strain.

OBJECTIVE: To improve the production of A40926, a combined strategy of constructing the engineered strain and optimizing the medium was implemented. RESULTS: The engineered strain lcu1 with the genetic features of dbv23 deletion and dbv3-dbv20 coexpression increased by 30.6% in the production of A40926, compared to the original strain. In addition, a combined medium called M9 was designed to be further optimized by the central composite design method. The optimized M9 medium was verified to significantly improve the A40926 yield from 257 to 332 mg l-1. CONCLUSIONS: The engineered strain lcu1 could significantly promote A40926 production in the optimized M9 medium, which indicated that the polygenic genetic manipulation and the media optimization played an equally important role in increasing the A40926 yield.

Chemoenzymatic Synthesis and Antibody Binding of HIV-1 V1/V2 Glycopeptide-Bacteriophage Qß Conjugates as a Vaccine Candidate.

The broadly neutralizing antibody PG9 recognizes a unique glycopeptide epitope in the V1V2 domain of HIV-1 gp120 envelope glycoprotein. The present study describes the design, synthesis, and antibody-binding analysis of HIV-1 V1V2 glycopeptide-Qß conjugates as a mimic of the proposed neutralizing epitope of PG9. The glycopeptides were synthesized using a highly efficient chemoenzymatic method. The alkyne-tagged glycopeptides were then conjugated to the recombinant bacteriophage (Qß), a virus-like nanoparticle, through a click reaction. Antibody-binding analysis indicated that the synthetic glycoconjugates showed significantly enhanced affinity for antibody PG9 compared with the monomeric glycopeptides. It was also shown that the affinity of the Qß-conjugates for antibody PG9 was dependent on the density of the glycopeptide antigen display. The glycopeptide-Qß conjugates synthesized represent a promising candidate of HIV-1 vaccine.

Strategy for Producing the High-Quality Glycopeptide Antibiotic A82846B in Amycolatopsis orientalis Based on the CRISPR-Cas12a System.

Oritavancin is a new-generation semisynthetic lipoglycopeptide antibiotic used to prevent the spread of vancomycin-resistant Gram-positive bacteria. The glycopeptide A82846B is the direct precursor of oritavancin. Considering the structural similarity between A82846B and vancomycin, the vancomycin producer Amycolatopsis orientalis was used as a chassis for the construction of a strain producing high-quality A82846B. To construct the A82846B synthetic pathway, we established a highly efficient CRISPR-Cas12a system by optimizing the conditions of conjugation and by screening the regulatory elements in the A. orientalis, which is difficult to be genetically manipulated. The efficiency of gene knockout was almost 100%. The glycosyltransferases module (gtfDE) and glycosyl synthesis module (vcaAEBD) in the vancomycin gene cluster were replaced with the corresponding glycosyltransferases module (gtfABC) and glycosyl synthesis module (evaAEBD) in the A82846B cluster, respectively. A82846B was successfully produced by the artificially constructed synthetic pathway. Moreover, the titer of A82846B was increased 80% by expressing the pathway-specific regulatory strR. This strategy has excellent potential for remodification of natural products to solve antibiotic resistance.

Natural Glycoforms of Human Interleukin 6 Show Atypical Plasma Clearance.

A library of glycoforms of human interleukin 6 (IL-6) comprising complex and mannosidic N-glycans was generated by semisynthesis. The three segments were connected by sequential native chemical ligation followed by two-step refolding. The central glycopeptide segments were assembled by pseudoproline-assisted Lansbury aspartylation and subsequent enzymatic elongation of complex N-glycans. Nine IL-6 glycoforms were synthesized, seven of which were evaluated for in vivo plasma clearance in rats and compared to non-glycosylated recombinant IL-6 from E. coli. Each IL-6 glycoform was tested in three animals and reproducibly showed individual serum clearances depending on the structure of the N-glycan. The clearance rates were atypical, since the 2,6-sialylated glycoforms of IL-6 cleared faster than the corresponding asialo IL-6 with terminal galactoses. Compared to non-glycosylated IL-6 the plasma clearance of IL-6 glycoforms was delayed in the presence of larger and multibranched N-glycans in most cases.

Analytical comparability assessment on glycosylation of ziv-aflibercept and the biosimilar candidate.

