Calcareous deposits can also be abiotically formed in seawater at the surface of steel grids under cathodic polarisation. In this work, we showed that this mineral-rich alkaline environment harbours bacteria belonging to different genera able to induce CaCO3 precipitation. We previously isolated 14 biocalcifying marine bacteria from electrochemically formed calcareous deposits and their immediate environment. By microscopy and µ-Raman spectroscopy, these bacterial strains were shown to produce calcite-type CaCO3. Identification by 16S rDNA sequencing provided between 98.5 and 100% identity with genera Pseudoalteromonas, Pseudidiomarina, Epibacterium, Virgibacillus, Planococcus, and Bhargavaea. All 14 strains produced carbonic anhydrase, and six were urease positive. Both proteins are major enzymes involved in the biocalcification process. However, this does not preclude that one or more other metabolisms could also be involved in the process. In the presence of urea, Virgibacillus halodenitrificans CD6 exhibited the most efficient precipitation of CaCO3. However, the urease pathway has the disadvantage of producing ammonia, a toxic molecule. We showed herein that different marine bacteria could induce CaCO3 precipitation without urea. These bacteria could then be used for eco-friendly applications, e.g., the formation of bio-cements to strengthen dikes and delay coastal erosion.Marine bacterial biomineralisation by CaCO Among these metabolic pathways, urea hydrolysis is the best known and the most efficient biocalcification process for CaCO3 [Sporosarcina pasteurii and Bacillus sphaericus [Natural limestone structures formed under the action of microorganisms, such as beachrocks and stromatolites, are observed in the marine environment ,2. Indeeronments ,6,7. Theompounds ,6,7. Allor CaCO3 ,12,13,14haericus ,16,17,182 and carbonates, such as respiration, CO2 transport, and photosynthesis [3 precipitation [2 into HCO3− (Equation (6)) and increases pH by releasing carbonates and bicarbonates in large quantities [Another metabolic pathway for biocalcification involves the carbonic anhydrase (CA) enzyme ,20. Thisynthesis . Neverthpitation ,22. CA pantities .H2O + C3 [3 can be enhanced.Under alkaline conditions and in the presence of dissolved calcium, bicarbonates formed by CA can precipitate as CaCO3 ,23,24. T3 ,19. Owin3 precipitation also occurs in abiotic conditions on metal surfaces under cathodic polarisation in seawater. A calcareous deposit made of a mixture of CaCO3 and magnesium hydroxide Mg(OH)2 is formed because the cathodic reactions taking place at the metal surface, i.e., O2 reduction and/or H2O reduction, produce OH− ions and increase the pH at the metal/seawater interface [3-rich environment could host microorganisms and that it should be explored to isolate indigenous bacteria that can survive in extreme alkaline environments for efficient biocalcification. Fourteen marine bacteria able to induce CaCO3 precipitation were thus isolated from calcareous deposits developed by cathodic polarisation on steel grids and their immediate environment (mud and seawater).CaCOnterface . In the nterface ,27. In anterface , we show3 production for 7 days.In the present work, we carried out a thorough characterisation of these 14 strains while specifying the characteristics of the environment from which they were isolated. All the strains were identified based on molecular characteristics. Investigations were conducted to validate urease and/or CA activities and their role in biocalcification. Finally, the biocalcification capacity of each of the isolated marine bacteria was evaluated by monitoring bacterial growth, pH evolution, and CaCO−1, bacteriological peptone 5 g L−1, boric acid 0.022 g L−1, anhydrous calcium chloride 1.8 g L−1, disodium phosphate 0.008 g L−1, anhydrous magnesium chloride 8.8 g L−1, potassium bromide 0.08 g L−1, potassium chloride 0.55 g L−1, sodium bicarbonate 0.16 g L−1, sodium chloride 19.4 g L−1, sodium fluoride 0.0024 g L−1, sodium silicate 0.004 g L−1, sodium sulphate 3.24 g L−1, strontium chloride 0.034 g L−1, yeast extract 1 g L−1, and ferric citrate 0.1 g L−1, pH 7.6 ± 0.2, or in M1 medium  medium  containing ammonium nitrate 0.0016 g Ll., 2015 ) containAlcanivorax borkumensis DSM 11573 [−1, magnesium sulphate heptahydrate 5.8 g L−1, magnesium chloride dihydrate 6.16 g L−1, calcium chloride dihydrate 1.47 g L−1, sodium hydrogen phosphate heptahydrate 0.89 g L−1, sodium nitrate 5 g L−1, ferrous sulphate heptahydrate 0.03 g L−1, and sodium pyruvate 10 g L−1, pH 7.0–7.5. SM 11573 , a marinSporosarcina pasteurii DSM 33, a non-marine bacterial strain, also used as a control for its ability to biocalcify [−1, peptone from soymeal 5 g L−1, sodium chloride 5 g L−1, and urea 20 g L−1, pH 7.3.ocalcify ,9, was gEscherichia coli ATCC 25922, used as a control for its urease-negative and carbonic anhydrase-positive characteristics, was grown at 37 °C in Tryptic soy Broth medium (TSB) composed of tryptone 17 g L−1, papain digest of soybean meal 3 g L−1, glucose 2.5 g L−1, dipotassium phosphate 2.5 g L−1, and sodium chloride 5 g L−1, pH 7.3 ± 0.2. The non-marine bacterial strain −1 (20 mmol L−1) and/or urea 20 g L−1 (330 mmol L−1). Solid media were prepared by adding agar 12 g L−1. For liquid cultures, all bacterial strains were grown with shaking (130 rpm).The different media were supplemented or not with calcium chloride dihydrate 3.7 g L−1, calcium chloride dihydrate 1.54 g L−1, magnesium chloride hexahydrate 11.1 g L−1, sodium sulphate 4.09 g L−1, potassium chloride 0.69 g L−1, and sodium carbonate 0.23 g L−1.Artificial seawater (ASW) based on the standard ASTM D1141  was composed of sodium chloride 24.54 g L−2. Samples of about 10 mL of seawater, 10 g of mud and 8 g of calcareous deposit were collected in November 2018. Seawater was collected from above the polarised grid in a sterile bottle. Polarised grids were deposited in a container of seawater from their environment, transported to the laboratory, and immediately processed. The mud was then collected from the grids using a sterile scalpel and transferred to a sterile vial. The calcareous deposit was sterilely scraped from different points of the grids with a scalpel, homogenised by grinding, and transferred into a sterile bottle. A first half of each sample was then placed in 50 mL of MB, and the second half was placed in 50 mL of M1 medium, both media being supplemented with CaCl2 20 mmol L−1. All samples were thereafter incubated at 25 °C with shaking  for one week. For each culture condition, bacterial cultures were diluted in ASW using the serial dilution technique, and each dilution was plated on solid-media MB and M1, supplemented with CaCl2 20 mmol L−1, and incubated for 3 days at 25 °C. By observation with a binocular magnifying glass , mud (MD), and calcareous deposits (CD) in the Atlantic Ocean from two different sites in the La Rochelle area  . The samlass see , bacteri−1 of each primer . Polymerase chain reaction was performed in a thermal cycler under the following conditions: 95 °C for 2 min, 30 cycles of 30 s at 92 °C, 30 s at 54 °C, and 1.5 min at 72 °C. Amplicons were sent to GenoScreen  for purification and gene sequencing. Amplicons with insufficient sequence quality were remade by Genoscreen with their own primers (P8/P535 and/or 1040F/1040R) and resequenced. Genomic DNA was extracted from pure culture using the Genomic DNA from Tissue Kit  according to the instructions of the manufacturer. Amplification of about 1400 bp 16S rRNA gene was performed in 50 µL of PCR reaction mixture containing 50–100 ng template DNA, 0.2 µmol LCGACTT3′ , 320 µmohttp://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 10 March 2021)). Genus identification of these bacteria was confirmed using SILVA ACT ) and RDP II classifier (http://rdp.cme.msu.edu/classifier/classifier.jsp (accessed on 15 December 2021)). Sequences were then submitted to the GenBank database (accession numbers MW774399 to MW774412).To identify the isolated bacterial strains, the 16S rDNA sequences were compared with those in GenBank using Blast software (−1 CaCl2 (MB + CaCl2), and MB + CaCl2 supplemented with 330 mmol L−1 urea (MB + CaCl2 + urea). For each condition, 100 mL of culture media was inoculated with bacteria at 1% from an overnight culture in MB, and 500 µL was collected for analysis regularly over 7 days of incubation. The pH of each sample was measured using a WTW pH-meter 3310 with electrode SENTIX Mic-D (WTW). Bacterial enumeration was carried out by the method of serial dilution of the samples. The samples were diluted in ASW and plated on solid MB medium for 24 h at 30 °C.For each bacterial strain, growth kinetics were carried out during 7 days of incubation at 30 °C with shaking  in three different culture media: MB, MB supplemented with 20 mmol Lg for 5 min. The pellets, which included bacterial cells and calcium carbonate precipitates, were washed in 20 mL of sterile water, centrifuged again, and resuspended in 20 mL of sterile water. Samples were then frozen at −80 °C and lyophilsed by a free-drying process at a pressure below 450 mTorr at −80 °C (COSMOS 20K) for 4 days. Bacterial strains were grown under the same three conditions as for growth kinetics see . After 73 production was performed by thermogravimetric analysis (TGA) on a Setaram Setsys Evolution 16/18 apparatus. Quantities of 20 to 60 mg of dried samples were heated from 20 °C to 1000 °C at a 10 °C/min heating rate under a neutral argon atmosphere. CaCO3 content was deduced from the mass loss between 550 and 900 °C due to decarbonation. To obtain a more accurate temperature range, data were analysed through differential thermogravimetric curves.Quantification of bacterial CaCOThe morphology of colonies and cells was characterised. Gram staining was performed according to the standard method . g for 5 min, the cell pellets were resuspended in 1 mL of fresh indole urea medium. The presence of urease activity was determined by observation of the colour change (yellow to pink) induced by the increase in pH following the release of ammonium from urea. Escherichia coli ATCC 25,922 and Sporosarcina pasteurii DSM 33 were used as negative and positive controls of urease activity, respectively.Urease activity assay was performed using bacterial strains from an overnight culture in MB at 30 °C and indole urea medium  according to the instructions of the manufacturer. Two tests were performed at room temperature: (i) In the first assay, 100 µL of bacterial cultures was inoculated in 900 µL of indole urea medium; (ii) In the second assay, after a cell concentration step by centrifugation of the bacterial cultures at 2500× p-nitrophenyl acetate  as CA substrate [−1 of p-NPA and 20 mmol L−1 of sodium phosphate buffer (pH 7.5). Bacterial colonies grown on solid MB for 24 h were added to the reaction solution (1 mL) and incubated for one hour at room temperature. The esterase activity of CA was detected by a colour change of the solution, which turns yellow when p-NPA is hydrolysed. For each experiment, the reaction solution without bacteria was used as a negative control, and Escherichia coli ATCC 25955 was used as a positive control of CA activity.Esterase activity of carbonic anhydrase (CA) was detected using ubstrate . The rea−1) and EZ (1250 mmol L−1) were prepared in N,N-dimethylformamide (DMF). For inhibition experiments, solid MB medium in Petri dishes containing AZ or EZ at a final concentration of 100 µmol L−1 was inoculated on the surface with 10 µL of an overnight culture in MB. After 48 h of incubation at 30 °C, crystal enumeration on the surface of bacterial colonies was carried out using fluorescence microscopy .Mineralisation products were observed using fluorescence microscope  with magnification of 50× and 200× in brightfield and under fluorescence . ZEN software (ZEISS) was used for image acquisition, and images were analysed using Fiji/Image J software.−1.