Vibrio cholerae, infected by ICP1, a phage ubiquitous in clinical samples. This work identifies the ICP1-encoded holin, teaA, and antiholin, arrA, that mediate lysis inhibition. Further, we show that an MGE, the defensive phage satellite PLE, collapses lysis inhibition. Through lysis inhibition disruption a conserved PLE protein, LidI, is sufficient to limit the phage produced from infection, bottlenecking ICP1. These studies link a novel incarnation of the classic lysis inhibition phenomenon with conserved defensive function of a phage satellite in a disease context, highlighting the importance of lysis timing during infection and parasitization.Bacteria, bacteriophages that prey upon them, and mobile genetic elements (MGEs) compete in dynamic environments, evolving strategies to sense the milieu. The first discovered environmental sensing by phages, lysis inhibition, has only been characterized and studied in the limited context of T-even coliphages. Here, we discover lysis inhibition in the etiological agent of the diarrheal disease cholera,  Bacteriophages, or phages for short, are viruses that infect bacteria, take over the molecular machinery inside the bacterial cells and use it to make more copies of themselves. The bacteriophages then break open, or “lyse”, the bacterial cell, releasing the viral copies into the environment, ready to infect more bacteria nearby.Vibrio cholerae – which causes cholera – and its bacteriophage called ICP1. This analysis revealed that the ICP1 phage uses a gene called teaA as the first step in the lysis of bacterial cells. The ICP1 phage can also delay that lysis with a second gene called arrA. This “lysis inhibition” gives the bacteriophages more time to make copies of themselves inside the bacterium, so even more are released when the cell finally breaks open.Hays and Seed set out to understand how the timing of lysis can impact the bacteriophage, using the bacterium Vibrio cholerae cells can defend themselves against lysis inhibition using a single gene called lidI. This gene is part of a system that defends against bacteriophage attack called the PLE, which consists of several genes of previously unknown function. Hays and Seed saw that the lidI gene disrupts lysis inhibition, speeding up the bursting of infected bacterial cells, which in turn decreases the number of bacteriophages produced from each infected cell.Hays and Seed also found that the Escherichia coli. Now that researchers know that ICP1 bacteriophages also delay lysis in Vibrio cholerae, this might lead to more studies exploring this process in samples from cholera patients. Further studies could test to see if the phenomenon of lysis inhibition may also exist in yet more bacterial species.Lysis inhibition had previously only been observed in the bacterium  Escherichia coli’s T1 through T7 phages were widely accepted as model systems  found integrated in the chromosome of clinical V. cholerae isolates  which decrease but do not eliminate the production of progeny virions, PLE completely abolishes ICP1 production – a balancing act that likely requires the exploitation of select products at exact times during the 20 minutes before PLE-mediated accelerated cell lysis.Phages are abundant in natural environments including marine ecosystems  and gut  cholera . In bothutbreaks  leading utbreaks . The preutbreaks  that is ngladesh . Added iisolates . Previouoduction . Upon inoduction , replicaoduction , and accoduction . As no iming PLE . While tlication  and int,excision ). From tV. cholerae harboring PLE led us to investigate the prolonged infection of ICP1 in strains without PLE where we discovered that ICP1 exhibits lysis inhibition. In this work we report the first mechanistic characterization of archetypal LIN outside of E. coli and we reveal ICP1 LIN mechanisms in V. cholerae by identifying previously uncharacterized ICP1 genes with holin and antiholin activity, termed teaA and arrA respectively. Subsequently, we discovered a single PLE-encoded gene we call lidI for lysis inhibition disruption that is sufficient to collapse ICP1-mediated lysis inhibition. All PLEs encode LidI, highlighting a conserved strategy PLEs may use to antagonize an aspect of the phage lifecycle not previously known to be targeted by parasitic satellites. While we cannot be sure of LidI function in the context of the PLE, it alone is sufficient to decrease the yield of ICP1 from infected V. cholerae and impose an evolutionary bottleneck on phage populations.The accelerated cell lysis program in V. cholerae , ICP1 completes virion production within 20–25 minutes in PLE (-) cholerae . This ticholerae . This inabilized . Such lyabilized . These sE. coli cultures because cells stay intact instead of lysing , let phage adsorb for four minutes, then superinfected the culture with ICP1 . As expected of a phage exhibiting LIN, the culture optical density was stabilized, eliminating the early lysis event , collapses the proton motive force and subsequently disrupts LIN by T-even phages, causing rapid lysis of infected E. coli (V. cholerae infected at a high MOI (MOI = 5) to 2,4-dinitrophenol  V. cholerae in the absence of phage and probed its ability to collapse the proton motive force and be triggered by an energy poison. We measured proton motive force using 3,3’-diethloxacarbocyanine iodide (DiOC2(3)), a fluorescent green membrane stain that forms red fluorescent aggregates in the presence of intact proton motive force  did not  V. cholerae containing a spacer targeting arrA. We observed a mixture of edge phenotypes including clear-edged plaques  hindering tests of infection at high multiplicities of infection. Instead, we infected PLE (-) V. cholerae with ΔarrA ICP1, waited for approximately two cycles of infection to complete, and then, once lysis began, we observed a rapid crash in optical density   , ICP1 innfection . This acPLE 4/5 does not have homologs in PLEs 1 or 2, suggesting it is not a conserved player mediating accelerated lysis. Consequently, we focused on ORF26PLE 4/5. Although no homologs were immediately obvious, the synteny between PLEs suggested the presence of previously unannotated open reading frames in PLEs 1, 2, and 3 , which are homologous to ORF26PLE 4/5 and contain transmembrane domains. We subsequently named these genes lidI and the homologs cluster into two groups: PLE 1lidI and PLE 2lidI encode for a 66 amino acid long protein, while PLE 3lidI, PLE 4lidI and PLE 5lidI encode larger proteins at 121 amino acids  contain two ORFs with predicted transmembrane domains: ORF2 and ORF26. ORF2no acids . These OlidI genes, we endogenously tagged LidIPLE 1 and evaluated expression during ICP1 infection  V. cholerae. During infection with ICP1, LidIPLE 1 was sufficient to recapitulate the PLE-mediated accelerated lysis phenotype  V. cholerae does not change the efficiency of plaquing (EOP) by ICP1, an experiment that probes the number of successful initial infections at a low multiplicity of infection . Induction of PLE 1lidI at the time of infection or 20 minutes before infection resulted in decreased phage yield by one or two orders of magnitude, respectively, in comparison to strains with the empty vector control  V. cholerae constrains ICP1 evolution. To test if LidIPLE 1-mediated accelerated lysis is enough to impact diversity through the acquisition of random mutations in the progeny phage population, we exposed PLE (-) V. cholerae with and without PLE 1lidI to ICP1 (MOI = 0.1), collected the population of progeny phage, and looked for plaque formation on PLE (-) V. cholerae encoding a Type I-E CRISPR-Cas system  (V. cholerae strains (MOI = 0.01) with and without LidIPLE 1. After lysis, progeny phage were tested for their ability to plaque on PLE 1 V. cholerae, which is only possible if homologous recombination between the two variants restored a functional CRISPR-Cas system able to target PLE 1 (PLE 1 decreased the number of progeny phages per infection that recombined to reconstitute the CRISPR-Cas system and overcome PLE (While exposure to as ICP1)  and the et PLE 1 ; an expacome PLE . AlthougV. cholerae, in response to ICP1, the predominant phage isolated from clinical samples, and identify the relevant ICP1-encoded lysis machinery: the holin, TeaA, and the antiholin, ArrA. Consistent with T-even induced LIN, ICP1 LIN results in fuzzy-edged plaques and delayed lysis sensitive to cellular proton motive force. This work subsequently reveals that the disruption of LIN is part of PLE’s anti-phage repertoire. A single open reading frame, lidI, which is conserved through all five PLEs spanning the last 70 years, is sufficient to disrupt LIN and limit progeny phage populations when expressed outside its native context in PLE (-) V. cholerae. These two opposing forces, ICP1 LIN and accelerated lysis by PLE through LIN disruption (which our data shows LidI is capable of doing in isolation and yet other undiscovered PLE-encoded mechanisms redundantly accomplish), act in the midst of the ongoing evolutionary arms race between V. cholerae and its parasites.Here, we observe a previously unknown lysis inhibition (LIN) state in the globally relevant pathogen, V. cholerae reinforces lysis inhibition as a relevant form of environmental sensing and highlights the importance of phages tuning their infection parameters depending on host availability in the environment. Following ingestion, V. cholerae colonizes and blooms in the small intestine before being shed in stool, further contaminating aquatic reservoirs and promoting subsequent ingestion and infection cycles. If large numbers of ICP1 are co-ingested with small numbers of V. cholerae, it would be theoretically beneficial for ICP1 to demonstrate LIN in infected cells, bide time inside the cell while making more virions, and lyse in the small intestine after uninfected V. cholerae have had time to replicate, providing ample hosts for subsequent rounds of phage predation. This is consistent with outbreak models in which phage become more abundant in the environment towards the end of an epidemic and there is phage amplification within cholera patients  can function in a phage-concentration dependent manner , small apatients . Similarronments , and thefectious .E. coli systems , kanamycin (75 μg/mL), ampicillin (100 μg/mL), and streptomycin (100 μg/mL) when appropriate. Cell densities were measured at OD600 in tubes  and in 96-well plates ). To induce chromosomal constructs in both liquid and top agar, plasmid constructs in top agar, and the plasmid Ptac-arrA construct for complementation, 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and 1.5 mM theophylline were added to cultures while the remaining plasmid constructs were induced with 125 μM IPTG and 187.5 μM theophylline in liquid cultures. Plasmid constructs were used for all experiments other than phage infection yield experiments which utilized chromosomal constructs to decrease leaky expression in uninduced strains.Bacteria were propagated at 37°C via streaking from frozen glycerol stocks on solid LB agar plates and growth in Miller LB (Fisher Bioreagents) with aeration. Media was supplemented with chloramphenicol (2.5 μg/mL for V. cholerae hosts and prepped via polyethylene glycerol precipitation or concentration and media exchange on Amicon Ultra – 15 (Millipore) centrifugal filters  was determined by calculating the number of plaque forming units and varying that with the number of colony forming units of V. cholerae at a given optical density. This does not take into account virions that adsorb but do not successfully form plaques. Consequently, all reported MOIs do not address multiplicity of adsorption which could vary and impact initial changes in optical densities during experiments. Mechanical lysis of cultures infected with phage was accomplished by mixing chloroform into cultures then letting cultures stand at room temperature for ten minutes before