Ziv-aflibercept (aflibercept) is a recombinant fusion protein which combines the portions of human vascular endothelial growth factor receptors extracellular domains fused to the Fc portion of human IgG1. It is a highly sialylated glycoprotein with 5 N-glycosylation sites. In this study, a comprehensive strategy for comparability study of the complex glycosylation was developed between aflibercept and the biosimilar candidate including the investigations on N-glycosylation sites, site occupancy, site-specific glycoforms, released glycans and sialic acids. The results indicated that same N-glycosylation sites were identified, site occupancy were 100% except N68 site, site-specific glycoforms and released glycans showed similar glycan species, contents of NANA were at a same level for two products. Minor differences were found between two products. The biosimilar candidate presented lower level of aglycosylation, lower level of glycans containing one terminal sialic acid, higher level of glycans containing two terminal sialic acids, higher level of G0F and Man5, lower level of G1F and G2F compared with aflibercept. However, further studies exhibited no differences were observed in the cell-based biological potency and Fc effector function. Moreover, the biosimilar candidate showed a similar pharmacokinetics curve and bioequivalence compared with aflibercept.

Modulation of immune cell reactivity with cis-binding Siglec agonists.

Inflammatory pathologies caused by phagocytes lead to numerous debilitating conditions, including chronic pain and blindness due to age-related macular degeneration. Many members of the sialic acid-binding immunoglobulin-like lectin (Siglec) family are immunoinhibitory receptors whose agonism is an attractive approach for antiinflammatory therapy. Here, we show that synthetic lipid-conjugated glycopolypeptides can insert into cell membranes and engage Siglec receptors in cis, leading to inhibitory signaling. Specifically, we construct a cis-binding agonist of Siglec-9 and show that it modulates mitogen-activated protein kinase (MAPK) signaling in reporter cell lines, immortalized macrophage and microglial cell lines, and primary human macrophages. Thus, these cis-binding agonists of Siglecs present a method for therapeutic suppression of immune cell reactivity.

Vasopressin and copeptin release during sepsis and septic shock.

Sepsis is defined as a potentially fatal organ dysfunction caused by a dysregulated host response to infection. Despite tremendous progress in the medical sciences, sepsis remains one of the leading causes of morbidity and mortality worldwide. The host response to sepsis and septic shock involves changes in the immune, autonomic, and neuroendocrine systems. Regarding neuroendocrine changes, studies show an increase in plasma vasopressin (AVP) concentrations followed by a decline, which may be correlated with septic shock. AVP is a peptide hormone derived from a larger precursor (preprohormone), along with two peptides, neurophysin II and copeptin. AVP is synthesized in the hypothalamus, stored and released from the neurohypophysis into the bloodstream by a wide range of stimuli. The measurement of AVP has limitations due to its plasma instability and short half-life. Copeptin is a more stable peptide than AVP, and its immunoassay is feasible. The blood concentrations of copeptin mirror those of AVP in many physiological states; paradoxically, during sepsis-related organ dysfunction, an uncoupling between copeptin and AVP blood levels appears to happen. In this review, we focus on clinical and experimental studies that analyzed AVP and copeptin blood concentrations over time in sepsis. The findings suggest that AVP and copeptin behave similarly in the early stages of sepsis; however, we did not find a proportional decrease in copeptin concentrations as seen with AVP during septic shock. Copeptin levels were higher in nonsurvivors than in survivors, suggesting that copeptin may work as a marker of severity or sepsis-related organ dysfunction.

TF-containing MUC1 glycopeptides fail to entice Galectin-1 recognition of tumor-associated Thomsen-Freidenreich (TF) antigen (CD176) in solution.

Aberrant Mucin-1 (MUC1) glycosylation with the Thomsen-Friedenreich (TF) tumor-associated antigen (CD176) is a hallmark of epithelial carcinoma progression and poor patient prognosis. Recognition of TF by glycan-binding proteins, such as galectins, enables the pathological repercussions of this glycan presentation, yet the underlying binding specificities of different members of the galectin family is a matter of continual investigation. While Galectin-3 (Gal-3) recognition of TF has been well-documented at both the cellular and molecular level, Galectin-1 (Gal-1) recognition of TF has only truly been alluded to in cell-based platforms. Immunohistochemical analyses have purported Gal-1 binding to TF on MUC1 at the cell surface, however binding at the molecular level was inconclusive. We hypothesize that glycan scaffold (MUC1's tandem repeat peptide sequence) and/or multivalency play a role in the binding recognition of TF antigen by Gal-1. In this study we have developed a method for large-scale expression of Gal-1 and its histidine-tagged analog for use in binding studies by isothermal titration calorimetry (ITC) and development of an analytical method based on AlphaScreen technology to screen for Gal-1 inhibitors. Surprisingly, neither glycan scaffold or multivalent presentation of TF antigen on the scaffold was able to entice Gal-1 recognition to the level of affinity expected for functional significance. Future evaluations of the Gal-1/TF binding interaction in order to draw connections between immunohistochemical data and analytical measurements are warranted.

Differential splicing of the lectin domain of an O-glycosyltransferase modulates both peptide and glycopeptide preferences.