μ-Raman spectroscopy analysis was used for crystal identification and performed at room temperature on a Jobin Yvon high-resolution Raman spectrometer  equipped with a microscope , a Peltier-based cooled, charge-coupled detector (CCD), and a near-infrared diode laser (785 nm). The acquisition time was variable and depended on fluorescence background of the spectrum due to the organic matter. It was generally equal to 60 s but could be increased up to 5 min to optimise the signal-to-noise ratio. At least 5 or 6 zones (diameter of approximately 2 μm) of a given deposit were analysed through a long-working-distance 50× objective, with a spectral resolution estimated at 0.5 cmp values were <0.05.All values presented in the Results and Discussion section are the averages of data from three independent experiments. Statistical analyses were performed using GraphPad Prism version 5.00 software using two-way ANOVA analysis and Bonferroni post hoc tests. Differences between a sample and the corresponding control were considered significant if 3 precipitation were isolated from CaCO3-rich environments: calcareous deposit and surrounding mud and seawater [−1 corresponding to the OH symmetric vibration mode of the magnesium hydroxide (Mg(OH)2) named brucite. Usually, brucite also exhibits vibration bands at 280 and 450 cm−1 [3, was identified via its Raman vibration bands at 155, 207, and 1086 cm−1. The vibration band around 1085 cm−1 corresponds to the symmetric stretching mode of the carbonate ion and is found at a similar position in all CaCO3 minerals. In contrast, the weaker bands found in the 100–300 cm−1 region are characteristic of the crystal structure, and the peak at 207 cm−1 is actually characteristic of aragonite [In previous work, fourteen marine bacteria able to induce CaCOseawater . In the 450 cm−1 . Howeverragonite . These rragonite ,35,36. Tragonite ,37. FurtThe fluorescence background that appeared on the Raman spectra reflected the presence of organic matter and thus the presence of organisms b. This i2 to detect calcifying bacteria), bacteria from calcareous deposit, mud, and seawater samples were isolated on their respective agar media  of the carbonate symmetric stretch could be due to the presence of magnesium in the calcite structure. The CaCO3 polymorphs resulting from biocalcification are typically calcite, aragonite, and vaterite, which are found in different shapes [3. Some researchers have suggested that the spheroidal and dumbbell shapes, as found here, reflect a microbial origin of the calcite and may serve as a bio-signature [Crystals of all isolated strains were analysed by µ-Raman spectroscopy . Spectra705 cm−1 . In our t shapes ,43. Calcignature ,44, wherignature . 2+ ions inhibit the growth of calcite [3 (development centre driving CaCO3 growth) therfore differs from that occurring in abiotic conditions and can lead to calcite instead of aragonite. It must be noted that cathodic polarisation in seawater leads mainly to aragonite ,35,36, w calcite . Biocalc3 precipitation [Previous studies have shown that a wide variety of microbial genera are involved in biocalcification in various natural environments, such as soils, freshwater, oceans, salt lakes, and marine sediments ,48,49,50pitation . These cThe 14 biocalcifying bacterial strains were identified by sequencing the near-complete 16S rRNA gene and comparing with GenBank, SILVA ACT, and RDP II sequences. All sequences matched with at least one identified strain with more than 98.5% identity .Pseudoalteromonas genus and CD3 to the Pseudidiomarina maritima species. These sequences were related to Alcanivorax borkumensis, a marine strain selected for its ability to biocalcify and used as a reference in this study [Epibacterium mobile species. The Firmicutes included four isolates  close to Sporosarcina pasteurii, a soil bacterium, also used as a control for its ability to biocalcify [Bhargavaea sp., and the two isolates, CD6 and CD8, were affiliated with Virgibacillus halodenitrificans and Planococcus maritimus species, respectively. We mainly identified Gram-negative bacilli  but also Gram-positive bacilli  and Gram-positive cocci , microorganisms that are quite phylogenetically distant but all capable of mineralising , Alphaproteobacteria (21.4%), and Firmicutes 28.6%) . In the is study . The Gam.6% . In ocalcify . In thisralising .Pseudoalteromonas sp. was found to be the main cultured species (six strains). Possessing Gram-negative cell walls, all Pseudoalteromonas strains formed rod-shaped cells and possessed aerobic and chemoheterotrophic metabolism, confirming previously described the characteristics of this microorganism [Pseudoalteromonas genus was commonly isolated from various marine environments, such as seawater, algae, and marine invertebrates [Pseudoalteromonas lipolytica was studied to protect steel from corrosion in seawater via the formation of a biomineralised film [Pseudoalteromonas sp. was also shown to precipitate other minerals, such as barite (BaSO4), in conjunction with EPS production [Pseudoalteromonas isolates  were characterised by highly mucoid colonies, suggesting significant production of EPS associated with CaCO3.organism . The Psetebrates ,53,54. Ttebrates  and theitebrates . Howeversed film ,58. The Pseudidiomarina maritima species, also known as Idiomarina maritima, was isolated for its ability to biocalcify. The CD3 strain was Gram-negative and rod-shaped and shared many phenotypic characteristics with other heterotrophic, oxidative, and marine members of the Gammaproteobacteria group. The Pseudidiomarina genus is widely distributed in marine and hypersaline habitats [Idiomarina bacteria were related to biomineralisation processes by investigating their production of Mg-rich minerals (struvite (NH4MgPO4·6H2O) and kutnahorite (CaMg(CO3)2)) under seawater salinity conditions. Idiomarina sp. was also shown to precipitate barite under experimental conditions [Pseudidiomarina/Idiomarina bacteria.In our work, only one strain (CD3) of the habitats . Previouhabitats  showed tnditions . Herein,Epibacterium mobile isolates , formerly known as Ruegeria mobilis [Epibacterium mobile species belongs to the Roseobacter clade, which constitutes up to 25% of the total bacterial community in coastal areas [In the Alphaproteobacteria class,  mobilis , were Gral areas ,62. WithVirgibacillus halodenitrificans , Bhargavaea beijingensis (formerly known as Bacillus beijingensis) and Bhargavaea ginsengi (formerly known as Bacillus ginsengi) were all related to Bacillus genus. Bacillus bacteria are widely distributed in natural environments and have been broadly described for their ability to precipitate calcium carbonate [Virgibacillus and Bacillus genera have already been isolated from brine and seawater and have been shown to form calcium carbonate minerals [Planococcus maritimus species, to our knowledge, so far, no work has demonstrated its capacity to biocalcify.All Gram-positive bacilli and cocci identified in the Firmicutes phylum were facultative and anaerobic and exhibited oxidase and catalase activities. Among these strains, arbonate ,63. Its arbonate . Severalminerals . Concern3 precipitation. The presence or absence of urease activity in the 14 bacterial strains was determined by an enzymatic colorimetric assay (indole urea medium). A first analysis had shown that some strains possessed urease activity, thanks to a colorimetric test inducing a more or less intense pink coloration in the presence of urease [Proteobacteria and Firmicutes. These results are consistent with a broad distribution of ureolysis metabolism among bacteria [3 in the absence of urea  were performed for the 14 biocalcifying strains .3 crystals for the majority of strains  was performed in the following liquid culture media: MB, MB + CaCl2, and MB + CaCl2 + urea , it would therefore appear that the nucleation sites are not on the cell walls but rather correspond to the diffuse EPS outside the cells.Obviously, no changes in pH or crystal formation were detected in the control experiments without bacteria. In the liquid media, all strains induced calcite precipitation (except the CD4 strain in MB) and grew rapidly to reach maximum growth between 8 and 16 h of incubation, regardless of culture conditions . Since nelements . Nucleatelements . The EPSelements ,81,82. Ug2+ ions ,60. In o2+ concentration, the cell survives by allowing the entry and accumulation of calcium ions, resulting in an excessive expulsion of protons. Subsequently, the cells actively export calcium and compensate for the loss of protons. A low concentration of protons and a high concentration of Ca2+ in the microenvironment is required for secretion of carbonate ions, while supersaturation of carbonate induces precipitation of calcium carbonate on the surface of the cell [3 precipitation [3 precipitated in the two calcium-enriched conditions. Thus, in the presence of urea, the urease-positive CD6 strain exhibited pH values (pH 9) and CaCO3 production (5.1 mg mL−1) significantly higher than those of the other strains. These results, together with the urease activity assay result  produced 1.65 mg mL−1. Their differing ability to biocalcify in this environment may be due to their different metabolic capacities. The absence of urea in the medium suggests that CA and/or a metabolic pathway other than urease are effective in inducing CaCO3 precipitation. Urea enrichment (MB + CaCl2 + urea) was used to assess the biocalcification capacity in the presence of urea for both urease-positive and urease-negative strains , CaCO3 production in the presence of urea was significantly higher for only two strains—CD6 (5.1 mg mL−1) and CD9 (1.5 mg mL−1) . Sporosarcina pasteurii is an efficient ureolytic strain extensively studied in CaCO3 production mechanisms and in various biomineralisation applications [Virgibacillus halodenitrificans CD6 urease-positive strain was the most efficient marine strain to induce calcium carbonate precipitation, with an ability equivalent to that of the related bacterium Sporosarcina pasteurii, the well-known biocalcifying bacterium described in the literature. Recently, Arias et al. [−1) biocalcification capabilities than those of CD6. These isolates were identified as Bacillus (related to Sporosarcina), Salinivibrio, and Halomonas species, already known to be biocalcifying [3 production with CA activity was overall lower than that of CD6 in the presence of urea, while the Pseudoalteromonas SW3 strain was, nevertheless, as efficient as CD6 under conditions without urea (1.7 mg mL−1)  —confirmiications ,15,85. Ts et al.  also deslcifying . Concernmg mL−1) . This re pathway .3 may act as a bio-cement [3 could be used as a natural cement, binding sediments, sand, shells, etc., giving rise to a solid agglomerate. In recent years, cementing bacteria have been widely studied for applications in multiple engineering fields, including geotechnical, construction, and environmental engineering, where they are used as a means of bio-cementation for the strengthening and consolidation of soils and sand or for the protection and restoration of stone and concrete structures [Finally, the metabolic properties of these biocalcifying marine bacteria lead to the formation of precipitated CaCOo-cement . Indeed,ructures ,86,88. Tructures . Potenti3 precipitation. The ability to grow in an alkaline and saline environment is crucial for calcium-carbonate-precipitating bacteria to be used in such environments. We showed that the composition of the medium, in particular the concentration of CaCl2, influenced the amount of CaCO3 produced by the 14 bacterial strains. Moreover, urease-positive bacteria showed higher CaCO3 production than urease-negative bacteria, as observed for the more efficient strain, Virgibacillus halodenitrificans CD6, in the presence of urea. Carbonic anhydrase—and potentially other enzymes—would be also involved in biocalcification and could act in synergy with urease metabolism, depending on the strain. Thus, these marine bacteria could be promising candidates for eco-friendly technological applications, such as the formation of bio-cements in synergy with the electrochemical process of calcareous deposit formation, in order to reinforce marine structures and to delay coastal erosion. In conclusion, we showed that calcareous deposits formed on a steel grid under cathodic polarisation in marine environment harbour bacteria belonging to different genera that are able to induce CaCO