spinning at 5000 x g for 15 minutes at 4°C and removing the supernatant for further analysis.Bacteriophages were propagated on PLE (-)  filters . Stocks  filters . BrieflyV. cholerae were accomplished through natural transformation of linear DNA created via splicing by overlap extension PCR (V. cholerae engineered to include an inducible Type 1-E CRISPR-Cas system (CRISPR-Cas (+) V. cholerae). This system in the host strain was induced for 20 minutes before ICP1 infection for plaque assays on 0.5% LB top agar containing antibiotics to maintain the CRISPR array plasmid. Plaques were picked into STE , purified on the same host twice, and genomic DNA was prepped for PCRs with the DNeasy Blood and Tissue Kit (Qiagen). Phages were subjected to PCR of the targeted gene and subsequent Sanger sequencing. Clean knockouts were accomplished by adding a repair template of homologous sequence containing the desired deletion and flanking DNA to the plasmid containing the CRISPR array as previously described  plate reader every two minutes with one minute of shaking between each read while the machine incubated cultures at 37°C. Assays were interrupted for the addition of phage, inducers, 2,4-dinitrophenol , or ethanol (2 μL of 80% ethanol) during which samples were removed from the plate reader briefly before measurement was resumed. This enabled superinfection of cultures , addition of DNP in ethanol or ethanol alone to ICP1 MOI = 5 infected cultures 25 minutes post-infection, and DNP addition to induced cultures after 20 minuntes of growth with the inducers.V. cholerae were grown. The first was grown to an OD600-tube=0.15, inducer was added, and the culture was returned to the incubator; this culture served as the pre-induced culture. All cultures were grown for an additional 20 minutes to OD600-tube=0.3 at which point ICP1 MOI = 5 was added. Inducer was added to one tube at this time; this culture served as the culture induced at time zero. Tubes were returned to the incubator for 5 minutes for phage adsorption. Cultures were spun at 5000 x g for 3 minutes to pellet cells. Unadsorbed phage was aspirated off and cells were washed once with 1 mL of prewarmed LB with or without inducer. Cells were spun again and resuspended in media with and without inducer at which point OD600-tube was determined. Cultures (150 μL) were moved to 96-well plates with one well designated to the pre-induced culture, one well devoted to the culture induced at time zero, and three wells filled with uninduced culture. The plate was returned to the 37°C incubator to shake at 230 RPM. Inducer was added to two wells of the uninduced cultures 20 and 40 minutes post-infection respectively, leaving one uninduced control. The experiment was ended with mechanical lysis of cultures and subsequent quantification of phage titers.For each strain, three 2 mL cultures of V. cholerae containing the specified plasmids were grown to OD600-tube=0.2. Inducers (125 μM IPTG and 187.5 μM theophylline) were added and cells were grown at 37°C with aeration for 30 minutes before 0.5 mL were pelleted at 5000 x g for 3 minutes and resuspended in 0.1 mL of phosphate buffered saline  containing 20 μM 3,3’-diethloxacarbocyanine iodide (DiOC2(3); Sigma-Aldrich). Fluorescence measurements were completed in black 96-well half-volume plates (Corning) in the SpectraMax i3x (Molecular Devices) with 480(508) and 488(650) excitation(emission) wavelength settings.V. cholerae strains were grown to mid-log before plaquing assays. For EOP experiments, plasmid constructs were induced for 20 minutes prior to infecting and plating with antibiotic and inducer in the 0.5% LB top agar. For plaque edge analysis, plasmids were maintained with antibiotics but not induced prior to plating on 0.5% top agar containing antibiotics and inducer. Plates solidified at room temperature prior to incubation at 37°C. For spot plates, V. cholerae was mixed with 0.5% top agar prior to infection, vortexed, and poured onto an LB agar plate to solidify before 3 μL spots of phage dilutions were overlaid on the agar. Plates were allowed to dry prior to incubation at 37°C. To visualize plaques, plates were scanned on the EPSON Perfection V800 Dual Lens scanner.PLE 1 were grown to OD600-tube=0.2 then induced with 1 mM IPTG and 1.5 mM for 40 minutes before 0.5 mL samples were taken. To observe expression during infection, strains were grown to OD600-tube=0.3, infected with ICP1 MOI = 2 with 4 mL samples taken at the labeled timepoints. Samples were prepared and visualized as previously described , vortexed, and boiled for 10 minutes before being run on 4–12% Bis-Tris SDS gels (Bio-Rad Criterion XT). Gels were transferred via the Trans-Blot Turbo (Bio-Rad) and visualized with rabbit-α-FLAG  primary and goat-α-rabbit-HRP conjugated secondary antibodies on the ChemiDoc MP Imaging System (Bio-Rad).Plasmid empty vector (EV) and FLAG-LidIescribed . BrieflyV. cholerae harboring an empty vector control or LidIPLE 1 plasmid were grown to OD600-tube=0.15 before being induced and returned to the incubator to grow with aeration until OD600-tube=0.3. At this point 150 μL of culture were added to 96-well plates and infected with ICP1 MOI = 0.1. After lysis 90 minutes post-infection, any remaining cells were mechanically lysed and the resulting phage population was plaqued on CRISPR-Cas (+) V. cholerae as previously described (V. cholerae harboring CRISPR array plasmids were induced 20 minutes before phage populations were titered on each specified V. cholerae strain. EOPs were determined by dividing the number of PFU/mL on V. cholerae containing spacers by the PFU/mL on the CRISPR-Cas (+) V. cholerae that did not contain a spacer against the phage (denoted as spacer ‘none’).escribed . BrieflyV. cholerae and PLE 1 V. cholerae. Control infections with only one phage never formed plaques on PLE 1 V. cholerae. The proportion of phages that successfully recombined was determined by the dividing the PFU/mL of each phage population on PLE 1 V. cholerae divided by the PFU/mL on PLE (-) V. cholerae.Coinfection experiments were completed in the same manner as the CRISPR-Cas targeting of phage populations described above with minor alterations: instead of infection with wild type ICP1, cultures were coinfected in the plate with CRISPR*-Cas ICP1 and CRISPR-Cas* ICP1 each at an MOI = 0.01, observed for 200 minutes in the plate reader, and, after mechanical lysis, phage populations were plaqued on PLE (-) Transmembrane domains were predicted by conversion of all predicted open reading frames to amino acid sequence by CLC  before a In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:V. cholerae. Lysis inhibition, first discovered about 70 years ago, serves to increase phage burst size and protect phage progeny from adsorbing to cells that are already infected. Prior to this work, lysis inhibition had only been well-characterized in E. coli T-even phages, although it was also reported in coliphage N4. In these T4 phages, lysis inhibition is mediated by antiholins that inhibit the activity of phage holin proteins and slow progression towards cell lysis in the presence of high phage concentrations.In this work, Hays and Seed characterize a new phage lysis inhibition phenotype encoded by the ICP1 phage in the important human pathogen Decision letter after peer review:Vibrio cholerae phage exhibits lysis inhibition sensitive to disruption by a defensive phage satellite" for peer review at eLife. Your article has been evaluated by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Wendy Garrett as the Senior Editor.Thank you for sending your article entitled "Dominant V. cholerae. Lysis inhibition, first discovered about 70 years ago, serves to increase phage burst size and protect phage progeny from adsorbing to cells that are already infected. Prior to this work, lysis inhibition had only been well-characterized in E. coli T-even phages, although it was also reported in coliphage N4. In these T4 phages it is mediated by antiholins that inhibit the activity of phage holin proteins and slow progression towards cell lysis in the presence of high phage concentrations.In this work Hays and Seed characterize a new phage lysis inhibition phenotype encoded by the ICP1 element in the important human pathogen E. coli. Phage ICP1, a lytic phage that infects V. cholerae, is the predominant phage in cholera patient samples. The parasitic phage satellite PLE is integrated into the clinical V. cholerae isolates and it abolishes ICP1 phage production. When ICP1 infects, PLE is excised from the chromosome, replicates, and accelerates cell lysis; this results in release of PLE, and no infectious ICP1. One open question was how PLEs restrict ICP1, as no single PLE gene had been shown to be necessary for inhibition. Hays and Seed show that ICP1 inhibits lysis inhibition – they discover previously uncharacterized ICP1 genes with holin and antiholin activity (teaA and arrA), and they characterize a single PLE-encoded gene, lidI, which collapses ICP-mediated lysis inhibition. LidI is the first PLE-encoded ORF shown to negatively impact ICP1 phage yield. They show that all PLEs encode LidI, thereby revealing a conserved strategy to antagonize the phage lifecycle and inhibit progeny production.This work provides the first study of lysis inhibition outside of There are a number of technical questions that need to be clarified, after which the reviewers feel confident that the authors have shown that a genuine LIN state is induced in ICP1 infections and aborted by PLE.Major concernsteaA genes are associated with arrA genes. In cases where no ArrA homologue is identified, is there some other transmembrane-containing protein encoded next to teaA where arrA is expected to be?1) The bioinformatic analyses could be improved with a more systematic investigation of the TeaA and ArrA homologues. For example, the BLASTp hits for TeaA and ArrA are noted to be present "throughout marine phage and bacterial genomes". However, the figures appear to show that they are only found in Vibrio spp. The statement made in the paper makes it sound as though they are not restricted to Vibrio, and it should be made clear with respect to where these homologues are found. Also, an indication of how many homologues were found for each of the proteins and a table listing the hits (if this is a manageable number) with the associated ArrA homologues would be useful. It seems that not all – "the presence of ArrA homologues without TeaA homologues" – where is this data presented?PLE 1 – and + samples. This is approximately the same difference that is noted between the LidIPLE 1 – and + samples in each of the three CRISPR targeting samples . It's not clear to me how the conclusion that fewer phage progeny from LidIPLE 1V. cholerae were able to overcome targeting than progeny from strains without LidIPLE 1 (subsection “LidI lowers phage yield”). It seems that the difference between phages produced in +/- LidIPLE 1 strains remains constant in the absence of CRISPR targeting or in the presence of the three spacers. Why is the targeting so poor for the spacers, particularly in the case of spacer A, which shows less than 10-fold decrease in phage plaquing? How were the three spacers chosen? Do the authors always find such poor targeting with CRISPR spacers and ICP1? I also had difficulty following the line of reasoning presented at the very end of the Results, centered on the reconstitution of the CRISPR system and the impact on evolution in infected cells.2) There are some confusing issues that should be resolved in the CRISPR-targeting experiments presented in the last section of the Results. In the absence of a spacer, there is approximately 10-fold difference in pfu/mL produced by LidI3) There is some concern about the importance of both Figures 5 and 6. The authors have no real evidence that LidI is the LIN collapsing protein. It is an integral membrane