Mucin-type O-glycosylation is an essential post-translational modification required for protein secretion, extracellular matrix formation, and organ growth. O-Glycosylation is initiated by a large family of enzymes (GALNTs in mammals and PGANTs in Drosophila) that catalyze the addition of GalNAc onto the hydroxyl groups of serines or threonines in protein substrates. These enzymes contain two functional domains: a catalytic domain and a C-terminal ricin-like lectin domain comprised of three potential GalNAc recognition repeats termed α, ß, and γ. The catalytic domain is responsible for binding donor and acceptor substrates and catalyzing transfer of GalNAc, whereas the lectin domain recognizes more distant extant GalNAc on previously glycosylated substrates. We previously demonstrated a novel role for the α repeat of lectin domain in influencing charged peptide preferences. Here, we further interrogate how the differentially spliced α repeat of the PGANT9A and PGANT9B O-glycosyltransferases confers distinct preferences for a variety of endogenous substrates. Through biochemical analyses and in silico modeling using preferred substrates, we find that a combination of charged residues within the α repeat and charged residues in the flexible gating loop of the catalytic domain distinctively influence the peptide substrate preferences of each splice variant. Moreover, PGANT9A and PGANT9B also display unique glycopeptide preferences. These data illustrate how changes within the noncatalytic lectin domain can alter the recognition of both peptide and glycopeptide substrates. Overall, our results elucidate a novel mechanism for modulating substrate preferences of O-glycosyltransferases via alternative splicing within specific subregions of functional domains.

Glutathione: A powerful but rare cofactor among Actinobacteria.

Glutathione (γ-l-glutamyl-l-cysteinylglycine, GSH) is a powerful cellular redox agent. In nature only the l,l-form is common among the tree of life. It serves as antioxidant or redox buffer system, protein regeneration and activation by interaction with thiol groups, unspecific reagent for conjugation during detoxification, marker for amino acid or peptide transport even through membranes, activation or solubilization of compounds during degradative pathways or just as redox shuttle. However, the role of GSH production and utilization in bacteria is more complex and especially little is known for the Actinobacteria. Some recent reports on GSH use in degradative pathways came across and this is described herein. GSH is used by transferases to activate and solubilize epoxides. It allows funneling epoxides as isoprene oxide or styrene oxide into central metabolism. Thus, the distribution of GSH synthesis, recycling and application among bacteria and especially Actinobacteria are highlighted including the pathways and contributing enzymes.

The fully synthetic glycopeptide MAG-Tn3 therapeutic vaccine induces tumor-specific cytotoxic antibodies in breast cancer patients.

Cancer is one of the main causes of mortality worldwide and a major public health concern. Among various strategies, therapeutic vaccines have been developed to stimulate anti-tumoral immune responses. However, in spite of extensive studies, this approach suffers from a lack of efficacy. Recently, we designed the MAG-Tn3 vaccine, aiming to induce antibody responses against Tn, a tumor-associated carbohydrate antigen. The Tn antigen is of interest because it is expressed by several adenocarcinomas, but not normal cells. The fully synthetic glycopeptide vaccine MAG-Tn3 is composed of four arms built on three adjacent Tn moieties associated with the tetanus toxin-derived peptide TT830-844 CD4+ T-cell epitope. This promiscuous CD4+ T-cell epitope can bind to a wide range of HLA-DRB molecules and is thus expected to activate CD4+ T-cell responses in a large part of the human population. The MAG-Tn3 vaccine was formulated with the GSK-proprietary immunostimulant AS15, composed of CpG7909, MPL, and QS21, which has been shown to stimulate both innate and humoral responses, in addition to being well tolerated. Here, seven patients with localized breast cancer with a high-risk of relapse were immunized with the MAG-Tn3 vaccine formulated with AS15. The first results of phase I clinical trial demonstrated that all vaccinated patients developed high levels of Tn-specific antibodies. Moreover, these antibodies specifically recognized Tn-expressing human tumor cells and killed them through a complement-dependent cytotoxicity mechanism. Overall, this study establishes, for the first time, the capacity of a fully synthetic glycopeptide cancer vaccine to induce specific immune responses in humans.

Effects of alcohol consumption on copeptin levels and sodium-water homeostasis.

