Herein, kinetic investigations reveal that low-molecular-weight phenolic compounds naturallypresent within wetland ecosystems  are accepted by five recombinantly expressedwetland tyrosinases (TYRs) as substrates. Investigations of the interactionsbetween TYRs and wetland phenolics reveal two novel mechanisms thatdescribe the global impact of TYRs on the wetland carbon cycle. First,it is shown that o-quinones (produced by TYRs fromlow-molecular-weight phenolic substrates) are capable of directlyinactivating hydrolytic enzymes. Second, it is reported that o-quinones can interact with high-molecular-weight phenolicpolymers (which inhibit hydrolytic enzymes) and remove them throughprecipitation. The balance between these two mechanisms will profoundlyaffect the fate of wetland carbon stocks, particularly in the wakeof climate change.Wetlands are globallydistributed ecosystems characterizedby predominantlyanoxic soils, resulting from water-logging. Over the past millennia,low decomposition rates of organic matter led to the accumulationof 20–30% of the world’s soil carbon pool in wetlands.Phenolic compounds are critically involved in stabilizing wetlandcarbon stores as they act as broad-scale inhibitors of hydrolyticenzymes. Tyrosinases are oxidoreductases capable of removing phenoliccompounds in the presence of O Two novel mechanismsdescribing the impact of tyrosinaseson carbon storage in wetland ecosystems have been identified usingfive recombinantly expressed tyrosinases produced by bacteria indigenousto globally distributed wetland ecosystems. Phenolic compounds act asunspecific enzyme inhibitors and are naturally abundant within wetlandecosystems13 due to plant secondary metabolism. Enzymescapable of oxidatively removing phenolic compounds in the presenceof molecular oxygen (or H2O2 for peroxidases)are often grouped under the umbrella term “phenol oxidases”,and include, among others, tyrosinases, laccases, and peroxidases.4 In wetlands, the activity of phenol oxidases is restricted by oxygenscarcity, resulting from water-logging.13 Climate change, which will lead to increased temperatures and reducedrainfall, threatens water tables in wetlands and will, therefore,promote the aeration of previously anoxic wetland soils. This, inturn, will lead to increased levels of phenol oxidase activity, aconsecutively reduced concentration of phenolic compounds, and anincreased activity of organic matter degrading hydrolases.14 As a consequence, the stability of wetland carbon stores is at risk,and vast amounts of carbon will potentially be emitted back into theatmosphere, which itself will further promote climate change.Wetlands are ecosystems characterizedby permanently or seasonallywater-logged soils in combination with plant growth.on sinks 1A. Accor18 however, contradictory observations led to the development of complementarymechanisms describing the processes controlling carbon cycling withinwetland ecosystems.23 In a recent study, several enzyme groups involved in anoxic phenol metabolism in wetland soils have been identified,which are not affected by the “latch mechanism”.19 Furthermore, Wang et al. demonstrated in analpine wetland that the enzymatic breakdown of soil organic matterand phenolic compounds is dependent on the relative concentrationsof Fe(II) and Fe(III) ions, which potentially counteract the “latchmechanism”.20 Consequently, it wasconcluded that several mechanisms  in concertcontrol carbon cycling in wetland ecosystems.25Ample experimental evidence supporting the centralhypothesis ofthe “latch mechanism” 1A has be23 investigations focused on theprecise enzymes controlling these processes are scarce. A recent reviewshowed that a variety of bacteria capable of producing tyrosinase enzymes (TYRs) are present in globallydistributed wetland ecosystems .4 Thus, it has been concluded that TYRs are among the key enzymescontrolling carbon cycling within wetland ecosystems .4 The relative impacts of TYRs, laccases, peroxidases,and enzymes involved in anoxic phenol metabolism on carbon cyclingin wetland ecosystems require further investigation.While numerous studies simulated the effects of climate changeon carbon cycling within wetland ecosystems on a macroscopic scale,28 bacteria,29 fungi,30 plants,31 and animals,32 including humans.33 They catalyze the o-hydroxylation of monophenolsto o-diphenols (EC 1.14.18.1), as well as the subsequentoxidation of o-diphenols to o-quinones(EC 1.10.3.1), which is coupled to the reduction of molecular oxygento water , l-tyrosine (monophenol), dopamine (diphenol),and l-DOPA (diphenol) often used as standard substrates fortesting mono- and diphenolase activities.4 Several phenolic compounds naturally abundant within wetland ecosystems have been reported to be acceptedby bacterial TYRs as substrates.4 Structuralinvestigations of TYRs revealed a conserved architecture of the activecenter while the overall folds of TYRs show little conservation.38 In the active center, two copper ions are coordinated by three