To investigate the replication of influenza A virus A/Puerto Rico/8/34 (H1N1) in pulmonary microvascular endothelial cells and its effect on endothelial barrier function.Human pulmonary microvascular endothelial cells were infected with influenza A/Puerto Rico/8/34 (H1N1) virus. Plaque reduction assay, real-time quantitative PCR, immunofluorescence staining, and western blot were used to elucidate the replication process of virus-infected endothelial cells. In addition, real-time quantitative PCR was used to detect the relative expression levels of mRNA of some inflammatory factors. The endothelial resistance assay was used to determine the permeability of the endothelial monolayer. Excavation and analysis of data from open databases, such as the GeneCards database, DAVID Bioinformatics Resources, STRING search tool, and DGIdb database determined the genes, proteins, and signal pathways related to microvascular leakage caused by the H1N1 virus, and predicted the drugs that could be effective for treatment.In vitro experiments showed that the influenza virus can infect endothelial cells, leading to a significant increase in the permeability of pulmonary microvascular endothelial cells and the release of pro-inflammatory cytokines, but does not efficiently replicate in endothelial cells. A total of 107 disease-related target genes were obtained from the Gene-cards database. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that these genes mainly affected the pathways related to “Inflammatory bowel disease” (IBD), “Chagas disease” (American trypanosomiasis), “Influenza A”, and also played a key role in anti-inflammation and regulation of immunity. After enrichment analysis, 46 hub genes were screened. A total of 42 FDA-approved drugs corresponding to the hub genes were screened from the DGIdb database, and these could be formulated for topical application. In addition, these drugs can be used to treat other diseases, including cancer, inflammatory diseases, immune system disorders, and cardiovascular diseases.H1N1 influenza virus affects the barrier function of endothelial cells indirectly. Combined with bioinformatics tools, we can better understand the possible mechanism of action of influenza A (H1N1) virus causing pulmonary microvascular leakage and provide new clues for the treatment of pulmonary microvascular leakage. The spread of the influenza virus to the lower respiratory tract can cause viral pneumonia, which can then develop into acute lung injury and acute respiratory distress syndrome (ARDS). ARDS causes a marked increase in pulmonary microvascular leakage leading to pulmonary edema, hypoxemia, and respiratory failure . Recent Previous studies have used umbilical vein endothelial cells or pulmonary microvascular endothelial cells to study the replication kinetics and host response of some influenza A virus strains . Other sIn this study, we used human lung microvascular endothelial cells to understand the replication dynamics and effects of the influenza virus strain A/Puerto Rico/8/34 (H1N1) on pulmonary microvascular endothelial cells. We used bioinformatics tools to conduct text mining of biomedical literature, identify corresponding and targeted genes, proteins, and signal pathways from previous studies, and establish a variety of network relationships between drugs/molecules/targets and diseases. In addition, the the connection between the new genes and pathology was revealed. The computational prediction of multi-target drugs not only helped to discover more effective drugs but also provided great possibilities for studying new pharmacological targets and promoting drug repositioning and development in the pharmaceutical industry.vitro, Influenza A/Puerto Rico/8/34 (H1N1) virus can infect the pulmonary microvascular endothelium, and the infection can lead to the loss of barrier integrity. Finally, we used bioinformatics tools to explore the mechanism by which vascular leakage may increase, and predict some drugs that can reduce vascular leakage.In this study, we demonstrated that, in Influenza A/Puerto Rico/8/34 (H1N1) virus was donated by the Guangzhou Institute of Respiratory Diseases. The virus was propagated in 9 days-old specific pathogen-free (SPF) embryonated hen eggs. Each chicken embryo was inoculated with 0.1ml of 100-fold diluted virus solution, and the hole was sealed with wax liquid. Following successful inoculation of the chicken embryos with the virus, they were incubated at 37 °C, for 48 h. The allantoic fluid was collected, centrifuged at 3000 rp for 5 min, to obtain the supernatant. The viral titer was detected by plaque assays . The vir2 . Unless otherwise stated, and in the priming experiment, influenza virus was added to the MDCK cells and HULEC-5a cells in serum-free media at a multiplicity of infection (MOI) of 0.00001, 0.0001, 0.001, and 0.01 for two hours. The virus was removed, and DMEM containing 2 µg/ml tosylphenylalanyl chloride methyl ketone (TPCK) treated trypsin and 1% bovine serum albumin was added. All infections occurred within 48 hours (Madin-Darby canine kidney (MDCK) cell was a gift from the Institute of Respiratory Diseases of Guangzhou Medical University and the State Key Laboratory of Respiratory Diseases . MDCK cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 U/ml streptomycin at 37 °C under 5% CO2 . The hum48 hours .5 cells/well) were seeded in 12-well plates and cultured at 37 °C under 5% CO2 for 24 h  and incubated at 37 °C for 3 days. The cell monolayers were fixed with 4% paraformaldehyde for 2 h, the agarose overlays were then removed, and the cell monolayers were stained with 2%(w/v) crystal violet  according to the manufacturer’s instructions. RNA was reverse transcribed into cDNA using the reverse transcription kit . SYBR Green real-time PCR amplification and detection were performed using the ABI 7500 system . The PCR system comprised of, 25 µl reaction buffer . 18s rRNA was regarded as a house-keeping gene .IL-1β (forward primer) 5′-AGCTGATGGCCCTAAACAGA-3′IL-1β (reverse primer) 5′-TGGTGGTCGGAGATTCGTAG-3′TNF-α (forward primer) 5′-TGTAGCCCATGTTGTAGCAAA-3′TNF-α (reverse primer) 5′-CAAAGTAGACCTGCCCAGACT-3′NP (forward primer) 5′-GCACCAAACGGTCTTACGAA-3 ′NP(reverse primer) 5′-TTTGGATCAACCGTCCCTCA-3 ′18s rRNA(forward primer) 5′-GATGGAAAATACAGCCAGGTCCTA-3′18s rRNA(forward primer) 5′-TTCTTCAGTCGCTCCAGGTCTT-3′IL-4(forward primer) 5′-AGCTGATCCGATTCCTGAAAC-3 ′IL-4 (reverse primer) 5′-AACGTACTCTGGTTGGCTTC-3 ′IL-6(forward primer) 5′-CCACTCACCTCTTCAGAACG-3 ′IL-6(forward primer) 5′-CATCTTTGGAAGGTTCAGGTTG-3 ′5 cells/well) and MDCK cells (1 ×105 cells/well) were cultured on coverslips for 24 h. After infection with the influenza virus (MOI 0.01), they were incubated for 48 h. The cells were washed three times with phosphate buffer saline (PBS), fixed with 4% (v/v) paraformaldehyde in PBS for 15 min, and permeabilized with 0.5% Triton X-100 (prepared in PBS) permeate at room temperature for 20 minutes (HULEC-5a (1.5 ×10 minutes . For imm minutes . CoverslLysates were prepared using the Radio Immunoprecipitation Assay (RIPA) lysis buffer and separated by 10% polyacrylamide gels. Proteins were transferred to nitrocellulose membranes, blocked for 1 h using 5% BSA, and probed overnight with a primary antibody at 4 °C. After washing, the blots were incubated with HRP-conjugated secondary antibodies for 1 h, washed, and visualized by advanced chemiluminescence (Amersham). Band intensity was quantified using Image J (NIH) and normalized to the loading control after background correction . The NP 5 cells/well) were seeded on a 24-hole polyester transfer hole (Costar) with a 0.4 mm pore size for 24 h. The cells were then infected with the influenza virus, MOI 0.01. Endothelial permeability was evaluated by measuring trans-endothelial electrical resistance (TEER) at 0, 8, 16, 24, and 48 h after stimulation using the EVOM2 Epithelial Voltohmmeter . Untreated HULEC-5a cells were used as a negative control, and medium alone was used as a blank control. Relative TEER was expressed as follows: [ - ]/[(resistance in untreated HUVECs)-]; resistance was expressed in ohms.HULEC-5a cells (3 ×10https://www.genecards.org/Search/Keyword?queryString=Pulmonary%20microvascular%20lea kage%20caused%20by%20H1N1). The GeneCards is a searchable, integrative database that provides comprehensive, user-friendly information on all annotated and predicted human genes. The knowledgebase automatically integrates gene-centric data from 150 web sources, including genomic, transcriptomic, proteomic, genetic, clinical, and functional information. The keyword was “Pulmonary microvascular leakage caused by H1N1”. All targets were restricted to human origin.We performed text mining in the GeneCards database (version 5.0) (https://david.ncifcrf.gov/tools.jsp) was used to perform GO analysis, including biological process (BP), cellular component (CC), molecular function (MF), and KEGG Pathway analysis. The terms with a p-value of less than 0.05 were selected for functional annotation and signaling pathway clustering  and Kyoto Encyclopedia of Genes and Genomes (KEGG). DAVID Bioinformatics Resources (version 6.8) provides a comprehensive set of functional annotation tools for investigators to understand the biological meaning behind a large list of genes. DAVID (ustering .https://www.string-db.org/cgi/input?sessionId=bvSr4bpeU0nK&input_page_active_form=multiple_sequences), and the dates were saved as “CSV”. Cytoscape app was used for subsequent analysis. The molecular complex detection (MCODE) algorithm, was used to discover the closely related areas in the PPI network, by calculating the node information of each node in the network. The dates were run on the MCODE and STRING modules in Cytoscape, and the top one protein interaction module was saved for the next analysis  among the potential targets was constructed using the STRING search tool (version 11.0)  was used to search for potential targets with existing associations with existing drugs or small organic compounds. We selected the genes of the top one protein interaction module and click “Find Drug-Gene Interactions” to identify the drugs. Here, we set the preset filter to all approved drugs (DGIdb database (version 3.0)  influenza virus for 48 h. Pathological changes of endothelial cells under the microscope were not obvious . Immunof0 PFU/ml . We dete0 PFU/ml . Finally0 PFU/ml , File S2The fused HULEC-5a cells formed a compact monolayer on the semipermeable membrane, and the initial resistance value was measured before the cells were infected with the virus. Afterward, the old medium in the upper and lower chambers was removed, and influenza virus was added to the upper chamber at a multiplicity of infection (MOI) of 0.01, and maintenance solution was added to the lower chamber. Two hours after infection with the virus, the liquid in the upper and lower chambers was withdrawn, rinsed with PBS, and maintenance liquid added to both the upper and lower chambers. Resistance was determined at 0, 8, 16, 24, and 48 h after virus removal. A decrease in TEER was associated with increased permeability, while increased TEER suggested enhanced integrity of HULEC-5a cells . We obseCytokines are considered to be useful indicators of the severity of influenza infection and lung pathological characteristics, and endothelial cells are at the center of cytokine expansion during influenza virus infection . Real-tiThe GeneCards database was used to perform text mining, and search for genes related to “pulmonary microvascular leakage caused by H1N1”, Through text mining, a total of 107 genes related to pulmonary microvascular leakage caused by H1N1  were ideThe top five BP, CC, MF, and KEGG pathways were determined using DAVID. For CC, the genes were mainly enriched in the extracellular space, extracellular region part, cell surface, and vesicle lumen . For BP To understand the relationship between the targets more intuitively, 107 targets were imported into the SRING database to construct a gene functional association network, and the minimum score of protein interaction was set to 0.400. The network contained 105 nodes, 1808 edges, and a network graph with an average node degree of 34.4. The nodes represented the target protein, and the edges represented the protein-protein interaction relationship. STRING was used to analyze the protein-protein interaction of the