Alcohol consumption influences sodium-water homeostasis. However, the effect of alcohol on vasopressin levels is controversial. The aim of the present study was to evaluate physiological changes of alcohol consumption on the stable vasopressin surrogate marker copeptin. In addition, we aimed at investigating the effect of additional sodium and/or water consumption on plasma sodium, osmolality, and copeptin levels. Ten healthy men underwent four interventions in random order: 1) beer consumption only, 2) beer consumption with additional water, 3) beer consumption with additional stock, or 4) water consumption only. Fluid consumption was equal between interventions and calculated to reach a blood alcohol concentration of 0.8‰ in the beer interventions. Blood and urinary samples were taken at six time points over the observation period of 720 min. The primary end point was the mean difference in copeptin levels 90 min after the start of fluid consumption, which showed no in-between group differences (P = 0.4). However, a higher total urinary volume excretion in all alcohol compared with water interventions was observed (P = 0.01). Furthermore, plasma copeptin, sodium, and urinary osmolality levels increased significantly at the end of the observation period in all alcohol compared with water-only interventions (P = 0.02). In conclusion, initial copeptin suppression does not differ between alcohol or water interventions but seems to be prolonged in the alcohol interventions. This leads to increased volume loss followed by a counterregulation with increased copeptin levels and water retention after 720 min in alcohol compared with interventions. Additional sodium and/or water consumption with alcohol did not change the observed alcohol-induced effects.

Identification of a five-mRNA signature as a novel potential prognostic biomarker in pediatric Wilms tumor.

BACKGROUND: The aim of this study was to generate a prognostic model to predict survival outcome in pediatric Wilms tumor (WT). METHODS: The data including mRNA expression and clinical information of pediatric WT patients were downloaded from the Therapeutically Available Research to Generate Effective Treatments (TARGET) database. The differentially expressed genes were identified and a prognostic signature of pediatric WT was generated according to the results of univariate and multivariate Cox analysis. Receiver operating characteristic (ROC) curve was used to evaluate the five-mRNA signature in pediatric Wilms tumor patients. Bootstrap test with 500 times was used to perform the internal validation. RESULTS: We identified 6,964 differentially expressed mRNAs associated with pediatric WT, including 3,190 downregulated mRNAs and 3,774 up-regulated mRNAs. Univariate and multivariate Cox analysis identified five mRNAs (SPRY1, SPIN4, MAP7D3, C10orf71, and SPAG11A) to establish a predictive model. The risk score formula is as follows: Risk score = 0.3036*SPIN4 + 0.8576*MAP7D3 -0.1548*C10orf71 -0.7335*SPRY1 -0.2654*SPAG11A. The pediatric WT patients were divided into low-risk group and high-risk group based on the median risk score (value = 1.1503). The receiver operating characteristic (ROC) curve analysis revealed good performance of the 5-mRNA prognostic model (the area under the curve [AUC] was 0.821). Bootstrap test (Bootstrap resampling times = 500) was used to perform the internal validation and revealed that the AUC was 0.822. REACTOME, KEGG, and BIOCARTA pathway analyses demonstrated that these survival-related genes were mainly enriched in ErbB2 and ErbB3 signaling pathways, and calcium signaling pathway. CONCLUSION: The five-mRNA signature can predict the prognosis of patients with pediatric WT. It has significant implication in the understanding of therapeutic targets for pediatric WT patients. However, further study is needed to validate this five-mRNA signature and uncover more novel diagnostic or prognostic mRNAs candidates in pediatric WT patients.

Phylogenetic evidence of the intercontinental circulation of a Canine distemper virus lineage in the Americas.

Canine distemper virus (CDV) is the cause of a multisystem disease in domestic dogs and wild animals, infecting more than 20 carnivore and non-carnivore families and even infecting human cell lines in in vitro conditions. Phylogenetic classification based on the hemagglutinin gene shows 17 lineages with a phylogeographic distribution pattern. In Medellín (Colombia), the lineage South America-3 is considered endemic. Phylogenetic studies conducted in Ecuador using fragment coding for the fusion protein signal peptide (Fsp) characterized a new strain belonging to a different lineage. For understanding the distribution of the South America-3 lineage in the north of the South American continent, we characterized CDV from three Colombian cities (Medellín, Bucaramanga, and Bogotá). Using phylogenetic analysis of the hemagglutinin gene and the Fsp region, we confirmed the circulation of CDV South America-3 in different areas of Colombia. We also described, for the first time to our knowledge, the circulation of a new lineage in Medellín that presents a group monophyletic with strains previously characterized in dogs in Ecuador and in wildlife and domestic dogs in the United States, for which we propose the name "South America/North America-4" due its intercontinental distribution. In conclusion, our results indicated that there are at least four different CDV lineages circulating in domestic dogs in South America: the Europe/South America-1 lineage circulating in Brazil, Uruguay, and Argentina; the South America-2 lineage restricted to Argentina; the South America-3 lineage, which has only been reported in Colombia; and lastly an intercontinental lineage present in Colombia, Ecuador, and the United States, referred to here as the "South America/North America-4" lineage.