protein with 2 TMDs and its overexpression will likely cause depolarization of the membrane and that would collapse LIN, just like DNP. Unfortunately, the lidI knockout has no phenotype. Regarding Figure 5, if one starts with low MOI, it takes longer to see LIN just because most cells are not infected. Recommend deleting these figures. Major concerns1) The bioinformatic analyses could be improved with a more systematic investigation of the TeaA and ArrA homologues. For example, the BLASTp hits for TeaA and ArrA are noted to be present "throughout marine phage and bacterial genomes". However, the figures appear to show that they are only found in Vibrio spp. The statement made in the paper makes it sound as though they are not restricted to Vibrio, and it should be made clear with respect to where these homologues are found. Also, an indication of how many homologues were found for each of the proteins and a table listing the hits (if this is a manageable number) with the associated ArrA homologues would be useful. It seems that not all teaA genes are associated with arrA genes. In cases where no ArrA homologue is identified, is there some other transmembrane-containing protein encoded next to teaA where arrA is expected to be?"the presence of ArrA homologues without TeaA homologues" – where is this data presented?The trees for ArrA and TeaA are in Figures 3E and Figure 3—figure supplement 1, respectively. Figure 3E shows the presence of ArrA homologs without TeaA homologs (squares without yellow dot). We have expanded the bioinformatics analyses on these homologs in the revised manuscript (available in Supplementary files 2 and 3). These newly added supplementary files include the GenBank Description and number of predicted transmembrane domains for each homolog as well as for the adjacent genes as per reviewer request.and ArrA as originally written). ArrA homologs are indeed limited to phages, specifically phages that infect the Vibrionaceae family (limited to members of the Vibrio and Entrovibrio genera) as we note in the text:We note that the homologs for TeaA are found throughout marine phage and bacterial genomes as shown in Figure 3—figure supplement 1. This figure shows that TeaA homologs are found in phages that infect Vibrionaceae (indicated by filled colored boxes) as well as in a number of bacteria  and multi-species metagenomic samples (signified in grey box outlines). We have clarified the statement to specify this is true for TeaA (and not for TeaA “ArrA yielded fewer homologs than TeaA (Supplementary file 3), however, using less stringent search parameters, we found that some organisms containing TeaA homologs also contain ArrA homologs though these were limited to vibriophages ”.We do not expect all TeaA homologs to be associated with ArrA homologs because holins are fully functional without antiholins present. We think what is more interesting is that we find antiholins (i.e. ArrA homologs) in organisms without a holin (TeaA) homolog because antiholins function to regulate holins. As such, we displayed this information as yellow circles on both trees and clarified this in the text:“This suggests that there are potential homologous lysis inhibition systems – complete with both holin and antiholin – present in phages other than ICP1. In contrast, the presence of ArrA homologs without TeaA homologs raises the question: what are antiholins doing on their own? Perhaps they have evolved functionality with holins divergent enough to no longer be considered homologous to TeaA under our search parameters, or they have been coopted for divergent functions much like holins .”PLE 1 – and + samples. This is approximately the same difference that is noted between the LidIPLE 1 – and + samples in each of the three CRISPR targeting samples . It's not clear to me how the conclusion that fewer phage progeny from LidIPLE 1V. cholerae were able to overcome targeting than progeny from strains without LidIPLE 1 (subsection “LidI lowers phage yield”).2) There are some confusing issues that should be resolved in the CRISPR-targeting experiments presented in the last section of the Results. In the absence of a spacer, there is approximately 10-fold difference in pfu/mL produced by LidIReviewers are correct that LidI decreases the PFU/mL produced and this finding is the major result of figure panels 6A and B and Figure 6—figure supplement 1D. These panels demonstrate that LidI expression decreases the yield of phage from infected cultures. This is confirmed again in the samples with and without spacers targeting the phage in Figure 6D.This effect is further quantified in Figure 6—figure supplement 4A where the efficiency of plaquing of LidI + strains in respect to LidI – strains is ~0.1 for all spacers and the control meaning that, though the absolute number of phages has been impacted, the frequency of escapes has not.7 more phages per mL can plaque on spacer A from a LidI– infection in comparison to a LidI+ infection.While the proportion of progeny phage that can overcome the spacer remains the same , the total number of phage that can overcome a spacer from a LidI + infection is less than the total number of phage that can overcome a spacer from a LidI– infection . For example, an average of ~6.13x10We have worked to make this clear in the text:PLE 1V. cholerae were able to overcome targeting than progeny from infections of strains without lidIPLE 1 . This defect is due to the LidI-mediated decrease in the population of phages as the frequency of phage escaping stays the same . Consequently, because less phage are produced from lidIPLE 1-expressing V. cholerae, an order of magnitude fewer phages can overcome the spacer in the population .”“For each spacer, fewer phage progeny from lidIPLE 1 strains remains constant in the absence of CRISPR targeting or in the presence of the three spacers.It seems that the difference between phages produced in +/- LidIThis is exactly the case and is shown conclusively Figure 6—figure supplement 4A. Our interpretation of these data is in agreement with these comments and we have expanded the text as stated above that indeed the frequency of escapes remains the same +/-LidI.Why is the targeting so poor for the spacers, particularly in the case of spacer A, which shows less than 10-fold decrease in phage plaquing? How were the three spacers chosen? Do the authors always find such poor targeting with CRISPR spacers and ICP1?Spacer A shows ~10-fold drop in phage while spacers B and C show ~1000-fold drop. These decreases are within the range of previously published values and are linked to the frequency of escapes . The additive effect that LidI has on the total number of phage that can overcome targeting is consistent across spacers. We chose these spacers from the lab’s collection and could add more if reviewers think it would be helpful hoewever we do not expect the results to change – spacers will work to varying degrees while LidI expression will decrease the population of phages that overcome the spacer by ~10-fold.I also had difficulty following the line of reasoning presented at the very end of the Results, centered on the reconstitution of the CRISPR system and the impact on evolution in infected cells.Vibrio cholerae and targets the phage (“we exposed PLE (-) V. cholerae with and without lidIPLE 1 to ICP1 (MOI=0.1), collected the population of progeny phage, and looked for plaque formation on PLE ; an expanded schematic of this experiment is available in Figure 6—figure supplement 2.”).We have provided additional supplementary schematics to clarify the experimental set up and better contextualize these experiments for the reviewers. For Figures 6C and D and further explained in Figure 6—figure supplement 2, the CRISPR system discussed is encoded by V. cholerae chromosome   and the other lacking Cas2-3 (CRISPR-Cas* ICP1).”). These phage with inactivate CRISPR systems can only plaque on PLE+ strains if homologous recombination occurred reconstituting an active CRISPR system in the phage .In contrast, for Figures 6E and F inactive CRISPR systems are encoded by two phage strains and target the PLE within the These assays probe the evolution of the phage by measuring how many successful recombinants are formed in the presence and absence of LidI as shown in Figure 6—figure supplement 3. The key finding is that by limiting the population size as a whole, the number of phage that can evolve to overcome some selection pressure  is also limited.3) There is some concern about the importance of both Figures 5 and 6. The authors have no real evidence that LidI is the LIN collapsing protein. It is an integral membrane protein with 2 TMDs and its overexpression will likely cause depolarization of the membrane and that would collapse LIN, just like DNP. Unfortunately, the lidI knockout has no phenotype. Regarding Figure 5, if one starts with low MOI, it takes longer to see LIN just because most cells are not infected. Recommend deleting these figures.Figure 6 shows the decreased yield that results from LidI expression and the consequent collapse of lysis inhibition. This is an important finding in line with literature about T4 lysis inhibition and we observe the decreased phage production consistently across all tested conditions:yield after phage infection in with LidI from both PLE 1 and PLE 4 induced at various times in respect to infection at high MOI (MOI=5) in Figure 6A and BV. cholerae encoded-CRISPR targeting after low MOI infection conditions (MOI=0.1) in Figure 6Dphage capable of overcoming phage capable of recombining to reconstitute a functional phage encoded-CRISPR-system after low MOI infection conditions (MOI=0.01)) in Figure 6Fand we have now included three very low MOI infections of LidI expressing strains in Figure 6—figure supplement 1  at the reviewer’s requestThis finding was identified by the reviewers as important in the summary statement where, “LidI is the first PLE-encoded ORF shown to negatively impact ICP1 phage yield” and in the minor concern number 5 where a reviewer stated, “the authors designed a nice experiment to measure phage yield presented in Figure 6A where a specific high MOI was used. The two log decrease in yield is impressive under these conditions.” We believe this series of experiments marks the finding as consistent, reproducible, and significant, as such we do not agree to the recommendation to delete these figures.In Figure 5, we do not claim that LidI is the protein that collapses lysis inhibition but instead that it is a protein sufficient to collapse lysis inhibition, which the data shows conclusively in Figures 4A, 5A, and Figure 6—figure supplement 1A-C. We further show that the PLE 4 LidI homolog is also sufficient to disrupt lysis inhibition in Figure 4E. We are careful to state that lysis inhibition collapse still happens in the absence of LidI .At this time, we can propose many molecular mechanisms for LidI activity: it likely could depolarize the membrane directly upon expression, which we show occurs after phage infection  as the reviewer suggests, alternatively it could stabilize holin pore formation, destabilize the antiholin, inhibit holin-antiholin binding, or work through some other unforeseen mechanism. At this time we cannot distinguish between these hypotheses beyond stating that LidI expression alone does not depolarize membrane in the absence of phage infection  yet still is sufficient to collapse lysis inhibition under the same induction conditions .Additionally, the reviewers communicated the following via email: The authors still leave the impression that LidI is the protein that collapses LIN. "LidI is the first PLE-encoded ORF shown to negatively impact ICP1 phage yield". This sounds like LidI actually does this but there is no evidence to this effect. Indeed, by earlier standards of phage genetics, we would conclude that it does not have this function, since there is no phenotype. The language still needs to be softened to avoid this false impression.As stated above, we are careful not to say that LidI is the protein that collapses lysis inhibition in the context of PLE in the revised manuscript, but that it is sufficient to collapse lysis inhibition outside of the context of PLE. We conclusively show that LidI is the first PLE-encoded ORF demonstrated to negatively impact ICP1 phage yield (by orders of magnitude) in Figure 6 and Figure 6—figure supplement 1D. It is true that LidI could function differently in the context PLE, however, even if the function of LidI is a “fluke” in that LidI normally completes a different function in the cell, this “fluke” conveniently phenocopies the accelerated lysis we see in PLE+ cells. Although redundancy is perhaps not expected for mobile genetic elements with restricted genome size (like PLE), the most parsimonious explanation is that accelerated lysis is mediated by product(s) with redundant function, LidI being one of them (now stated in subsection “PLE accelerates ICP1-mediated lysis”). The earlier standards noted by the reviewer apparently do not allow for redundancy, nonetheless our investigation of this system is consistent with such redundancy and we believe this is even more of an interesting biological system because of it. The data demonstrating this redundancy has been added and can be found in Figure 4—figure supplement 3.We have clarified language throughout the manuscript to impress upon the reader that the LidI characterization we have done is outside the context of PLE.