histidineresidues each .41 Second shell amino acids located in a distance of ∼5–15Å around the active center are responsible for substrate selectionand orientation and, therefore, govern the substrate scope and substratepreferences.44 Bacterial TYRs (except TYRs from Streptomyces and Bacillus species) are expressed in their latentstates and require an activation step to develop catalytic activity. In vivo, this activation is proposedly achieved by an autolyticmechanism,45 which can be mimicked in vitro by the addition of suitable molarities of SDS.48TYRs aretype III copper proteins featuring a dicopper center andare ubiquitously distributed in nature among archaea,to water 1B.27 KinCanSTYR, CabSTYR, SinATYR, PseSTYR, and ChrSTYR) identifiedwithin the genomes of bacteria indigenous to wetland ecosystems andoriginating from a phylogenetically diverse set of host organisms showedthat phenolic compounds naturally present within wetland ecosystemsare commonly accepted as substrates. Furthermore, in-depth investigationsof the interactions between SinATYR and wetland phenolics have been performed. SinATYR representsa planctomycetal TYR from Singulisphaera acidiphila, identified within a peatland in Russia. Importantly, these investigationsrevealed two novel and competing mechanisms that describe how TYRsimpact the stability of organic carbon stored in wetland ecosystems.First, wetland carbon stores will be stabilized by increased TYR activitydue to the inactivation of hydrolytic enzymes by quinones , which are naturallypresent within wetland ecosystems. Second, results presented hereindemonstrate that high-molecular-weight phenolic polymers, responsiblefor the inhibition of hydrolytic enzymes, can be partially removedvia precipitation as a result of increased TYR activity. IncreasedTYR activity in wetland soils has become a likely scenario in recentyears due to climate change.50 The balance between the two novel mechanisms presented herein willimpact the fate of wetland carbon stores and will, consequently, affectthe future development of the global climate itself.This study has been performed to straddle the previously disparateresearch areas of macroscopic investigations of carbon fluxes withinwetland ecosystems and biochemical investigations of TYR enzymes.Biochemical investigations of five recombinantly expressed TYR enzymes using protocols developed and optimized for eachof the five TYRs. Optimized parameters included the purification tag,expression time, expression temperature, and lysis buffer composition.Expression conditions as well as expression yields of the respectiveactive and purified enzymes are reported in Table S2. Bacterial TYRs have previously been expressed using a His-tag,63 while several expression protocols of plant and fungal TYRs utilizea GST-tag.66 Both of thesetags (His-tag and GST-tag) were successfully employed in this studyto produce active enzymes in high yields and purity (Table S2). Expression yields for purified and active TYRsranged from 4.1 mg/L of expression medium (CabSTYR)to 65 mg/L of expression medium (CanSTYR). In comparison,the literature-reported expression yields of bacterial TYR enzymesvaried between 10 mg/L expression medium (Streptomycescastaneoglobisporus)59 and80 mg/L expression medium (Bacillus megaterium).60CanSTYR, CabSTYR, and SinATYR showed maximum yieldswhen expressed at low temperatures  and displayed significantly reducedyields (less than 10%) at expression temperatures of 20 °C orhigher. Previously reported expression temperatures for bacterialTYRs ranged between 16 °C (Bacillus aryabhattai)61 and 37 °C (B.megaterium).60 Althoughhigher expression temperatures allow for shorter expression times,we propose that low-temperature expression provides a suitable wayto increase the yield of recombinantly expressed bacterial TYRs.Genes codingfor wetland TYRs have been obtained bywhole genesynthesis , and the correspondingTYR enzymes 1 have bep-coumaricacid37 and p-hydroxybenzoic acid34), diphenols (protocatechuic acid34 and caffeic acid35), triphenols, methoxylated phenols , and flavonoids. Substrate scopeassays  were acceptedby all investigatedTYRs , p-hydroxybenzoic acid , and quercetin  were partiallyaccepted (S1) and can, therefore, be expectedto be involved in the “latch mechanism” to a variableextent dependent on the substrate scope of TYRs present in the respectivewetland ecosystem. In a previous study, a TYR from a Streptomyces species isolated from globally distributed wetlands (located inAustria67 and China68) has been demonstrated to accept phenolic compounds naturallypresent within its habitat. Accordingly, the results presented hereindemonstrate that TYRs present within the genomes of phylogeneticallydiverse and globally distributed host organisms indigenous to wetlandecosystems accept a broad scope of phenolic (nonmethoxylated) compoundspresent within their natural environment  were determined for recombinantlyexpressed TYRs , diphenols arepreferred over monophenols, which is in accordance