genes using a protein interaction network diagram. We saved the file in “TSV” format and used Cytoscape to analyze the file (Supplement 9). The levels of TNF, IL6, TLR4, VEGFR, ALB, IL-10, IL-1β, and CXCL8 were significantly higher than other targets that play an important role in the PPI network. The highest target in this network was TNF, with a degree value of 82, which may be the most important potential target for H1N1 in causing pulmonary microvascular leakage. We used the MCODE plug-in in Cytoscape to perform module analysis on the file and obtained 39.111 points, ranking first, cluster-1 with 46 core genes , Fig. 4Bp-value, the first three BP, CC, MF, and KEGG were identified. CC enrichment was mainly enriched in the extracellular space and the extracellular area. For BP the enrichment was closely related to “defense response”, “inflammatory response” and “immune response”. For MF, the enrichment was mainly related to “receptor binding”, “cytokine activity” and “cell Factor receptor binding”. The top 3 most enriched signal pathways were “Chagas disease (American trypanosomiasis), ”Influenza A”’ and “Inflammatory bowel disease (IBD)” ” . In summThe DGIdb tool was used to search and screen 46 genes in cluster-1 for drug-gene interaction analysis . As showInfluenza A virus lacks a complete enzymatic system and has no raw materials and energy to synthesize its components. Therefore, the virus must invade susceptible host cells and rely on the host cell’s enzymatic system, raw materials, and energy to complete replication and produce progeny viruses. The process of influenza A virus replication is divided into five steps: Adsorption, penetration, uncoating, biosynthesis, assembly, and release, also known as the replication cycle . InfluenWe also studied the permeability of virus-infected endothelial cells and cytokine production. The permeability of endothelial cells was found to increase after influenza virus infection , and theTo further understand the mechanism of vascular leakage caused by the influenza A virus, we used bioinformatics tools to screen for the genes related to H1N1-induced pulmonary microvascular leakage, explore the potential molecular pathways and understand protein-protein interaction relationships. The results showed that 107 potential targets and biological processes of core genes were mostly related to inflammation and immunity. The CC enrichment results were mainly related to the extracellular region and vesicle lumen, the BP enrichment results were mainly related to immune response, and the MF enrichment results were primarily associated with “receptor binding”, “cytokine activity”, “cytokine receptor binding”, “growth factor receptor binding” and “identical protein binding”. The KEGG pathway enrichment was mainly involved in pathways such as “inflammatory bowel disease” (IBD), “Chagas disease” (Chagas disease), and “influenza A”, suggesting that the reason why influenza virus causes increased permeability of vascular endothelial cells may be related to the activation of these pathways.Combining the current literature and the results of this study, the possible reasons for the influenza virus to increase the permeability of vascular endothelial cells are as follows: (1) The influenza virus can infect endothelial cells, activate signaling pathways, and induce endothelial cell apoptosis, thereby destroying the epithelial-endothelial barrier. The signaling pathway associated with increased vascular endothelial permeability caused by the influenza virus may be the signaling pathway enriched by KEGG, but this still needs further experimental verification. (2) The influenza virus can induce endothelial cells to produce inflammatory cytokines, aggravate the cytokine storm, and increase damage to endothelial cells. These results indicate that, after infection with the A/Puerto Rico/8/34 (H1N1) influenza virus, endothelial cells could not effectively replicate to produce progeny virions, but caused vascular leakage and cytokine production and release. This finding can exclude the direct impact of the virus on endothelial cells and be used to study other mechanisms that cause increased endothelial cell permeability.vitro without changing the level of the virus titer (In addition, the currently studied drugs for enhancing vascular barrier, such as Slit2N, can enhance the adhesion of VE-cadherin at the endothelial cell–cell junction in us titer ; Doxycycus titer ; Vasculous titer ; There aus titer . Most ofIn summary, pulmonary endothelium plays an important role in the pathogenesis of severe influenza. The study of the replication kinetics and host response of the influenza A virus is of great significance for pathogenesis and treatment. This study findings reveal that enhancing the integrity of the endothelial barrier is an attractive treatment option, since it does not directly target the virus, and it is less vulnerable to viral mutations. Therefore, in clinical practice, vascular barrier enhancers should be used in combination with antiviral drugs.10.7717/peerj.11892/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.11892/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.11892/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.11892/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.11892/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.11892/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj.11892/supp-7Supplemental Information 7Click here for additional data file.10.7717/peerj.11892/supp-8Supplemental Information 8Click here for additional data file.10.7717/peerj.11892/supp-9Supplemental Information 9Click here for additional data file.10.7717/peerj.11892/supp-10Supplemental Information 10Click here for additional data file.10.7717/peerj.11892/supp-11Supplemental Information 11Click here for additional data file.10.7717/peerj.11892/supp-12Supplemental Information 12Click here for additional data file.10.7717/peerj.11892/supp-13Supplemental Information 13Click here for additional data file.10.7717/peerj.11892/supp-14Supplemental Information 14Click here for additional data file.10.7717/peerj.11892/supp-15Supplemental Information 15Click here for additional data file.10.7717/peerj.11892/supp-16Supplemental Information 16Click here for additional data file.


COVID-19 cases have been increasing since the epidemic started. One of the major concerns is how clinical symptomatology would behave after coinfection with another virus.In this case report, a pediatric native patient from Estado de Mexico (EDOMEX), MEX had severe DENV-2 and acute SARS-CoV-2 at the same time. The clinical features were severe thrombocytopenia, secondary septic shock, cerebral edema, pericardial effusion, fluid overload that exhibited bipalpebral edema in all four extremities, hemophagocytic lymphohistiocytosis (HLH), coronary artery ectasia (CAE), multisystemic inflammatory syndrome in children (MIS-C), and probable COVID-19 pneumonia or acute respiratory distress syndrome (ARDS) that triggered patient intubation. The patient presented unusual symptomatology according to the literature. After 15 days of intubation and 15 more days under surveillance, he was released without respiratory sequelae and without treatment after major clinical improvement.The aim of this manuscript is to present clinical challenges that coinfection may cause in pediatric patients, even though COVID-19 in children does not tend to be as severe as in other sectors of the population. In late 2019, many cases of atypical pneumonia were reported in the province of Wuhan, Hubei, China; later, these cases were later linked to a new virus, SARS-CoV-2 [Dengue virus (DENV) is endemic in Mexico, and climatic diversity provides fertile ground for the transmission of DENV, with four serotypes circulating in the territory . At the In August 2020, a 7-year-old male patient presented with clinical symptoms of difficulty swallowing, fever of 39 °C, and headache and was diagnosed with bacterial pharyngitis by a primary health care provider. Antibiotics (amoxicillin) and ibuprofen were prescribed. He was subsequently treated at two more private practices as symptoms did not improve, first for gastroenteritis medicated with trimethoprim/sulfamethoxazole and ibuprofen and later with clindamycin, ibuprofen, and nimesulide. Although a slight improvement was seen, the headache worsened and was accompanied by adynamia and asthenia, which were present for at least 5 days. Finally, after 6 days of failed treatments, the patient was admitted to the “Hospital Materno Infantil,” ISSEMYM, EDOMEX, MEX.3/μL) of 16,820/μL, low lymphocyte count (RR 15–61%) of 11.2% (1260/μL), and a normal monocyte count (RR 0–5%) of 3%. Based on clinical suspicion, COVID-19 and DENV RT-qPCR were requested.During physical examination upon the patient’s admission , petechia was seen in the face, armpits, and lower limbs; he also presented with dehydration and odynophagia. Laboratory tests indicated severe thrombocytopenia (reference range (RR) 150,000–450,000/μL) with a platelet count of 15,000/μL, long prothrombin time (RR 12.2–15.5 s) of 10.7 s, normal hemoglobin (RR 11.5–15.5 g/dL) of 13.9 g/dL, elevated leucocytes . During this time, he also had severely stiff limbs and myoclonus, leading to suspicion of encephalitis. A CT scan of the brain showed cerebral edema, which was treated with phenytoin sodium (20 mg/kg loading dose maintained at 7 mg/kg per dose); later, the patient responded to treatment for myoclonus, and he did not present abnormal movements afterwards.d-dimer. He was treated with immunoglobulin gamma (1 g/kg per dose with 2 doses), which is the most effective therapy for MIS-C, from day 7 of illness to day 12 of illness.On day 2 of hospital admission, the nasopharyngeal swab result was positive for SARS-CoV-2. Nucleic acid extraction was performed with the chemagic 360 instrument  using 300 µL of sample in viral transport medium (VTM), amplification was done with SARS-CoV-2 RT-PCR vitro master diagnostica® , and viral load was quantified in a QX ONE Droplet Digital PCR (ddPCR) System  which calculated 9.11 copies/µL. The patient was treated with 3rd generation cephalosporin and meningeal doses of ceftriaxone and acyclovir intravenously for 5 days for COVID-19. On the same day, a pediatric cardiology service ran echocardiography, which detected right coronary ectasia and pericardial effusion without hemodynamic compromise; physicians suggested 3 mg/kg acetylsalicylic acid every 24 h, although due to thrombocytopenia, treatment was delayed until D11. He also showed fluid overload, exhibiting bilateral periorbital edema and edema in all four extremities, hepatomegaly, and pericardial effusion and was administered furosemide (D2–16). In addition, on illness day 7, the patient presented with respiratory distress (oxygen saturation 90%), and noninvasive ventilation was initiated due to its clinical evolution. As the inflammatory response continued, multisystemic inflammatory syndrome in children (MIS-C) was suspected due to persistent fever (1 week), headache, cytopenia, thrombocytopenia, anemia, hyperferritinemia, generalized petechia, and elevated Following D4 (day 9 of illness), his condition became unstable due to ventilatory deterioration . To prevent worsening, mechanical ventilation was started while he was sedated with opioid analgesia (midazolam 20 mcg/kg/min) until D14 of hospital admission. By D5, clindamycin was added to the COVID-19 treatment.By D7, the patient had high blood pressure (above the 90th percentile) and was treated with hydralazine (0.2 mg/kg per dose every 6 h) while norepinephrine was stopped, although he presented low cardiac output along with grade III cardiomegaly and was treated with levosimendan (0.1 μg kg/min until D12). Additionally, the patient had electrolyte imbalance (hypernatremia) which was corrected immediately with the Holliday-Segar equation.On D9 of hospital admission, the patient had hypophosphatemia (RR mild from 2 to 2.5 mg/dL) at 2.3 mg/dL, hyponatremia (RR < 135 mEq/L) at 133 mEq/L, hypomagnesemia (RR < 1.5 mg/dL), and hypokalemia (RR < 3 mEq/L) which were corrected immediately with the Holliday-Segar equation.On D10, hemophagocytic syndrome was suspected due to the presence of cytopenia, persistent