“Through lysis inhibition disruption a conserved PLE protein, LidI, can limit the phage produced from an infection, bottlenecking ICP1.”“Subsequently, we discovered a single PLE-encoded gene we call lidI for lysis inhibition disruption that is sufficient to collapse ICP1- mediated lysis inhibition.”PLE 1 expression during ICP1 infection, we next sought to characterize LidIPLE 1 function in PLE (-) V. cholerae. During infection with ICP1, LidIPLE1 was sufficient to recapitulate the PLE-mediated accelerated lysis phenotype.”“After confirming lidIPLE 1 recapitulates PLE-mediated accelerated lysis, we wanted to determine if it was also necessary for this phenotype. Interestingly, however, when we deleted lidIPLE 1 from PLE 1 V. cholerae the lysis kinetics were unchanged .”“Having demonstrated that LidIPLE 1 can function through lysis inhibition disruption.”“LidIPLE1 is sufficient to accelerate lysis but does not phenocopy what we expect of a holin, we wanted to determine the mechanism of LidIPLE 1-mediated accelerated lysis in the absence of PLE.”“As LidIPLE 1 disrupting lysis inhibition, lidIPLE 1 expression in PLE (-) V. cholerae does not change the efficiency of plaquing (EOP) by ICP1, an experiment that probes the number of successful initial infections at a low multiplicity of infection . Consistent with this, the phenotypic change in plaque morphology expected of disrupted lysis inhibition is the loss of fuzzy plaque edges, which we see in PLE (-) V. cholerae expressing lidIPLE 1in trans . It is important to note that these data showing that LidI disrupts lysis inhibition when expressed alone do not reveal the molecular mechanism underlying this activity or ensure that the gene serves the same function in the context of PLE, even though it successfully phenocopies PLE-induced accelerated lysis.”“Congruent with LidI“These data reveal LidI as the first PLE-encoded ORF that can singlehandedly negatively impact ICP1 phage yield.”V. cholerae constrains ICP1 evolution.”“Hence, we hypothesize that PLE-mediated accelerated lysis decreases the ability of ICP1 to evolve in the face of PLE. However, because our data support a model in which accelerated lysis is redundantly encoded, it is not currently possible to test the impact of delayed lysis on ICP1 evolution in the context of the PLE. We can, however, interrogate how the lidI-mediated collapse of lysis inhibition and concomitant decrease in phage production in PLE (-) V. cholerae.”“A single open reading frame, lidI, which is conserved through all five PLEs spanning the last 70 years, is sufficient to disrupt lysis inhibition and limit progeny phage populations when expressed outside its native context in PLE (-) V. cholerae and its parasites.”“These two opposing forces, ICP1 lysis inhibition and accelerated lysis by PLE through lysis inhibition disruption (which our data shows LidI is capable of doing in isolation and yet other undiscovered PLE-encoded mechanisms redundantly accomplish), act in the midst of the ongoing evolutionary arms race between Surprisingly, this is so important that the means to disrupt lysis inhibition and execute lysis for PLE’s own selfish benefit are apparently redundantly encoded within the PLE – LidI can collapse lysis inhibition in the absence of PLE, though DlidI PLE still shows accelerated lysis.We also further state that PLE may encode redundant ways to accelerate lysis subsection “PLE accelerates ICP1-mediated lysis”: “This accelerated timescale could result from any combination of processes such as PLE deploying its own lysis machinery, PLE modulating the expression or stability of ICP1’s lysis machinery, or PLE inhibiting or collapsing lysis inhibition.” and show that inhibition of plaque formation and accelerated lysis phenotypes are robust to knocking out each individual ORF in Figure 4—figure supplement 3.In respect to the importance of Figure 5, Figure 4 shows the reader that LidI is a conserved protein sufficient to cause accelerated cell lysis, that LidI is expressed during infection, and is not functioning as a holin in the absence of phage infection. The question that logically follows is how can LidI achieve acceleration of lysis? We do not specify molecular mechanism for the reasons explained above and instead check the obvious characteristics of lysis inhibition probing MOI , checking the plaque edge phenotype, and efficiency of plaquing. The resulting data are consistent with disruption of lysis inhibition so much so that the reviewer raising this concern has already hypothesized the mechanism by which lysis inhibition is collapsed: depolarization of the membrane.


This study aimed to establish the nature of the mediator(s) responsible for EFS‐induced contractions in HUCV. Rings with or without endothelium from human umbilical artery (HUA) or vein (HUV) were mounted in organ bath chambers containing oxygenated, heated Krebs‐Henseleit's solution. Basal release of dopamine (DA), noradrenaline, and adrenaline was measured by LC‐MS‐MS. Cumulative concentration‐response curves were performed with dopamine in the absence and in the presence of L‐NAME or of dopamine antagonists. EFS studies were performed in the presence and absence of L‐NAME, the α‐adrenergic blockers prazosin and idazoxan, and the dopamine antagonists SCH‐23390 and haloperidol. Tyrosine hydroxylase (TH) and dopa‐decarboxylase (DDC) were studied by immunohistochemistry and fluorescence in situ hybridizations. Basal release of dopamine requires an intact endothelium in both HUA and HUV. TH and DDC are present only in the endothelium of both HUA and HUV as determined by immunohistochemistry. Dopamine induced contractions in HUA only in the presence of L‐NAME. Dopamine‐induced contractions in HUV were strongly potentiated by L‐NAME. The EFS‐induced contractions in both HUA and HUV were potentiated by L‐NAME and inhibited by the D2‐like receptor antagonist haloperidol. The α‐adrenergic antagonists prazosin and idazoxan and the D1‐like receptor antagonist SCH‐23390 had no effect on the EFS‐induced contractions of HUA and HUV. Endothelium‐derived dopamine is a major modulator of HUCV reactivity  DDCdopa decarboxylaseEFSelectric field stimulationHUAhuman umbilical arteryHUCVhuman umbilical cord vesselsHUVhuman umbilical veinLC‐MS‐MSLiquid chromatography/tandem mass spectrometryL‐NAMENω‐nitro‐L‐arginine‐methyl esterTHtyrosine hydroxylase1Crotalus durissus terrificus and Bothrops jararaca,Panterophis guttatus,Chelonoidis carbonariaElectrical field stimulation (EFS) is a technique in which an electrical stimulus is applied uniformly to an isolated tissue in short pulse width waves. in vitro.In this study, the nature of the mediator was identified by liquid chromatography coupled to tandem mass spectrometry (LC‐MS‐MS), followed by a pharmacological characterization of the EFS‐induced contractions in both HUA and HUV22.1Participants over the age of 18, undergoing the natural or cesarean delivery from Santa Casa de Vinhedo (Vinhedo‐SP) and Campinas Maternity Hospital (Campinas‐SP), were invited to take part in the study. The women were normotensive, and did not have preeclampsia, pregestational, or gestational diabetes mellitus and none were on regular medication. Written consent was obtained from those who agreed to participate. Umbilical cords from 67 volunteers aged 18‐47 years were used .The investigation conformed to the principles outlined in the Declaration of Helsinki and the protocol was approved by the Ethics Committee of the Institute of Biomedical Sciences of the University of São Paulo—ICB/USP (protocol number 3.165.417).2.2ω‐Nitro‐L‐arginine methyl ester hydrochloride (L‐NAME), H‐oxadiazoloquinoxalin‐1‐one (ODQ), and SCH‐23390 were purchased from Sigma‐Aldrich Chemicals Co. . Haloperidol was bought from Nallin Farmácia e Manipulação Ltda . Dopamine‐d3 hydrochloride, DL‐noradrenaline‐d6 hydrochloride, and adrenaline‐d6 hydrochloride were acquired from CDN Isotopes . Aluminum oxide and Harris’ Hematoxilin were purchased from Dinamica Quimica Contemporanea Ltda . Sodium chloride (NaCl), potassium chloride (KCl), calcium chloride (CaCl2), magnesium sulfate (MgSO4), sodium bicarbonate (NaHCO3), potassium phosphate monobasic (KH2PO4), glucose, and entellan were bought from Merck KGaA . Acetonitrile was obtained from J.T Baker , and formic acid (HPLC grade) was purchased from Mallinckrodt . Anti‐human calretinin (code: IS627) was purchased from DAKO . Anti‐tyrosine hydroxylase (code: ab76442), anti‐dopa decarboxylase (code: ab211535), and the secondary antibody (a goat anti‐chicken IgY [code: ab150169]) were purchased from Abcam . The tertiary antibody, a rabbit anti‐goat IgG (code: AP106P), was purchased from Sigma/Merck . NovoLink™ Max Polymer Detection System (code: RE7280‐k) and 3,3′ diaminobenzidine (DAB) were purchased from Leica Biosystems (UK). Citrate buffer, pepsin, 2XSSC, and DAPI (code: Z‐2028‐20) were bought from ZytoVision kit .Adrenaline, noradrenaline, dopamine, adenosine 5′‐triphosphate (ATP), N2.32 (2.5), MgSO4 (1.2), NaHCO3 (25), KH2PO4 (1.2), and glucose (5.6) gassed with a mixture of 95%O2: 5% CO2 (pH 7.4) at 37°C. After a period of 30 minutes, an aliquot of 2 mL of the supernatant was transferred to an Eppendorf tube and stored at −20°C until the time for analysis.This study was carried out on human umbilical cord specimens obtained from 12 different placentae/patients. A segment of the umbilical cord—10‐20 cm from the insertion point in the placenta and 5 cm from the umbilicus—was removed by the obstetrician and placed in a container with Krebs‐Henseleit's solution. The Wharton's jelly was removed and the umbilical arteries (HUA) and the umbilical vein (HUV) were dissected. Vessel rings  were incubated in 10 mL organ baths containing Krebs‐Henseleit's solution: (mM) NaCl (118), KCl (4.7), CaCl2O3 was added and left for incubation for 20 minutes in an orbital agitator (Centrifuge 5810/ 5810 R). The tubes were then centrifuged at 2000 g for 4 minutes at 4°C and the supernatant discarded. The residue was washed 4 times with 2 mL of deionized water. After the final wash, 200 µL of a solution containing trifluoroacetic acid 0.1% in HCN/H2O  was added. After vortexing for 40 seconds, the Eppendorf tubes were centrifuged for 2000 g for 5 minutes and the supernatant transferred to the vials for injection. The samples were analyzed by liquid