fever, hypertriglyceridemia, and hyperferritinemia.From D4 to D11, the patient was transfused multiple times due to active bleeding with platelet apheresis or platelet concentrates and was observed to be improving until D11. On D12, clindamycin was still administered as a result of a persistent inflammatory response.By D12, an increase in D-dimer was detected, and the pediatric hematology service suggested low molecular weight heparin with enoxaparin 1 mg/kg every 24 h. By D14 of hospital admission, the edema had resolved. However, chest X-ray still showed pulmonary congestion, cardiomegaly, and hepatomegaly. At this point, the patient was extubated with supplementary oxygen along with nasal cannulas (1 LPM) and nebulized with ipratropium bromide.On D15 after hospital admission, the bleeding stopped, but a grade 2 pressure ulcer was noted on the occipital region. A serum sample result was positive for DENV-2. Because of the increase in COVID-19 samples, the state laboratory was at full capacity and samples other than COVID-19 were delayed. Nucleic acid extraction was performed with the chemagic 360 instrument using 300 µL of serum sample, amplification was done with a one-step superscript III and CDC DENV-1–4 Real-Time RT-qPCR Multiplex Assay, and viral load was quantified by a QX ONE Droplet Digital PCR (ddPCR) System (Bio-Rad) and calculated to be 6.36 copies/µL.3/μL) at 14.84 × 103/μL, high neutrophils at 82%, low lymphocytes (RR 15–61%) at 12%, low erythrocytes at 3.60 × 106/μL, low hemoglobin (RR 11.5–15.5 g/dL) at 11.0 g/dL, and low hematocrit (RR 35–45%) at 30%. By D20 after hospital admission, the patient was moved from the COVID area to the general pediatric ward; at this time, the patient was on supplementary oxygen delivered via a nasal cannula.On D17 after hospital admission, the patient had pain upon physical examination, and he was also weak and needed support for daily living activities. Petechiae were still present, and hair loss was noted. Some laboratory parameters were still abnormal, including high leucocytes  and bleeding had stopped. Upon discharge, acetylsalicylic acid (3 mg/kg per dose every 24 h) and prazosin (0.4 mg/kg per dose) 2 mg every 6 h were continued for 2 weeks. After 2 weeks, the patient had fought off the infections and did not have respiratory sequelae, so he was released without treatment and experienced major clinical improvement over 3 months of follow-up.A few cases of coinfection with SARS-CoV-2 and DENV-2 have been reported in different countries for adult patients –6, yet t3, leukopenia, positive tourniquet, myalgia/arthralgia, petechia, gastrointestinal bleeding, elevation in serum glutamic oxaloacetic transaminase (SGOT), hepatomegaly, fluid leak, and pleural effusion [Plasmodium vivax, Plasmodium falciparum, scrub typhus, invasive aspergillosis, herpes simplex virus, respiratory syncytial virus, or Epstein‐Barr virus [According to previous reports –10, the effusion  are commeffusion  and has rr virus ), longerrr virus . It was rr virus . In the rr virus , 17. Carrr virus , 19. Likrr virus . Both vid-dimer [In the case of COVID-19, clinical symptoms in pediatric patients are not well documented, although various studies report fever and cough as the main symptoms; others include rhinorrhea, sore throat, headache, fatigue/myalgia, and gastrointestinal symptoms. It was also shown that infants 0–1 years old are more likely to be affected . COVID-1d-dimer –26.COVID-19 has also been related neurological symptoms . One of A few cases of COVID-19 and dengue have been reported. In a study of 178 adult patients (> 18 years of age) in Brazil, 63% were diagnosed as positive for SARS-CoV-2 after RT-qPCR confirmation; from this percentage, 38.4% of patients were also positive for dengue coinfection by a dengue IgM test. A higher rate of pulmonary impairment and hospitalization were demonstrated in coinfection. The patients also showed lower levels of blood lymphocytes (30.52% vs 26.37%) and monocytes (7.76% vs 6.99%) [Neither COVID-19 nor DENV disease has been shown to cause coronary artery ectasia (CAE). Degradation of the medial arterial layer by activation of serine proteinase in addition to matrix metalloproteinase (MMP) in the arterial smooth muscle cells generates CAE ; a studyOther relevant points are the high values of white blood cells (WBCs), as DENV disease causes leukopenia according to diverse studies , 41. In Infection caused by SARS-CoV-2 has been a major challenge for physicians due to its wide range of clinical manifestations. Diverse clinically approved drugs have been tested as potential anti-SARS-CoV-2 candidates . AntivirIt is worthwhile to address contraindications of nonsteroidal anti-inflammatory agents (NSAIDs), as these drugs can potentially increase bleeding risk . The patThe patient in this case report did not have all the symptomatology required for MIS-C caused by SARS-CoV-2, and he presented a coinfection with DENV-2, which may have aggravated the severity in both diseases resulting in effects not usually seen in children.As the COVID-19 vaccine is still in trials and DENV is a national public health concern in Mexico, we may see more cases of coinfection of SARS-CoV-2 and DENV in pediatric patients in the near future; thus, the intention of this paper is to yield insight on behavior, management, and preparation for coinfection. As observed in this case report, not all medication given to this patient was appropriate, thus some symptoms could have been avoided. As clinicians, it is our duty to research the best options for our patients and their wellbeing, especially with a novel virus that is not well characterized in different scenarios. We hope that this case report will help physicians better treat coinfection between DENV and SARS-CoV-2 in pediatric patients.


However, in vivo delphinidin is poorly absorbed, thus its modest bioavailability and stability reduce its anti-angiogenic effects. The present work takes advantage of small extracellular vesicle (sEV) properties to enhance both the stability and efficacy of delphinidin. When encapsulated in sEVs, delphinidin inhibits the different stages of angiogenesis on human aortic endothelial cells (HAoECs). (2) Methods: sEVs from immature dendritic cells were produced and loaded with delphinidin. A method based on UHPLC-HRMS was implemented to assess delphinidin metabolites within sEVs. Proliferation assay, nitric oxide (NO) production and Matrigel assay were evaluated in HAoECs. (3) Results: Delphinidine, 3-O-β-rutinoside and Peonidin-3-galactoside were found both in delphinidin and delphinidin-loaded sEVs. sEV-loaded delphinidin increased the potency of free delphinidin 2-fold for endothelial proliferation, 10-fold for endothelial NO production and 100-fold for capillary-like formation. Thus, sEV-loaded delphinidin exerts effects on the different steps of angiogenesis. (4) Conclusions: sEVs may be considered as a promising approach to deliver delphinidin to target angiogenesis-related diseases, including cancer and pathologies associated with excess vascularization. Polyphenols are found mainly in plant-derived foods and beverages and provide the taste and color of plant foods. Moreover, epidemiological studies have reported a greater reduction in cardiovascular risk and cancer associated with diets rich in polyphenols ,2,3.Delphinidin chromenylium-3,5,7-triol) is an anthocyanin abundantly identified in pigmented vegetables and fruits, particularly berries and red grapes. We previously reported that delphinidin possesses the same pharmacological profile as a total extract of red wine polyphenolic compounds to promote the increase of intracellular calcium concentration and activation of tyrosine kinases [Finally, we demonstrated that delphinidin displays anti-angiogenic properties, both on in vitro and in vivo angiogenesis models, and reduces in vivo tumor growth of melanoma ,7,8,9,10However, delphinidin is less potent to induce these beneficial effects compared to total red wine polyphenol extracts, especially in inducing endothelium-dependent NO-mediated vasodilatation . Indeed,One strategy to overcome such problems is the use of extracellular vesicles (EVs) as a drug delivery system. We recently found that EVs, including large and small EVs (sEVs), are nanostructures originating from different subcellular compartment properties, overcoming the limitations of classical nano-formulations. sEVs decrease instability and immunogenicity, improve bioavailability and target selectivity . Some reIn the present study, we took advantage of sEV properties to enhance both the stability and efficacy of delphinidin. sEV-loaded delphinidin induced angiogenesis inhibition using human aortic endothelial cells (HAoECs). Delphinidin content in terms of metabolites within these EVs was also determined.2 in endothelial cell growth medium MV2 (Promocell) supplemented with 1% penicillin/streptomycin . Cells were trypsinized at 70/80% confluence and were used between passage 3 and 6 for all experiments.HAoECs  were cultured at 37 °C and 5% CO2 in a complete culture medium composed of alpha minimum essential medium  containing ribonucleosides and desoxyribonucleosides and supplemented with 20% fetal bovine serum (FBS) , 4 mM L-glutamine (Lonza), 1 mM sodium pyruvate (Lonza), 1% penicillin/streptomycin  and 5 ng/mL murine GM-CSF . Cells were trypsinized at 70/80% confluence and were used between passage 8 and 16 for all experiments.The JAWS II dendritic cell line was purchased from the American Type Culture Collection . JAWS II cells were grown at 37 °C and 5% CO6 cells in a T175 cell culture flask in complete growth medium, and they were starved in FBS before any isolation. Cell medium was centrifuged at 300× g and 2000× g for 10 min to remove cells and cell debris, respectively. The resultant supernatant was centrifuged at 20,000× g for 30 min to exclude large EVs. The supernatant was centrifuged at 200,000× g  for 2 h to pelletize sEVs. Then, sEVs were washed in phosphate-buffered saline (PBS)  and recentrifuged at 200,000× g for 2 h. Finally, sEV pellets were resuspended in 1 mL of PBS and stored at 4 °C until subsequent use. The amount of sEVs was determined using the method of Lowry, with bovine serum albumin (Sigma-Aldrich) as the standard. sEVs were used at 10 µg/mL.JAWS II cells were seeded at a density of 5 × 10g, the obtained pellet was reconstituted in 1 mL of 0.1% DMSO or PBS. Delphinidin absorbance was measured at 530 nm, and a standard curve with different concentrations (0.1 to 10 µg/mL) of free delphinidin was performed. The percentage of the efficacy of the loading of sEVs was 9%, independently of the concentration of delphinidin used (data not shown). Thus, the amount of delphinidin was adjusted to obtain the desired concentration (0.1 to 5 µg/mL) within 10 µg/mL sEVs. To remove free delphindin, these vesicles were washed twice.Delphinidin was prepared in water at pH = 2 with 0.1% DMSO in order to reach the concentration of 10 µg/mL. The sEVs were added (2 mg), and the solution was stirred and then vortexed for 10 min. After 2 h of ultracentrifugation at 200,000× Stokes-Enstein equation.sEV samples were diluted in sterile NaCl 0.9%, and size distribution was analyzed using the NanoSight NS300 . Videos were recorded. NTA software determined the size distribution using the g centrifugation for 70 min. sEVs were deposited on copper grids for 2 min and negatively stained with 20 μL of uranyl acetate 5% (diluted in ethanol 50%) for 30 s. Grids were then observed with a Jeol JEM 1400 microscope  operated at 120 keV.sEVs were first fixed overnight at 4 °C with 2.5% glutaraldehyde  in 0.1 M PBS. Then, sEVs were washed two times in PBS by 100,000× g, 10 min, 4 °C) and evaporated in a miVac duo concentrator . The dry extract was reconstituted with 200 µL LC-MS grade water containing 1% formic acid. The mixture was subjected to a second centrifugation  prior to ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS) analysis in order to analyze delphinidin metabolites with accurate mass measurements.Sample preparation was as follows: 250 µL methanol (MeOH) was added to 10 µg sEVs reconstituted in PBS, and samples were subjected to a 20 min ultrasonication. Two hundred µL of MeOH was further added, and samples were centrifuged . Mobile phases consisted of H2O in channel A and acetonitrile in channel B, both containing 0.1% formic acid. The elution gradient  was as follows: hold initial conditions 95:5 for 2 min, followed by a linear gradient from 95:5 to 0:100 over a 6 min period, hold at 0:100 for 3 min, return to initial conditions 95:5 and hold these conditions for 3.5 min. A constant flow rate of 0.300 mL/min was used; the injection volume was 10 μL.The chromatographic separation was achieved with a Kinetex6 ions and a maximum ion injection time (IT) of 200 ms. Data-dependent MS/MS experiments were acquired in ‘Top5′ data-dependent mode.Full scan and targeted SIM mass spectra were acquired in positive ionization mode, using resolution 70,000 Full Width at Half Maximum (FWHM) with automatic gain control (AGC) target of 3 × 10Metabolites reported in the literature ,17,18 weDaily instrument calibration was performed by infusion of Pierce LTQ Velos ESI positive/negative calibration kits as recommended by the manufacturer. Xcalibur 2.2 software  was used for data acquisition, and TraceFinder 3.0 software (Thermo Fisher Scientific) was employed for data processing.4 HAoECs were seeded onto a 96-well plate and cultured for 24 h and treated with delphinidin (1 to 10 µg/mL). Then, 5 μg/mL of 3--5-(3-carboxymethoxyphenyl)- 2-(4-sulfophenyl)-2H- tetrazolium  was added into each well and incubated at 37 °C for 120 min. The absorbance was measured on a CLARIOstar®  spectrophotometer at 490 nm.1 × 104 cells were seeded in a 96-well plate. Cells were serum-starved for 2 h and then treated with delphinidin, native sEVs or sEVs loaded with delphinidin at different concentrations. After 24 h of incubation, cells were washed with PBS, and dye-binding solution was added. Cells were incubated at 37 °C for 30 min. A fluorescent microplate reader  with filters for 485 nm excitation and 530 nm emission was used for fluorescence measurement.Proliferation assays were conducted using CyQUANT Cell proliferation Assay kit  according to the manufacturer’s recommendations. Briefly, 1.5 × 104 cells per well  in 300 μL of medium. At 70–80% confluence, cells were stimulated for 24 h with delphinidin, native sEVs or sEV-loaded delphinidin. Adenosine triphosphate (ATP) was used as a positive control  to stimulate the production of NO. After 24 h, medium of each well was removed, and the diaminofluoroscein diacetate (DAF-2 DA) probe was added . Then, the wells were washed with PBS. Cells were fixed with paraformaldehyde . Fluorescence was read by confocal microscopy . Four pictures were acquired, and ImageJ software was used for quantification.HAoECs were seeded on a 8-well slide  at a rate of 3 × 10® . Briefly, 10 µL of liquid Matrigel® was placed in each well of a 15-well Ibidi µ-slide Angiogenesis plate (Ibidi) and then incubated for 45 min at 37 °C to form a gel. HAoECs were then seeded and incubated at 37 °C and 5% CO2 for 45 min before treatments with either delphinidin, native sEVs or sEV-loaded delphinidin, followed by an incubation of 12 to 14 h at 37 °C and 5% CO2. The formation of “capillary-like structures” was observed with an optical microscope (Olympus CK40). Quantification was performed by measuring the number of capillary-like structures using Image J software.HAoECs were seeded in wells coated with Matrigelp-values of < 0.05 were considered significant.Results are expressed as mean ± SEM. Significance of the differences between groups was determined by analysis of variance (ANOVA), followed by Tukey’s multiple comparisons test. In agreement with the literature, delphinidin loaded within sEVs did not induce changes in size of the vesicles, being 118.7 ± 2.9 and 111.7 ± 1.8 nm for empty sEVs and delphinidin-loaded sEVs, respectively, as determined by Nanoparticle Tracking Analysis A and con+ m/z = 463.12404) (+ m/z = 611.16121)  B and del1.16121) C were deDelphinidin alone or loaded in sEVs did not modify the viability of HAoECs for 24 h at concentrations of 1 to 10 µg/mL A.VEGF (20 ng/mL), used as a positive control, non-significantly increased endothelial cell proliferation B. NativeAs shown on HAoECs restructured and formed capillary-like structures A,B. NatiThe current study shows that the delphinidin loaded within sEVs was obviously more potent than free delphinidin regarding its ability to release endothelial NO, to inhibit endothelial proliferation and to reduce capillary-like structures. In addition to delphinidin found into sEVs, the analysis of delphinidin metabolites within the sEVs showed the presence of two metabolites  present in delphinidin samples. Thus, delphinidin degraded into the same metabolites in its free form or when loaded into sEVs; however, these were more potent in acting on endothelial cells. Of importance, when encapsulated within sEVs, delphinidin  was 2-fold, 10-fold and 100-fold more potent than free delphinidin regarding endothelial proliferation, endothelial NO production and capillary-like formation. Thus, sEV-loaded delphinidin exerts effects on different steps leading to angiogenesis. These results indicate that sEVs may be considered as a promising delivery of delphinidin as an innovative approach to target diseases associated with increased angiogenesis, including cancer, atherosclerosis and diabetic retinopathies.The encapsulation of polyphenols to protect them from degradation is a natural phenomenon. Indeed, it has been shown that plants rich in polyphenols produce EVs carrying these molecules. For instance, the flavonoid glycoside naringin and its metabolite, naringenin, are found in grapefruit-derived EVs . Moreove2+ concentrations via the increase of superoxide anion formation. This was associated with increased tyrosine phosphorylation of several intracellular proteins, resulting in endothelium-dependent vasodilatation [We previously reported that, in bovine aortic endothelial cells, delphinidin stimulates NO release by increasing intracellular Calatation ,25. Delplatation . In the latation .We previously reported that upregulation of the NO pathway is not responsible for the antiproliferative effect of delphinidin. Indeed, delphinidin inhibits endothelial cell proliferation by the activation of ERK-1/-2 pathway, leading to cell cycle arrest and accumulation of cells in the G0/G1 phase via down-regulation of cyclin A and D1 expression and an upregulation of p27kip1 ,7. We alIn concordance with these findings, we show that delphinidin decreases capillary-like formation in an experimental model of angiogenesis. Interestingly, when encapsulated within sEVs (even at a loading as low as 9%), delphinidin was 100-fold more potent than free delphinidin in decreasing capillary-like formation.Limitation of the study: The anti-angiogenic potential exhibited by many natural compounds contained in many Mediterranean diet constituents, including delphinidin, makes this dietary pattern especially interesting as a source of chemopreventive agents, defined within the angioprevention strategy. This has been recently reviewed by Martinez-Podeva et al. . DelphinIn summary, sEV-loaded delphinidin increased the efficacy of delphinidin 100-fold for proliferation, 10-fold for NO and 2-fold for capillary-like formation. Thus, sEVs either protected delphinidin and its metabolites from degradation or some unidentified delphinidin metabolites contained in the sEVs were more potent. The differential potency obtained for proliferation, NO production and angiogenesis supports the hypothesis that delphinidin-loaded sEVs exert effects on different steps leading to angiogenesis. Nevertheless, we provide evidence that we optimized delphinidin efficacy, probably by reducing its degradation and increasing its delivery when encapsulated in EVs. Thus, delphinidin-loaded sEVs represent a powerful delivery system to decrease angiogenesis in endothelial cells, with no unwanted side effects, knowing the low bioavailability of this compound. We underscore an innovative therapeutic strategy based on bio-engineered EVs as vectors of delphinidin in helping to increase its potential health benefit to target angiogenesis-related diseases, including cancer, which could eventually be extended to further diseases with excess vascularization.


Noncanonical (giant) AnkG isoforms play a key role in neuronal membrane biogenesis and excitability, with evidence for human neurologic disease when aberrant. However, the role of giant AnkG in cardiovascular tissue has yet to be explored. Here, we identify giant AnkG in the myocardium and identify that it is enriched in 1-week-old mice. Using a new mouse model lacking giant AnkG expression in myocytes, we identify that young mice displayed a dilated cardiomyopathy phenotype with aberrant electrical conduction and enhanced arrhythmogenicity. Structural and electrical dysfunction occurred at 1 week of age, when giant AnkG was highly expressed and did not appreciably change in adulthood until advanced age. At a cellular level, loss of giant AnkG results in delayed and early afterdepolarizations. However, surprisingly, giant AnkG cKO myocytes display normal INa, but abnormal myocyte contractility, suggesting unique roles of the large isoform in the heart. Finally, transcript analysis provided evidence for unique pathways that may contribute to the structural and electrical findings shown in giant AnkG cKO animals. In summary, we identify a critical role for giant AnkG that adds to the diversity of ankyrin function in the heart.Cardiovascular disease (CVD) remains the most common cause of adult morbidity and mortality in developed nations. As a result, predisposition for CVD is increasingly important to understand. Ankyrins are intracellular proteins required for the maintenance of membrane domains. Canonical ankyrin-G (AnkG) has been shown to be vital for normal cardiac function, specifically cardiac excitability,  Cardiovascular disease (CVD) characterized by heart failure (HF) and arrhythmia currently impacts >6 million people in the United States each year, with anticipation of growth to 9 million by 2030 , 2. It iv channel (Nav1.5) targeting, and in concert with βIV spectrin and CaMKII, for Nav1.5 membrane regulation  has been investigated first in neurologic tissue and later in the heart. The function of canonical AnkG (220 kDa) in neurologic tissue is well described, where it is important for regulating membrane and intracellular protein complexes necessary for normal neurologic function , 4 In thnel Nav1. targetingulation , 7, 8, 9ANK2 and ANK3 encode in canonical and “giant” isoforms. In neurologic tissue, “giant” AnkG (∼400 kDa) in particular has been studied and found to function in conductance and electrical signaling. At the axonal initial segment (AIS), giant AnkG operates as a membrane-associated adaptor protein that has been implicated in organizing voltage-gated sodium channels (Nav), βIV spectrin, KCNQ2/3, and neurofascin  and adult-aged animals (22 weeks old). To define the role of giant AnkG in the heart, we created a new model of giant AnkG deficiency in postnatal myocytes (αMHC-Cre). Mice deficient in giant AnkG display a dilated left ventricle, gross posterior wall thinning, and systolic dysfunction, characteristic of a dilated cardiomyopathy (DCM) phenotype in the neonatal period. In addition, these mice exhibit electrical dysfunction including ventricular arrhythmia and high-degree (third-degree and complete) heart block. Notably, unlike canonical AnkG, these phenotypes appear to be independent of NaANK3 in human, Ank2 in mice, 190 kD, Fig. 1A) is essential for normal ion channel targeting in neurologic tissue  have been identified in neurologic tissue, where they function in the assembly of the AIS and nodes of Ranvier  mice and cKO mice in the neonatal period (day of life 7) and adulthood (22 weeks of age).To both directly investigate the expression of giant AnkG in the heart (requires robust negative control) and test a potential escribed . The MHCescribed . We studC–F) consistent with eccentric hypertrophy. Notably, immunoblots illustrate that giant AnkG is easily detected in heart lysate of mice at 1 week of age . This expression in young hearts is significantly reduced in cKO; absolute cKO heart loss of giant AnkG is likely due to incomplete activity of the αMHC promoter during