chromatography coupled to a triple quadrupole mass spectrometer, LCMS‐8050 (Shimadzu).The dopamine, noradrenaline, and adrenaline concentrations in the Krebs‐Henseleit's solution were determined by liquid chromatography coupled to tandem mass spectrometry (LC‐MS/MS). The extraction procedure was similar to that described for extracting methyldopa from plasma.The separation of catecholamines was performed on a 100 × 4.6 mm Lichrospher RP‐8 column (GL Sciences Inc) using acetonitrile/water  with 0.1% formic acid as mobile phase at a flow rate of 0.4 mL/min. The mass spectrometer operated in positive electrospray ionization mode (ES+) for catecholamine detection. The analyses were executed in selected Multiple Reaction Monitoring (MRM) detection mode. The transitions and retention times employed are described in the table below  stainings.1,2; the epitope was not specified by the manufacturer; dilution: 1:200 in PBS; DAKO/Agilent, USA), (2) anti‐tyrosine hydroxylase , and (3) anti‐dopa decarboxylase  as previously determined by the manufacturer using overlapping synthetic peptides; and dilution: 1:100 in PBS; Abcam, Cambridge, UK). Detection of tyrosine hydroxylase required the use of secondary and tertiary antibodies. The secondary antibody was a goat anti‐chicken IgY . The tertiary antibody was a rabbit anti‐goat IgG . The slides were incubated for 1 hour at room temperature with the secondary antibody, followed by 1 hour incubation with the tertiary antibody. The detection system was the NovoLink™ Max Polymer Detection System , following the protocol described by the manufacturer. Thereafter, 3,3′ diaminobenzidine (DAB) was used as chromogen. Finally, the slides were dehydrated, counterstained with Harris´ hematoxylin and cover slipped in Entellan (Sigma/Merck). Negative controls consisted of the omission of the primary antibody and incubation with the primary antibody diluents  and were performed in all immunohistochemistry assays (one negative control per section) to identify any background staining. All solutions  were prepared for a single use on the same day of the immunohistochemistry assay, and kept at 4ºC until use. All slides were examined and photomicrographed using a trinocular Eclipse 50i microscope (Nikon) coupled to a 10MP CMOS digital camera . Positivity was assessed by an experienced MD, PhD pathologist (AAS), who was blind to the presence/absence of the primary antibody on the sample under examination (the observer did not know whether a test sample or an omission control was being assessed). Blinding was achieved by covering the slide labels with a removable occluding sticker.Tissue sections were deparaffinized and rehydrated in graded alcohols to distilled water, and then they were incubated in 3% hydrogen peroxide for 10 minutes to block the endogenous peroxidase. Antigen retrieval was performed by heating slides in citrate buffer  at 95°C for 20 minutes (in a steamer set). Subsequently, they were left to cool down at room temperature and rinsed with PBS. Each slide was then incubated for 2 hours at room temperature with one of the primary antibodies. The primary antibodies used were (1) anti‐human calretinin  at 80ºC for 1 hour. After this, they were incubated with pepsin for 8 minutes at room temperature. The slides were washed with 2XSSC , dehydrated in a sequence of ethanols , and then air dried. The slides were incubated with 100µL of the TH mRNA probe  for 10 minutes at 75ºC and overnight in a Dako Hybridizer  at 37ºC. The TH mRNA probe sequence was as follows: 5′‐ AACCGCGGGGACATGATGGCCT‐3′ (RNA Tm = 77.8°C) . The probe was labeled with fluorescein 6‐FAM in the 5’ region. The next day, the slides were placed in a UREA/0,1Xssc solution at 45°C for 30 minutes, and then, they were washed with a 2xSSC solution for 2 minutes. After this, the slides were dehydrated in 75%, 85%, and 100% ethanols for 2 minutes each, and air dried. Finally, the slides were mounted with 15 µL of a DAPI containing mounting medium (from the ZytoVision kit) and cover slipped . Negative controls consisted of the omission of the probe and were performed in all FISH assays (one negative control per section) to control for any significant autofluorescence. All FISH slides were examined and photomicrographed using a trinocular DM4000 B LED microscope  coupled to a 1.4 MP DFC 310 FX camera .2.52:5% CO2 (pH 7.4) at 37°C, and coupled to isometric transducer. The initial smooth muscle tension was set at 10 mN.This study was carried out on human umbilical cord specimens obtained from 47 different placentae/patients. The Wharton's jelly was removed and the umbilical arteries (HUA) and the umbilical vein (HUV) were dissected. Vessels rings (3 mm) were suspended vertically between two metal hooks in 10 mL organ baths containing Krebs‐Henseleit's, gassed with a mixture of 95%OFollowing a 90‐min stabilization period, the rings were precontracted with 5‐HT (1 µM), and the integrity of the endothelium in both HUA and HUV was evaluated by the addition of ATP to cause relaxation (10 µM).The HUA and HUV rings were submitted to EFS at 60V for 30 seconds, at 8‐16 Hz in square‐wave pulses, 0.3 ms pulse width, and 0.1 ms delay, using a Grass S88 stimulator . Electrical field simulations were performed in the presence and absence of L‐NAME, and in the presence and absence of the dopamine D1‐like receptor antagonist SCH‐23390 , of the dopamine D2‐like receptor antagonist haloperidol , of the adrenergic alpha‐1 receptor antagonist prazosin , and of the adrenergic alpha‐2 receptor antagonist idazoxan . The effect of the antagonists was evaluated always in the presence of L‐NAME (100 μM).2.6t test was used and a P‐value < .05 was considered as significant. In the pharmacological experiments, the number of experiments is expressed as x/y, where x represents the number of umbilical vessels and y the number of rings employed in the experiment. For Emax analysis and pEC50, unpaired Student's t test was used and a P‐value of < .05 was considered as significant.Data are expressed as mean ± (SEM) of the number of experiments (n = or>5). Paired Student's 50 was carried out using GraphPad Prism  with the constraint that Φ = 0. All concentration‐response data were evaluated for a fit to a logistics function in the form: E = Emax/([1 + (10c/10x)n] +Φ. The values of pEC50 data represent the mean ± SEM. Values of Emax were represented by mN.Nonlinear regression analysis to determine the pEC33.1Dopamine, noradrenaline, and adrenaline calibration curves were linear for concentrations of 0.1‐10.0 ng/mL, with a correlation coefficient higher than 0.99. The limit of quantification was 0.1 ng/mL. The method was fully validated, and the results reported elsewhere.3.2 = 8, Figure Tyrosine hydroxylase was detected by immunohistochemistry only in endothelial cells, in all samples of both HUA . Tyrosine hydroxylase mRNA was consistently detected in endothelial cells of all tested samples (n = 5), both in HUA  in umbilical cord samples, with special attention to the vessel walls. Calretinin was not found in any samples of either HUA  and in endothelium‐denuded HUA . There was no significant difference in the Emax, but the pEC50 2.9 of dopamine‐induced contractions in endothelium‐denuded HUA presented a significant right shift when compared to either L‐NAME‐ or ODQ‐treated HUA.Dopamine alone induced contractions in L‐NAME (100 µM)‐treated HUA  significantly increased the EFS (8 Hz and 16 Hz)‐induced contractions of both HUA Figure  and HUV 3.41‐adrenoceptor antagonist, had no effect in the EFS‐induced contraction of the HUA , a selective α2‐adrenoceptor antagonist, had no effect in the EFS‐induced contraction of the HUA , a selective α3.550 3.9 ± 0.1 [n = 5/10] and 4.2 ± 0.3 mN [n = 5/10], P < .05, without and with SCH‐23390, respectively; Figure 50 4.6 ± 0.1 [n = 5/10] and 3.3 ± 0.3 mN [n = 5/10], P < .05, without and with SCH‐23390, respectively; Figure In L‐NAME‐treated vessels, the dopamine D1‐like receptor antagonist SCH‐23390 (10 µM) caused reduction in dopamine‐induced contractions of HUA  caused significant reduction in EFS‐induced contraction of the HUA  abolished the contraction dependent of dopamine HUA 2) receptors has been assessed in smooth muscle tissue of rat cerebral, mesenteric and renal arteries.1 and D2 receptors have been localized in the endothelium.2 and D4 subtypes in cerebral and mesenteric vascular bed and D2 and D3 receptors in renal vasculature, with the D5 subtype predominantly residing as a smooth muscle receptor in the vascular beds of rats.Dopamine acts on selective receptors, belonging to the G protein–coupled receptor family. Five genes encoding DA receptors (DRs) have been identified. These receptors are divided into two subfamilies: the D1‐like receptor subtypes (D1R and D5R), coupled to Gs, activating adenylyl cyclase and the D2‐like subfamily  coupled to Gi, inhibiting adenylyl cyclase.The finding that dopamine could only contract HUA in the presence of the NO synthesis inhibitor L‐NAME indicates a major interaction of dopamine with NO on this vessel. Indeed, similar results were obtained with the heme‐site inhibitor of soluble guanylyl cyclase ODQ and in endothelium‐denuded vessel. It is likely that the dopamine released in the circulation by EC would cause NO‐dependent vasodilatation through the action on D1 receptors expressed by EC. Indeed, the hemodynamic effects of dopamine depend on the dose administered; with intravenous infusions ranging from 1 to 10 µg/kg/min, dopamine increased cardiac contractility,2‐like receptor antagonists haloperidol revealed another important modulator role of the endothelium‐derived dopamine, acting as a vasoconstrictor through the D2‐like receptor. The finding that SCH‐23390 also had some inhibitory effect on dopamine‐induced contractions of L‐NAME‐treated HUA and HUV was possibly due to the antagonistic effect of this compound on D2 receptors at higher concentration. in vitro scenario. Domperidone and haloperidol applied as ophthalmic solutions in a rabbit ocular hypertensive model produced a marked increase in ocular blood flow.The inhibition of EFS‐induced contractions by the D1‐adrenergic receptor antagonist prazosin2‐adrenergic receptor antagonist idazoxan3H‐haloperidol binding to dopamine receptors in calf brain membranes.The alphaThe interaction between dopamine and NO should not be restricted to their pharmacological actions. Nitro‐catecholamines such as nitro‐dopamine, nitro‐noradrenaline, and nitro‐adrenaline have been found in rat brain.What is the possible physiological role of endothelial‐derived dopamine? Although cardiac output is defined by the product of heart rate and systolic volume, it is known that the pumping function of the heart has a permissive role in the determination of cardiac output.The authors of this manuscript declare that they have no conflicts of interest.Research data are not shared.Conceptualization: José Britto‐Júnior, Gilberto De Nucci. Data curation: Fabiola Z. Mónica, Edson Antunes, Gilberto De Nucci. Formal analysis: Gilberto De Nucci. Funding acquisition: Edson Antunes, Gilberto De Nucci. Investigation: José Britto‐Júnior, Gilberto De Nucci. Methodology: José Britto‐Júnior, David Halen Araújo Pinheiro, Alberto Fernando Oliveira Justo, Guilherme M. Figueiredo Murari, Rafael Campos, Fernanda Viviane Mariano, Valéria Barbosa de Souza, André Almeida Schenka, Edson Antunes, Fabiola Z. Mónica, Gilberto De Nucci. Project administration: Gilberto De Nucci. Supervision: Fabiola Z. Mónica, Edson Antunes. Visualization: Edson Antunes. Writing – original draft: José Britto‐Júnior, Gilberto De Nucci. Writing – review & editing: José Britto‐Júnior, Gilberto De Nucci.10.22541/au.158456487.79594821.This article has earned Open Data and Open Materials badges for making publicly available the digitally‐shareable data necessary to reproduce the reported results. The data and materials are available at DOI:


Growth of molecular imaging bears potential to transform nuclear cardiology from a primarily diagnostic method to a precision medicine tool. Molecular targets amenable for imaging and therapeutic intervention are particularly promising to facilitate risk stratification, patient selection and exquisite guidance of novel therapies, and interrogation of systems-based interorgan communication. Non-invasive visualization of pathobiology provides valuable insights into the progression of disease and response to treatment. Specifically, inflammation, fibrosis, and neurohormonal signaling, central to the progression of cardiovascular disease and emerging therapeutic strategies, have been investigated by molecular imaging. As the number of radioligands grows, careful investigation of the binding properties and added-value of imaging should be prioritized to identify high-potential probes and facilitate translation to clinical applications. In this review, we discuss the current state of molecular imaging in cardiovascular medicine, and the challenges and opportunities ahead for cardiovascular molecular imaging to navigate the path from diagnosis to prognosis to personalized medicine.The online version of this article (10.1007/s12350-020-02319-6) contains supplementary material, which is available to authorized users.The online version of this article (10.1007/s12350-020-02319-6) contains supplementary material, which is available to authorized users. As cardiovascular precision medicine embraces molecular-targeted therapies, the identification of at-risk and likely-to-respond patients takes on greater importance. Imaging to non-invasively quantify these molecular targets can provide incremental value in selecting appropriate patient populations for selective and expensive therapies. Accordingly, nuclear cardiology finds itself at a critical junction, where the pathway demarcated by image-guided oncology may direct the future of cardiovascular molecular imaging. Conventional nuclear cardiology assesses myocardial perfusion, viability, function, and scar—i.e., measurements of disease severity after initial insult.Inflammation critically contributes to development and progression of cardiovascular disease. After ischemic injury, cardiomyocyte death initiates release of pro-inflammatory factors, followed by leukocyte infiltration, remodeling, and repair. High circulating blood leukocytes are associated with higher mortality and adverse cardiac events among patients.3Contrary to the robust local inflammatory response after MI, non-ischemic cardiac diseases are characterized by diffuse myocardial inflammation, a greater challenge for imaging. The inflammatory response can be triggered by mechanical strain, neurohormonal activation, oxidative stress, fibrosis, and/or modest cardiomyocyte necrosis.618F-fluorodeoxyglucose, novel molecular radioligands portend the opportunity to distinguish specific cellular components of the inflammatory response.9As such, a number of molecular imaging agents have been explored for characterization of cardiac inflammation Table . While mMyocardial fibrosis is a common endpoint of cardiovascular disease, characterized by resident cardiac fibroblast transdifferentiation and activation, which produce fibrillary collagen and reorganize extracellular matrix. Reparative or replacement fibrosis after ischemic injury culminates in scar formation and stabilization of the infarct.12Non-invasive characterization of fibrosis typically relies on estimation of ventricle stiffness and filling via echocardiography or characterization of tissue differences via cardiac magnetic resonance imaging.16To date, therapies to directly mitigate cardiac fibrosis are lacking, though novel strategies including gene transfer to reprogram cardiac fibroblasts68Ga-labeled FAP inhibitor accumulated in the infarct territory at 6d after injury, receding to baseline subsequently. Signal specificity was confirmed by blocking and immunofluorescence staining. The density of FAP-positive fibroblasts was significantly higher in the infarct border zone compared to center or remote myocardium,The expansion of imaging approaches for early indicators of fibrosis has stimulated interest in applying FAP-targeted imaging for cardiovascular disease. After permanent coronary artery ligation in rats, The sympathetic nervous system is the primary extrinsic control of heart rate and contractility. Heightened sympathetic signaling compensates for the failing heart, leading to downregulation of adrenoceptors and excitation–contraction uncoupling. Beta-blocker therapy initially inhibits the over-stimulation of adrenoceptors, re-establishing homeostasis in autonomic regulation of contractile function.22Imaging of the cardiac sympathetic nervous system has been pursued over the last three decades, but the impact on clinical care has been underwhelming. The principal limitation of innervation imaging lies with the radiotracers themselves, which are largely subject to variable permutations of neuronal reuptake, vesicular packaging, active synaptic release, passive diffusion to the synaptic cleft, and metabolic degradation. Despite evidence supporting the role of denervated myocardium in sudden cardiac arrest and heart failure progression Figure , the limThe expansion of the molecular imaging radiotracer arsenal provides a number of opportunities for research and patient management Table . When thTo this end, targeted imaging and treatment of inflammatory and fibrotic mechanisms provide the opportunity to interrogate the intersection of these processes, which can further refine treatment strategies to benefit the individual patient. Moreover, the growing capacity to acquire images beyond the target organ, either through multiple bed positions or total-body PET, enables systems-based analysis, offering unique insights into the interaction of different organ systems. As such, cardiovascular molecular imaging can define the pathway to precision patient management, giving critical insights into disease processes, early prognosis, and response to therapy that can ultimately lead the right patient to the right therapy on the appropriate schedule.Supplementary material 1 (PPDX 2043 kb)Supplementary material 2 (M4A 3594 kb)Supplementary material 3 (DOCX 12 kb)Below is the link to the electronic supplementary material.


This process is influenced by a number of mutually and strongly interlaced entry parameters  and their participation in the resulting nanofiber quality. The appearance of nanofibers is a result of the necessary primary experimental parameter setting within an acceptable range. However, finer analysis of nanofiber quality depends on the proper choice of these individual factors. The aim of this contribution is to evaluate one of the key factors—polymer concentration—with respect to the presence or absence of bead formation. This passage can be approximated by rheological oscillatory measurements when a sudden decrease in phase angle indicates this change. It replaces otherwise time- and cost-consuming trial-and-error experiments. This approach was tested using three different materials: solutions of poly(vinylidene fluoride- In past decades the application of nanofibrous materials has shifted from classical filters to other uses such as protective clothing, antibacterial wound dressing, and tissue engineering, to name a few. In some applications the appearance of singularities along the nanofibers, so called beads characterized by an abrupt increase in nanofiber cross-section followed by a sudden decrease, is required as it substantially improves adhesion ,2. HowevThe electrospinning process ,5,6,7,8 This concerns, for instance, an appearance of beads along the individual nanofibers, which is mostly an unwanted phenomenon. However, in some cases an appearance of beads supports an application of nanofibers, as in the case of improving adhesion ,2. Bead One of the crucial parameters is represented by the polymer concentration. Its value contributes to distinguishing the visual properties of the final product to three distinct cases: electrospraying (discrete drops), continuous nanofibers, and the creation of spots produced by spinning due to incomplete solvent evaporation. De Gennes  set four(a)co-HFP) dissolved in N,N’-dimethylformamide (DMF),copolymer of poly(vinylidene fluoride) and hexafluoropropylene (PVDF-(b)poly(ethylene oxide) (PEO) dissolved in water, and(c)poly (PVB) dissolved in ethanol.The question is whether or not this method of evaluating an onset of concentrations for which beadless nanofibers are produced can be simplified. To this aim we chose three frequently used materials:ad (a)3 in comparison with polytetrafluoroethylene (PTFE)—2.2 g/cm3). Even more attractive is a copolymer of PVDF and hexafluoropropylene (PVDF-co-HFP), which is characterized by higher solubility, lower crystallinity, better mechanical strength, and higher hydrophobicity. In addition, this copolymer exhibits the highest dielectric constant and electroactive response, including piezoelectric, pyroelectric, and ferroelectric effects. All these properties predetermine PVDF-co-HFP as a very perspective material in membrane production. PVDF-co-HFP membranes are used especially in two spheres: membrane distillation  [Thermoplastic fluoropolymer poly(vinylidene fluoride) (PVDF) is widely used in various applications due to its unique properties compared to other polymers. It is resistant to aggressive conditions due to its high chemical, thermal, and UV stability. Further, PVDF exhibits very good mechanical strength, high hydrophobicity, thermal stability, and low density (only 1.78 g/cmosmosis)  and in eosmosis)  or in enosmosis) .ad (b)Poly(ethylene oxide) is among the most easily spinnable materials. Moreover, its excellent biodegradability, biocompatibility, and non-toxicity predetermine electrospun PEO nanofibrous mats to applications in biomedicine and the food industry. The relatively small participation of PEO (up to 2%) also enables spinnability of otherwise unspinnable or very problematically spinnable materials such as chitin or chitosan ,23,24,25ad (c)Poly exhibits features that are similar to those introduced above for PEO. PVB is an odorless, nontoxic polymer, fully biocompatible, and characterized by flexibility and good adhesion to various substrates. It predominantly serves as an interlayer material ,31 in caBased on oscillatory rheological measurements, the aim of this contribution is to show a close correlation between the onset of a starting concentration for which a presence of beadless nanofibers dominates, and a location of phase angle decrease, i.e., a sudden decrease from the constant course or only a moderate decrease in the curve phase angle vs. the concentration of polymer solution. ® 2801 (copolymer poly(vinylidene fluoride)-co-hexafluoropropylene), datasheet [N,N′-dimethylformamide (DMF) (p.a.) was purchased from P-LAB, a.s. . Kynar Flexatasheet , was purPoly Mowital B 75H, datasheet , was purPoly(ethylene oxide), datasheet , was purAll chemicals were used as obtained without further refinement.