this time of development and/or giant AnkG expression in nonmyocytes. Further, the expression of giant AnkG was significantly abolished in the cKO mice compared with the floxed littermates at 4 weeks of age, while canonical AnkG protein expression did not vary between the different genotypes (I and J). Relative expression of the large isoform (qPCR) confirmed near absence of giant AnkG in the 4-week cKO animals  and myocardial thinning  in cKO animals . Unlike control mice, cKO animals demonstrated increased heart weight when normalized to tibial length , indicative of the eccentric remodeling expected in DCM (D). Finally, we observed myocardial fibrosis in cKO animals, consistent with aberrant remodeling (E–G). Taken together, these findings provide evidence for an eccentric hypertrophy phenotype such as DCM, a disease process that requires fibrosis and abnormal remodeling, as necessary substrate for the development of both structural and electrical dysfunction.We examined gross and histologic specimens to evaluate myocardial structure and function in giant AnkG cKO and control mice (age- (22 weeks) and sex-matched). We observed mild dilatation of the LV chamber . Both structure and functional changes were evident at early ages (12 weeks) and did not appreciably change through 32 weeks of age . However, by 54 weeks of age, LV dilatation and dysfunction significantly worsened in the cKO animals   to determine if there was further impact upon heart structure and late cardiovascular function (54 weeks). Similar to the pathologic data, we observed thinning . In conc < 0.01) . Thus, gv1.5 function and recruitment of CaMKIIδ to the intercalated disc. Canonical AnkG loss leads to CaMKII-dependent regulation of INa,L resulting in both conduction defects and arrhythmia : 521 ± 41 versus 539 ± 54 bpm, p = 0.45, PR Interval: 33 ± 0.8 versus 33 ± 1.0 ms, p = 0.85, QTc: 59 ± 3 versus 63 ± 2 ms, p = 0.26) . However, with catecholamine exposure, high-degree atrioventricular block was observed more frequently in cKO, versus control mice  (C and E). The same animals displayed significantly more ventricular arrhythmia. However, this only occurred in response to epinephrine , as there were no ventricular arrhythmic events in either control or cKO animals in response to the beta-agonist isoproterenol (B and D).We performed conscious electrocardiographic monitoring at rest on both cKO and control animals. Electrocardiographic tracings revealed no significant differences in resting ECG parameters (cKO  = 0.26) , A, F–H. 0.0001) , C and Eoterenol , B and DA) or amplitude (B) at either pacing interval. Similarly, there was no difference in the maximum rate of voltage change (dV/dT) between control and cKO ventricular myocytes (C). We next measured myocyte electrical activity in response to specific protocols. During 0.5 Hz pacing, early afterdepolarizations (EADs) were observed in AnkG cKO myocytes and when present, EADs occasionally gave rise to a spontaneous action potential (D). Next, cardiac myocytes were subject to a S1-S5 protocol where they were paced at successively higher frequencies ranging from 0.5, 1.0, 2.0, 5.0, and 10.0 Hz for a period of 1 s at each frequency. After cessation pacing, giant AnkG cKO myocytes displayed runs of delayed afterdepolarizations (DADs); these were not observed in control myocytes (E). The presence of resting EADs and post-HFP DADs suggests that giant AnkG-deficient myocytes exhibit susceptibility to increased automaticity, likely impacted by a functional role that giant AnkG plays in myocyte stabilization required for normal electrical conduction and cardiac function.To assess the time-dependent transmembrane potential for clues to the cause of electrical dysfunction in giant AnkG cKO animals, (22 weeks), we paced ventricular myocytes at 0.5 and 1.0 Hz and measured action potential duration and amplitude. There was no significant difference in action potential duration A or amplmplitude B at eithmyocytes C. We nexotential D. Next, myocytes E. The prv channel , and time-dependent recovery of INa (A–D). The action potential and INa findings support that giant AnkG may at least in part function independent of the primary voltage-gated Nav channel in the heart.Canonical AnkG functions in concert with the voltage-gated Na channel . Cardiac channel . To detey of INa , A–D. ThINa, suggest that giant AnkG may have additional unique functions in the heart. We hypothesized that this could be influenced by calcium dysfunction and therefore analyzed sarcomere length and performed real-time cardiomyocyte functional and calcium studies in neonatal (1 week old) control and cKO animals (n = 4 each group). In the cKO group, myocyte sarcomeres were shorter at baseline  and demonstrated increased percent peak shortening  when stimulated, albeit with no change in relaxation time (A–C), consistent with increases in cytosolic Ca2+. To further evaluate potential etiologies for abnormal depolarization and slower Ca2+ reuptake in the cKO group, we performed computer simulations to predict mechanisms for DADs in the absence of overt changes to baseline action potential. We used the Bondarenko model of the murine ventricular action potential  in the sarcoplasmic reticulum (SR) reaches 11.5 (mM). The generated regression coefficient estimates predict that Ca2+ leak, release, and reuptake into SR have a marked effect on peak CSQN with a minimal effect on other action potential properties (D). To further test this hypothesis, these three scaling factors were then incrementally increased over a wide range (0.2 steps starting at 0.1–2.9) and evaluated for their effect on peak CSQN, action potential amplitude, APD75, and max derivative. Ca2+ leak and release from the SR had an inversely proportional relationship with peak CSQN, while increasing the maximal Ca2+ uptake into SR showed an increase in peak CSQN. (E) Action potential amplitude, APD75, and max derivative showed minimal responses to the dramatic changes in SR Ca2+ factors (F–H). Together, these data indicate that in the heart, giant AnkG functions independent from the primary voltage-gated Nav channel, with evidence for novel roles in Ca2+ metabolism.These data, in combination with the AP recordings, including display of abnormal depolarization (both DADs and EADs) in the setting of normal action potential duration and ion time , A–C, cootential , in whicoperties D. To furak CSQN. E Action  factors , F–H. ToINa. In the heart, there were 12,124 total transcripts analyzed. Of these, there were 113 coding transcripts that were either significantly either increased  or decreased  in cKO animals irrespective of sex and seven coding transcripts that were significantly increased in both male and female cKO animals  (A). Of the seven genes upregulated, four were found to be potentially relevant in CVD and included: Epsin 3 (EPN3) (GDF15), neuron-derived neurotrophic factor (NDNF) (PTPRN)  and female  animals. In part, we aimed to determine if unique pathways might explain the phenotype we observed, in the presence of normal action potential and animals) A. Of the3 (EPN3) , growth r (NDNF) , and pro (PTPRN) .Figure 8p < 0.05) in both male and female cKO animals  is affected by increases in Ca2+ uptake or alternatively decreases in Ca2+ release. This may suggest that giant AnkG in the heart has functional roles in maintaining physiologic levels of Ca2+. This may occur through traditional effects via the L-type Ca2+ channel or ryanodine receptor or from alteration in reuptake via SERCA or NCX  (Ank3 e37f/f floxed age- and sex-matched littermates were used as controls. Floxed wild-type (control) and giant AnkG cKO mice were studied in neonates (day of life 7) and both young (starting at 10 weeks) and aged (54 weeks) cohorts. All animal studies and surgeries were performed in accordance with the American Physiological Society Guiding Principles for Research Involving Animals and Human Beings and approved by the Institutional Animal Care and Use Committee at The Ohio State University.Mice with flanked exon 37 by loxP sites were crossed with αMHC-Cre knock in mice to produce animals specifically deficient in giant ankyrin-G exon 37 in the heart (kG cKO”) . Ank3 e33, 12 mM NaCl, 1% SDS, 1% BME)) were loaded into 3 to 8% precast gels (NuPAGE) and transferred to Tris-Acetate protein membranes, which were blocked for >1 h at room temperature in 5% milk and incubated in primary antibody overnight at 4 °C. We used the giant ankyrin-G antibody at a concentration of 1:1000 (Gift Paul Jenkins Laboratory) and Goat-anti-rabbit 1:10,000 (Jackson Laboratories) for secondary antibody  22-week murine whole-heart lysates . Continuous 2-h telemetry monitored recordings were performed to characterize the average resting HR 2 weeks after telemeter implantation. All ECG and telemetry tracings were analyzed with Labchart, ADinstruments (v.8).2 and 5% CO2. Anesthesia was maintained by administration of oxygen and approximately 1.25% isoflurane during the whole imaging procedure. HR was monitored throughout imaging and recordings taken at a HR less than 400bpm were excluded from the analysis. We obtained parasternal long-axis images inclusive of two-dimensional loops and freeze-frame end-diastolic images to measure septal thickness, end-diastolic LV cavity dimension , and posterior wall thickness . Additionally, we collected parasternal short-axis images at the level of the papillary muscles, inclusive of M-mode imaging, to determine the LV anterior wall dimension (LVAW), posterior wall (LVPW), and LVID in both systole and diastole. Functional data was derived from both parasternal long-axis two-dimensional real-time 3-beat loops and M-mode measurements, including fractional shortening (FS%) and ejection fraction (EF%).From 10 to 54 weeks of age, littermate and cKO animals underwent bimonthly noninvasive transthoracic echocardiography (GE Logiq e) with the L10-22 (mHz) transducer. Mice were anesthetized using 2.5% isoflurane in 95% OINa) were recorded at room temperature (20–22 °C) with pipette resistances of <2.0 MΩ when filled with pipette filling solution containing (in mM) five NaCl, 135 CsF (135), ten EGTA, five MgATP, and five HEPES at pH 7.2. The extracellular bathing solution contained (in mM) 5 NaCl, 1 MgCl2, 1.8 CaCl2, and 0.1 CdCl2. To assess the INa density, cells were held at −160 mV and stepped to various test potentials from −100 to 10 mV in 5-mV increments, with 200-ms duration pulses and 2800-ms interpulse intervals. Peak INa was assessed by an algorithm that calculates peak inward signal. As a result, any outward INa returns a zero value. Voltage dependence of inactivation was assessed by holding the cells from −140 to −40 mV for 300 ms, followed by a 30-ms test pulse to −40 mV; interpulse interval was 2700 ms. Recovery from inactivation was studied by holding cells at −160 mV and applying two 20-ms test pulses  to −45 mV, separated by increasing increments of 1 ms to a maximum S1-S2 interval of 50 ms. The S1-S1 interval was kept constant at 2000 ms.Whole-cell voltage- and current-clamp recordings were carried out in isolated adult mouse ventricular myocytes using standard patch-clamp techniques. Recordings were done using the Axopatch 200B amplifier (Molecular Devices). Sodium currents  148 NaCl, 0.4 NaH2PO4, one MgCl2, 5.5 glucose, 5.4 KCl, one CaCl2, 15 HEPES, and one EGTA, adjusted to pH 7.2 with NaOH. Appropriate corrections for access resistance compensation, as well as for leak and capacitive current subtractions, were made.Action potentials were elicited using square wave pulses , generated by a Axopatch 200B amplifier (Molecular Devices), and recorded at room temp with pipette solution containing (in mM) one MgCl2 was used to increase the calcium concentration stepwise with four additions in 10 min intervals, before spinning and resuspension in culture media for a final concentration of 1 mM CaCl2. Cardiac myocyte function was recorded using an Ion-Optix system using real-time sarcomere analysis. For intracellular calcium measurements, cells were loaded with 1 μg/ml Fura 2-AM for 20 min in culture media, then placed in culture media without FURA for 20 min before stimulation. Cells were stimulated in contractile buffer at 2 mM CaCl2 concentration using the Ion-Optix MyoPacer Field Stimulator equipped with a STIM-AT electrode (Cell Microcontrols) in stimulation chambers at 1 Hz for data collection. Collected cell data were then averaged and plotted using Graphpad Prism.Myocytes were isolated using a Langendorff-free method published in detail elsewhere, with slight modifications for 1-week-old mice . In brie2+-bound calsequestrin (peak CSQN) in the sarcoplasmic reticulum (SR) reaches 11.5 (mM). Therefore, peak CSQN was used as a surrogate for DADs throughout these studies. Partial least-squares regression analysis was performed, as previously described (75), and max derivative. A total of 200 single-cell simulations were performed with random perturbation  of scaling parameters for the following ion fluxes: L-type Ca2+ current (gCa(L)), Na+/Ca2+ exchanger (gNaCa), Na+/K+ ATPase (gNak), sarcolemmal Ca2+ pump (gCa(P)), Ca2+ leak flux from the SR (gleak), Ca2+ release from SR (grel), Ca2+ translocation from network to junctional SR (gtr), Ca2+ flux to troponin (gtrpn), Ca2+ uptake into SR (gup), and Ca2+ flux from the subspace volume to bulk myoplasm (gxfer). Following regression analysis, simulations were performed where a subset of the parameters were incrementally increased (0.2 steps starting at 0.1–2.9) and evaluated for their effect on peak CSQN, action potential amplitude, APD75, and max derivative. The cell model was paced using a stimulus of −60 mV with a 0.5 ms duration for 30,000 ms at 0.5 Hz, followed by an increase in pacing frequency every 10,000 ms to 1,2,5 and finally 10 Hz, after which pacing was abruptly stopped and the cell was allowed to return to baseline for 50,000 ms.Computer simulations were performed to predict mechanism for delayed after depolarizations (DADs) in the absence of overt changes to baseline action potential. The Bondarenko murine ventricular myocyte cell model  in the Lescribed , to deteWe collected whole heart specimens from cKO and floxed-control adult animals at 22 weeks of age to include: three male cKO, three male control, three female cKO, and three female control samples. Gene microarray analysis was conducted as previously described . Total RHPRT levels were used as a normalization control.Real-time PCR was performed as previously described . Brieflyt-tests (two-tailed) when evaluating single comparisons and with one-way analysis of variance in the case of multiple comparisons. Categorical variables were evaluated with Chi-Square and Shapiro–Wilks criteria were used to assess the distribution of data and whether or not nonparametric testing was indicated. An α level of <0.05 was considered statistically significant and all analyses were performed using JMP Pro, Version 12.2.0. SAS Institute Inc, 2015.Continuous variables were analyzed with unpaired Student’s All data is contained within the article and supporting information document.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Cerebral ventriculomegaly is an abnormal feature characteristic of myotonic dystrophy type 1 (DM1). This retrospective study investigated the morphologic changes accompanied by ventriculomegaly in DM1 on brain MRI.One hundred and twelve adult patients with DM1 and 50 sex- and age-matched controls were assessed. The imaging characteristics for evaluations included the z-Evans Index , callosal angle (CA), enlarged perivascular spaces in the centrum semiovale (CS-EPVS), temporo-polar white matter lesion (WML) on 3D fluid-attenuated inversion recovery (FLAIR), disproportionately enlarged subarachnoid-space hydrocephalus (DESH), and pathological brain atrophy. The “z-Evans Index” was defined as the maximum z-axial length of the frontal horns to the maximum cranial z-axial length. To determine the imaging characteristics and genetic information (CTG repeat numbers) that were associated with the z-Evans Index, we used binominal logistic regression analyses.The z-Evans Index was significantly larger in the patients than in the controls . The z-Evans Index was independently associated with the callosal angle (p < 0.01) and pathological brain atrophy (p < 0.01) but not with age, gender, CTG repeat numbers, or CS-EPVS. Of the 34 patients older than 49 years, 7 (20.6%) were considered to have DESH.Our MRI study revealed a normal pressure hydrocephalus (NPH)-like appearance as a morphologic finding accompanied by ventriculomegaly in DM1 that tends to occur in elderly patients. Myotonic dystrophy type 1 (DM1) is the most common disease that can cause muscle weakness and atrophy among adults . Neuropahttps://www.ninds.nih.gov/) reports that, in patients with suspected NPH, a variety of tests, including brain CT and/or MRI, a spinal tap or lumbar catheter, intracranial pressure monitoring, and neuropsychological tests are helpful to diagnose NPH and rule out other conditions. The study of NPH on Neurologic Improvement (SINPHONI) described important MRI features for the NPH diagnosis [Normal pressure hydrocephalus (NPH) is a syndrome of gait dysfunction and enlarged cerebral ventricles in the absence of another cause. It is frequently accompanied by dementia and bladder detrusor overactivity , 9. The iagnosis  as high-Ventriculomegaly is a characteristic MRI finding in DM1 . HoweverThe study was conducted in accordance with the Helsinki Declaration and Good Clinical Practice. The ethics review committees of Aomori Hospital approved the study . The ethics review committees approved this retrospective study, and the need to obtain informed consent from the patients was waived.We routinely perform screening brain MRI studies to assess DM1 patients. One radiologist (FN with 4 years of neuroradiology experience) reviewed a clinical database between April 2018 and May 2020 and selected adult 112 patients who had been diagnosed with DM1 based on a clinical examination, electromyography, and underlying genetic defects , the conventional MRI results were normal in all controls.. The imaging parameters (repetition time ms/echo time ms/inversion time/NEX/echo train length/matrix/field of view/imaging time) were 4,000/85/NA/2/9/226 × 384/23 cm/2 min and 12 s for 2D T2WI and 9,000/108/2,500/1/14/208 × 320/22 cm/3 min and 18 s for 2D FLAIR imaging, respectively. The 2D T2WI and 2D FLAIR images were acquired at a section thickness of 5 mm, an intersection gap of 1.0 mm. For 3D FLAIR, the following imaging parameters were used: 5,000/333/1800/1/235/248 × 256/24 cm/1.0 mm/2/5 min and 17 s .All studies were performed using a 1.5-T scanner  equipped with gradients with maximal gradient strength, 40 mTm − 1 and maximal slew rate, 200 mTm − 1/ms using a 20-channel head coil. All patients and controls underwent brain MRI according to our standard protocol, including axial T2-weighted imaging (T2WI), two-dimensional (2D) coronal fluid-attenuated inversion recovery (FLAIR) imaging, and three-dimensional (2D) sagittal FLAIR. For 3D FLAIR (3D fast spoiled gradient-echo imaging), we used three cross-section images  reconstructed from the images obtained on the sagittal planeAccording to the previous method , 15, quaIn the current study, on the coronal and sagittal FLAIR images, the radiologist measured the z-Evans Index, an alternative to the Evans Index. Yamada et al. found that, by using quantitative volumetric analyses, the volume expansion of the bilateral ventricles, especially at the frontal horns, was toward the z-axial direction, rather than the x-axial direction, in patients with NPH . They thTwo certified neuroradiologists  who did not take part in the image processing independently reviewed the MRI scans and categorized the findings  by consensus.The width of the Sylvian fissure was assessed on transverse and coronal sections. We used the following visual rating scale: 1, narrowed or normal; 2, mildly dilated ; and 3, severely dilated  on the coronal images  According to the previous reports , on T2WIFor the analyses, the age, CTG repeat numbers, z-Evans Index, and callosal angle were treated as continuous variables, while the sex, high convexity tightness, Sylvian fissure dilation, CS-EPVS, and temporo-polar WMLs were treated as categorical variables.A z-Evans Index of > 0.3 was arbitrarily defined as ventriculomegaly. For the subjective scale of high-convexity tightness and Sylvian fissure dilation, the “2 or 3” were defined as pathologic findings. Thus, high-convexity tightness (2 or 3) combined with Sylvian fissure dilation (2 or 3) accompanied by ventriculomegaly was defined as DESH , genetic information (CTG repeat numbers), and the patient age and gender that were associated with the z-Evans Index , we conducted the binominal logistic regression analyses. The analyses were performed by excluding the finding of “Sylvian fissure dilation”, “high-convexity tightness”, and “temporo-polar WML”, and because these findings are obvious confounding factors.P values < 0.05 were considered indicate statistical significance. All of the statistical analyses were performed using the R software program [We arbitrarily divided the DM1 patients into three age groups: ≤ 39, 40–49, and ≥ 50 years old  by considering our sample size and distribution. Because the continuous variables in this study showed non-normal distribution, we compared the clinical and imaging characteristics among the three age groups using the Steel–Dwass test for continuous variables and Ryan's procedure for categorical variables. All analyses,  program .Table Table Figure Table In the present study, we evaluated DM1 patients using the newly proposed z-Evans Index. Although the original Evans Index has been adopted as a criterion for discriminating the tap-test response , the z-E) (136.9 ± 8.2) [It is important to note that an increased z-Evans Index  was independently associated with the callosal angle. The callosal angle is a specific quantitative imaging biomarker in the diagnosis of NPH, allowing improved differentiation from other neurodegenerative disorders with ventriculomegaly, prediction of surgical shunt responsiveness, and sensitivity to ventricular changes following surgical shunt –27. Anot9 ± 8.2) . The mea) 16.9 ± 8.29 ± 8.2) . These r9 ± 8.2) . Three o9 ± 8.2) , the exaThe etiology and pathophysiology of the NPH-like appearance in DM1 are uncertain. One possible mechanism suspected by previous investigators involves widespread cell membrane defects caused by genetic abnormalities in DM1 , 31, leaFor diagnosis of NPH, the clinical evaluation still often relies on the clinical presentation and morphologic findings on neuroimaging only, without assessment of the intracranial pressure, nor the actual CSF manometric profile. Therefore, the exact pathophysiological mechanism of NPH remains unclear. In previous review article, Bret et al. proposed a more relevant nosographic designation for the NPH syndrome by renaming it "chronic hydrocephalus" without reference to age and CSF pressure . They alCostanzo et al. showed a significant negative correlation between the degree of EPVS and disease duration and suggested that EPVS might progressively disappear during the disease course . Our resIn DM1, the classic disease range of CTG repeat numbers is 50–4000, with repeat sizes of 50–80 associated with mild clinical phenotypes, while large repeat expansions of up to 4000 are often found in severe, mostly congenital forms of the disorder . Brain aThis study was limited by its retrospective design. Thus, neuropsychological tests were not performed in many cases, as neuropsychological deficits in cases of myotonic dystrophy can escape commonly applied neuropsychological test batteries for as-yet-unclear reasons. Moreover, no patients undergo lumbar puncture and were clinically diagnosed as having NPH. Second, our study included many qualitative analyses with subjective criteria. We arbitrarily defined ventriculomegaly as a z-Evans index of > 0.3 because an NPH-like appearance in DM1 patients is a relatively new finding.Our MRI study revealed an NPH-like appearance as a morphologic finding accompanied by ventriculomegaly in DM1 that tends to occur in elderly patients. Furthermore, because cognitive deficits are found in a high percentage of DM1 patients at the late stage, correlation analyses between neuropsychological deficits and the NPH-like appearances might be an interesting topic for a further study involving more patients.