® 2801). Polymer materials were dissolved in corresponding solvents  using a The concentrations of all prepared solutions and molecular weights of used polymers are summarized in The nanofibers were spun using our laboratory needleless device see  equippedThe three setup orientations for electrospinning are vertical top-down, vertical bottom-up, and horizontal. These orientations will change the mutual configurations between the gravitational and electric fields. Whilst it appears that the intensity of the gravitational field is negligible in comparison to the electric, some studies indicate that gravity should be taken into account. Yarin et al.  showed lThe individual materials were electrospun under the following conditions.co-HFP/DMF—The electrospinning process was carried out at a voltage of 18 kV with the fixed tip-to-collector distance of 100 mm at ambient conditions of 23 ± 1 °C and a relative humidity of 34 ± 1%.PVDF-PVB/ethanol—The following process parameters were fixed: the tip-to-collector distance was 100 mm, ambient temperature 21 ± 1 °C, and relative humidity 40 ± 1%. The voltage was fixed to 20 kV ensuring generation of fibrous mats without presence of blobs (sufficient rate of solvent evaporation).PEO/water—The electrospinning process was carried out at a voltage of 12–25 kV with the fixed tip-to-collector distance of 200 mm at ambient temperature of 22 ± 1 °C and a relative humidity of 39 ± 2%.G’ and viscous G’’ moduli, and for shear viscosity measurements (a range 0.01–300 s−1). The value of shear rate −1 belonging to a linear viscoelastic region was chosen for measurement of shear viscosity of polymer solutions with different polymer concentrations, and consequently applied to a determination of specific viscosity. The phase angle δ (tan δ = G’’/G’) was determined at a frequency of 10 Hz. The temperature was set to 25 °C. Each measurement was carried out at least three times and the individual runs were more or less identical.A rotational rheometer Physica MCR 501  equipped with the concentric cylinder geometry  was used both for oscillatory measurements (frequency sweep within 0.1–100 Hz at strain 1%) providing elastic The measured shear viscosities of all three solvents were compared with responsible literature  was used for characterization of nanofibrous mats. Prior to imaging, the samples were sputtered by a conductive coating layer using a sputter Quorum Q150R . In the following, interlacing of rheological characteristics of polymer solutions used in the process of electrospinning with the morphological characterization of electrospun nanofibers are presented with an emphasis to create nanofibers with a good (bead-free) quality. co-HFP, the specific viscosities were calculated using the relation ηsp = (η0–ηs)/ηs, where η0 is the zero-shear-rate viscosity (measured at −1) and ηs is the solvent (DMF) viscosity (=0.807 mPa·s). This measured value corresponds to the values presented in the literature [c* (≈12 wt.%) and entanglement concentration ce (≈19 wt.%). These two concentrations correspond to the intersection points of linear segments optimized with respect to the experimental points, see Based on measurement of shear viscosities for individual concentrations of PVDF-terature ,42. Consterature  were detG’ and viscous (loss) G” moduli, a course of the phase angle δ (tanδ = G”/G’) is depicted in Based on elastic (storage) co-HFP nanofibers was correlated with the rheological characteristics of measured solutions. c* ≈ 12 wt.%) no nanofibers were formed, and as a result, only a disordered set of blobs were received. Starting with the overlap concentration the primary forms of nanofibers were indicated mixed with the blobs, and finally a passage from bead to bead-free nanofibers was apparent around the entanglement concentration. It seems that an adequate quality of nanofibers was achieved for cstart = 22 wt.% and higher. This value closely corresponded to a concentration for which the phase angle started to decrease as elasticity began to manifest.The morphology of PVDF-ηs = 1.087 mPa·s corresponds to data in literature [The analogous experimental approach was also carried out with PVB dissolved in ethanol . Theterature .ηs = 0. 890 mPa·s according to [Mw = 637,500 g/mol) the polymer solutions also exhibited viscoelastic behavior for lower concentrations due to higher molecular weight. At first, the decrease in the relation phase angle vs. concentration was rather moderate. Then, at the concentration of approximately 6% a sudden drop was apparent. Hence, this value (cstart) indicates the region from which the nanofibers have a dominantly bead-free characteristic.The analogous experimental approach was also carried out with PEO dissolved in distilled water (rding to ). The reco-HFP and PVB solutions, for which the phase angle attains a constant value (90°) for lower concentrations. This also results in the relatively gradual passage of PEO nanofibers from beaded to beadless character. The determination of the starting concentration for the PEO solution is not so strict for the remaining two materials as PEO solutions also exhibit viscoelastic behavior for lower concentrations due to their relatively high molecular weight. A more pronounced decrease in the phase angle starts from an already mildly decreasing curve phase angle vs. concentration which contrasts to the PVDF-Possible electro-spinnability of polymeric materials and quality of the resulting nanofibers are subject to a number of entry parameters, which can be roughly distributed into four categories: polymer characteristics , solvent characteristics , polymer solution characteristics , and process characteristics .co-HFP (fixed molecular weight) is illustrated in These characteristics are mutually interlaced. This means that it is not possible to alter only one, as for instance, viscosity strongly depends on molecular weight and concentration. This is also connected with an increase in nanofiber diameter. The algebraic expression relating a mean nanofiber diameter with variable molecular weight and concentration for PVB is introduced in , and forAbove, it is implied that a specific polymeric material is only necessary to access experimentally in order to derive a suitable setting of entry parameters. However, for a given relatively acceptable set of entry parameters, there is a question as to whether a local tuning of one chosen parameter cannot be based on purely instrumental analysis , and thus eliminate time-consuming and expensive trial-and-error experimental methods. The explicit expressions that relate a distinct parameter to adjustable parameters—providing the first approximation—can serve to simplify and assist in the orientation of the whole problem. For example: to determine the nanofibers’ mean diameter, which is dependent on a variable molecular weight and concentration, as indicated above, and similarly, to determine the starting concentration cstart which is dependent on molecular weight and rheological characteristics. However, to derive such algebraic relationships, more experiments with the materials, each of them with variable molecular weight, would be necessary. Furthermore, specifics concerning molecular weight cannot be determined solely from the data provided by the producers.The topic of this study was a participation of polymer concentration on the appearance of singularities (beads) along the electrospun nanofibers. It is well known that characterization of polymeric materials differs from batch to batch . It resuco-hexafluoropropylene), poly, and poly(ethylene oxide).Determination of a starting concentration of electrospinnable polymer solutions for which the obtained electrospun nanofibers exhibit a bead-free surface is usually carried out by a trial-and-error method. The proposed method using oscillatory rheological measurements provided a very good approximation of the starting concentrations. For materials exhibiting first a constant behavior or moderate decrease in a phase angle for lower concentrations, an approximation of the starting concentration is given by a concentration value where a phase angle curve starts to decrease, apparently reflecting a more progressive viscoelastic nature. It was documented using solutions of three different polymeric materials, specifically poly(vinylidene fluoride-


Despite inguinal hernias being a common problem in public health, there is still scarce information about the epidemiology of the complications, especially incarceration, and their influence on the laparoscopic surgical methods considering the role of the learning process of the surgeon. Compare laparoscopic totally extraperitoneal (TEP) approach in the repair of incarcerated and non-incarcerated inguinal hernias from the perspective of technical difficulty for trained surgeons. We obtained data about sex, age, location and type of hernia, surgery duration, ASA score, postoperative complications, previous surgeries and BMI. Groups were descriptively analyzed and statistically compared to verify how similar the samples were. 265 (90.1%) patients had non-incarcerated hernias and 29 (9.9%) incarcerated. We observed that there was no significant difference in the pattern of location , sex, ASA, previous or complications between the two groups. Unilateral incarcerated hernias had longer operative times compared to non-incarcerated. No difference was found between bilateral hernias. We didn´t find significant epidemiological differences between incarcerated and non-incarcerated hernias. In our experience, with the limitation of a single-surgeon series, laparoscopic hernia repair achieved satisfactory results in terms of feasibility  and safety. Among abdominal hernias, the inguinal hernia is the most prevalent3, this type of hernia can suffer ischemia and strangulation, increasing the risk of postoperative mortality from 0.01 to 5%4.Incarcerated hernia is defined as inability to manually reduce it back into the abdomen without signs of strangulation. With a risk of 2.8% after 3 months and 4.5% after 21 months5, low length of postoperative hospital stay and less discomfort during recover when compared to open surgery. However, there is no significant difference in recurrence rates between the two approaches6. Due to the greater cost and technical difficulty8, the laparoscopic approach is the less common of the two, being used in the minority of procedures for hernia repair around the world7.In view of these complications, the most appropriate method for repair of incarcerated inguinal hernias has been extensively discussed in the literature. The laparoscopic approach has been proved to be a feasible method for non-complicated hernias, with a low rate of clinical postoperative complicationsThe aim of this study is to compare the laparoscopic totally extraperitoneal (TEP) approach in the repair of incarcerated and non-incarcerated inguinal hernias from the perspective of technical difficulty for trained surgeons. Therefore, being able to evaluate the technical feasibility of this procedure for this type of complication.9, Meyer et al.10 and Kassir et al.11. The procedures were carried out between May 2009 and July 2019 by a surgeon specialized in hernia repair in São Paulo, Brazil. A polypropylene mesh was used  per inguinal hernia12. The mesh was not trimmed for any patient.This is a retrospective observational study of patients who have undergone TEP for the repair of incarcerated and non-incarcerated inguinal hernias, previously described by Iuamoto et al.9) and cases operated by TAPP. 294 patients who were operated exclusively by TEP were included in our study.Our inclusion criteria were patients older than 18 years who underwent elective repair of inguinal hernias by videolaparoscopy and who gave consent for the data use. This cohort consisted of 380 patients. Our exclusion criteria were the first 65 cases , postoperative complications, previous surgeries and body mass index (BMI) of patients.Patients were followed for at least 1 one after the surgery. Complications were reassessed after 7, 15, 30, 182 and 365 days for all patients.Qualitative variables will be expressed by absolute (n) and relative (%) frequency. Quantitative variables by mean, median, standard deviation, amplitude (minimum and maximum) and quartile range (1st quartile and 3rd quartile).We will use the Student t-test or Mann–Whitney test to compare two groups of quantitative variables, depending on the assumption of data normality by the Kolmogorov–Smirnov test. To verify association in qualitative variables we will use Fisher's exact test. The adopted significance level was 5%.The project was approved by the ethics committee of Samaritano Hospital, SP—Brazil.All procedures performed in this study were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.Informed consent was obtained from all individual adult participants included in this study.2 and mean operative time was 46.34 min  with non-incarcerated hernias and 29 (9.9%) with incarcerated hernias. 288 patients (98%) were male. 155 (52.7%) cases were bilateral hernias. Patients with unilateral hernias were divided between 85 (28.9%) cases of hernias located on the right groin and 54 (18.4%) on the left. Only 9 (3.1%) patients were ASA 3, all others were ASA 1 or 2. 212 (72.1%) had no previous abdominal or pelvic surgeries. The mean age was 51.95 years old, mean BMI was 26.13 kg/m% with noComplications occurred in just seven patients: six cases of scrotal hematoma and another of umbilical hematoma and edema (Clavien–Dindo I), all in non-incarcerated hernias. Recurrence occurred in two patients after 1 year with non-incarcerated hernia . Linchtenstein conversion also occurred in two patients who had several adhesions after radical prostatectomy.Comparing incarcerated and non-incarcerated hernias we observed that there was no significant difference in the pattern of location , sex, ASA, previous surgeries or complications between the two groups. The most common previous surgery reported in both groups was unilateral hernia repair Table .Table 2DRegarding surgery duration, incarcerated and non-incarcerated hernias were divided into two groups: unilateral and bilateral. Unilateral non-incarcerated hernias had a significant lower operative time with a mean duration of 34.06 min versus 50.44 min in the incarcerated group  and there was no significant difference between these two groups in BMI or age , many studies indicate it as a viable and effective method for repair of inguinal hernias.Despite the laparoscopic technique still being little used worldwide  were compared to exclude differences in the epidemiological profiles that could interfere in the analysis of the operating times. In this comparison, there was no significant difference in the pattern of location , sex, ASA, previous surgeries or complications between the two groups. The groups were not statistically different in terms of epidemiological categories, a similar result to that found in the study of Wakasugui et al.19. Wakasugui et al.17 found a significant difference in operating time between unilateral incarcerated hernias and the control group, composed of non-incarcerated unilateral hernias (111 min vs 76 min). In our analysis also there was a significant difference between these groups.The duration of surgery performed by experienced surgeons presented in the international literature are similar to those found in our studyIn the analysis of bilateral hernias, Wakasugui et al. did not find statistically significant differences in operating time between incarcerated and non-incarcerated hernias, similar to our results.It is possible to elaborate some possible explanations for this similarity between our work and the literature. The time required in operating a unilateral incarcerated hernia is proportionally shorter than in bilateral incarcerated hernias, as only one side is incarcerated. Another possible explanation is that the surgeon only operates bilateral incarcerated cases after mastering the technique in unilateral cases.20 found that patients with BMI > 30 and unilateral inguinal hernias submitted to laparoscopic repair had fewer complications compared to open surgery, thus there was no impediment to laparoscopic surgery, despite longer operative times as BMI increased. In our study, the increase in BMI was also responsible for an increase in operative time but did not significantly increase the operative difficulty of incarcerated in relation to non-incarcerated hernias, as seen by the no difference in operative time between groups.Willoughby et al.21, inguinal hernia repair with glue fixation significantly decreased the frequency and intensity of the pain when compared with the pain before the surgery, showing the benefits of hernia correction for pain management. Asuri et al.23 showed that TEP was superior than TAPP with reduced postoperative pain up to 3 months reported by patients, but no difference in overall mortality or complications. Moreover, surgical fixation in TEP procedures has been shown not significant for postoperative pain24. The use of Infiltration of bupivacaine into the preperitoneal space and trocar incisions is a promising technique to reduce postoperative pain25.Moreover, postoperative pain is an important variable when deciding the best surgical technique. In TAPP procedures26 observed that the occurrence and intensity of pain was significantly higher in younger patients. Other characteristics may play a role, not only in pain, but also morbidity and mortality, this include smoking27, previous intestinal resection28 and concomitant diseases29.Besides the surgical technique, many factors are important for pain and mortality in hernia repair. Mitura et al.Among the limitations of this study are the small number of cases and the fact that it is based only on a series of procedures carried out by one surgeon. A multicenter randomized study would allow for broader conclusions.We did not find significant epidemiological differences between incarcerated and non-incarcerated hernias. In our experience, with the limitation of a single-surgeon series, laparoscopic hernia repair achieved satisfactory results in terms of feasibility  and safety. Few series about this topic were published. More prospective trials are needed.


Currently, Kaposi’s sarcoma (KS) is treated following the recommendations of international guidelines. These guidelines recommend esophagogastroduodenoscopy/colonoscopy for detecting multicentric KS of visceral lesions. Second primary malignancies (SPMs) are also a common KS complication; however, information on their detection and treatment is unfortunately not yet indicated in these guidelines. This paper reports on an 86-year-old man who suffered from quadruple primary malignancies: skin classic KS with colon adenocarcinoma, oral squamous cell carcinoma (maxilla), and well-differentiated stomach adenocarcinoma. Gastric cancer was incidentally detected during esophagogastroduodenoscopy, which was performed to detect visceral KS. We suggest that esophagogastroduodenoscopy/colonoscopy be routinely performed during the follow-up of patients with KS. As SPMs are crucial complications in patients with KS, these malignancies should be detected as early as possible. Kaposi’s sarcoma (KS) was first described by Moriz Kaposi in 1872 . Among tFor patients with KS, second primary malignancies (SPMs) are a serious problem ,10. SPMsAn 86-year-old male patient first noticed a lesion 8 months prior to the visit and underwent several biopsies at the Okinawa Prefectural Miyako Hospital . Angiosarcoma was histologically suspected, and he was subsequently referred to the Department of Dermatology, Ryukyu University Hospital, in August 2007. A physical examination indicated a 50 × 60 mm pedunculated red mass at the sole of his left foot  with parThe third malignancy, i.e., oral SCC, developed 3 years after the CKS surgery. The patient was referred to the Department of Oral and Maxillofacial Surgery at the Ryukyu University Hospital in September 2010 for further evaluation of the right gum of his upper jaw, in which a 22 × 18 mm mass was located. An EGD demonstrated no tumorous data. A well-differentiated SCC in the maxilla was pathologically diagnosed based on the oral mass biopsy. Neoadjuvant chemotherapy and surgical resection of the oral cancer were planned. The neoadjuvant chemotherapy included bleomycin  and “uracil and tegafur” (450 mg per day for approximately 1 month) and displayed a partial response in the tumor; however, the patient refused to undergo subsequent resection of the oral cancer and refused continuous chemotherapy. The patient was discharged and scheduled for follow-up for his oral cancer at Okinawa Prefectural Miyako Hospital.Helicobacter pylori antibodies were negative. Therefore, endoscopic submucosal dissection was performed (July 2011), diagnosing a well-differentiated adenocarcinoma Type IIa  histologically. No venous or lymphatic invasion was histologically found. The follow-up EGD was done in September 2011, and no tumorous lesion was found. Oral cancer examination revealed progression of the oral mass and mandibular lymphadenopathy. However, no treatment was administered due to the patient’s advanced age. No evidence of lung metastasis was found. In July 2012, oral cancer continued to progress, his condition continued to worsen, and he died of oral cancer at the Okinawa Prefectural Miyako Hospital. This report was submitted for ethical review to the Ethics Committee of the University of the Ryukyus , which waived the requirement for review per institutional protocol because the study did not contain content that requires ethical approval. The Ethics Committee approved the submission and publication of the manuscript in April 27, 2018. Written informed consent was obtained from the patient’s kin for the publication of this case report and the accompanying images. A copy of the written consent is available for review from the Editor-in-Chief of this journal.Although the patient continued to receive treatment for his KS with imiquimod cream at the Okinawa Prefectural Miyako Hospital, his KS exacerbated. Therefore, he was referred again to our institute in June 2011 for further evaluation and treatment. Physically, his tumor nodules had expanded and ulcerated on both soles of his feet and his right thigh . To deteThe following two important issues were noted in this case: (i) to our knowledge, the combination of quadruple PMs  has not been reported, and (ii) we suggest that EGD/CS be routinely performed to detect SPMs during the long-term follow-up of patients with KS.The current case was diagnosed as quadruple PMs using Warren and Gates criteria . To idenWe believe that life-threatening SPMs should be detected in the early stage in patients with CKS. In our case, early-stage gastric cancer was accidentally detected by EGD (3 years and 9 months after the treatment of CKS). However, no universal guidelines (NCCN) exist for patients with CKS. Conversely, European consensus-based interdisciplinary guidelines state that endoscopy is not strongly recommended for patients with CKS . In thesDespite no clinical gastric/colorectal symptoms, EGD/CS was performed in the current case for two reasons: first, to detect multicentric CKS of the visceral regions, and second, to examine colon cancer recurrence because the patient had a history of colon adenocarcinoma. Consequently, gastric SPM was incidentally detected. For patients with KS, SPMs as well as multicentric KS in other sites are reportedly serious complications ,28. Basen = 23) of 61 patients, SPM occurred ≥5 years after the diagnosis of CKS [n = 45) of 108 patients with CKS had SPM on long-term follow-up  [n = 35); among these, 82.9% (n = 29) occurred ≥ 1 year after the diagnosis of CKS [n = 13) experienced SPM > 1 year after the diagnosis of CKS [We think that if clinicians use the guidelines for patients with KS, SPMs cannot be detected at an early stage because clinical symptoms are basically expressed in advanced-stage disease (poor outcome) ,39. Tabls of CKS . Hiatt e8 years) . Remarka8 years) . Hjalgris of CKS . Among ts of CKS . No cancTo detect SPMs or visceral KS in patients with KS, EGD/CS is a more useful approach than positron-emission tomography (PET)/CT. PET/CT has been well-reported to detect SPMs in various body sites of patients with primary cancer ,43,44,45We report a rare case of CKS associated with quadruple PMs. We suggest that EGD/CS should be routinely performed during the follow-up of patients with KS. As SPMs are a crucial complication in patients with KS, they should be detected as early as possible.