with previous reports.29 When comparing the kcat values of correspondingmono- and diphenols , all investigated enzymes showed higheractivity levels toward the diphenolic substrate, compared to the correspondingmonophenolic substrate  per reaction cycle. In contrast, when monophenols areconverted, TYRs consume one molecule of O2 and generateonly one reaction product (one o-quinone) per reactioncycle  revealedthat the flavanol substrates (catechin and epicatechin) show higheractivity levels , while quercetin(flavonol) and taxifolin (flavanonol) exhibit higher levels of affinity  moreefficiently , while atlowconcentrations , quercetin and taxifolin areconverted more efficiently. This data proves that (besides diphenolsand monophenols) flavonoids, which are commonly encountered in wetlandssuch as mangrove forests,37 are accepted by a phylogeneticallydiverse set of TYRs as substrates.Analyzing kinetic data from flavonoid substrates  2. Thus, 76 and its activitycan be determined reliably and accurately using 4-methylumbelliferyl-β-d-glucopyranoside as a substrate.79 First, the direct inhibitionof β-glucosidase by low-molecular-weight phenolic TYR substrates has been investigated. For this purpose,β-glucosidase has been incubated with different concentrations, while β-glucosidase activity has been determinedafter 24, 48, 72, and 96 h. These measurements revealed only low-levelinhibition of β-glucosidase activity by any of the low-molecular-weightphenolic substrates , compared to β-glucosidase incubated without a low-molecular-weightphenolic compound under the same conditions. This suggests that low-molecular-weightphenolic TYR substrates do not contribute significantly to carbonstorage in wetland ecosystems by inhibiting β-glucosidase.Next, the impact of increasedTYR activity on the activities of hydrolyticenzymes was investigated to elucidate the effects of increased TYRactivity on the stability of wetland carbon stores. For this purpose,β-glucosidase has been chosen as a model enzyme since its involvementin the hydrolytic decomposition of soil organic matter is well establishedp-coumaric acid; Figure S1). This time,recombinantly expressed SinATYR has been added beforeincubating the samples  for 4 days withβ-glucosidase activity determined after 24, 48, 72, and 96 h. Moreover, since S. acidiphila is commonly encountered in wetlandecosystems and represents an aerobe organism, efficient expressionof SinATYR can be expected in its natural environment,following the aeration of previously anoxic wetland soils due to climatechange.80 In contrast to our previous experiments,β-glucosidase activity decreased significantly after the additionof SinATYR . This inhibition was dependent on the incubation time, the concentration,and the chemical structure of the low-molecular-weight phenolic TYRsubstrate , concentration-dependentinhibition has been observed. In the presence of SinATYR, p-coumaric acid exhibited the highest β-glucosidase-inhibitionpotential, followed by caffeic acid, tyramine, and catechin (Table S6), which is a well-documented phenomenon.81 It is caused by the reactivity of quinones towardamino groups and thiols , whichare commonly present in proteins. This leads to one or more of thefollowing reactions, all of which potentially reduce the enzymaticactivity: (a) the direct binding of the quinone to the protein,81 (b) the cross-linking of two enzyme molecules,81 and (c) the formation of a protein radical.81 In the context of TYRs in wetland ecosystems,these results are of significant relevance as they point toward theinactivation of hydrolytic enzymes (such as β-glucosidase) byquinones formed by wetland TYRs following the aeration of previouslyanoxic wetland soils. Since the reaction products (o-quinones) of TYR activity are responsible for this effect and differentTYR enzymes generate the identical reaction product from a respectivesubstrate,29 comparable results can be expectedfor other TYR enzymes, provided they accept phenolic compounds presentin their environment as substrates. This first mechanism might explaininvestigations reporting decreasing or unchanging emission rates fromwetland soils following aeration.83Next, the inhibition of β-glucosidase bylow-molecular-weightphenolic compounds in the presence of increased TYR activity has beeninvestigated. For this purpose, β-glucosidase has been mixedwith different concentrations  havebeen determined in the absence of TYR activity, which revealed thathumic acids have a more pronounced inhibitory effect on β-glucosidaseactivity, compared to lignin-derived polymers  by TYRs as substrates. To substantiatethis hypothesis, molecular docking studies have been performed, whichrevealed for all five investigated TYRs that high-molecular-weightphenolic polymers, in fact, cannot access the active center of TYRs. In TYRs, the hydroxy groupsof low-molecular-weight phenolic substrates are oriented toward thedicopper center in a distance of 2–3 Å ,86 which is a prerequisite for catalytic activity. Incontrast, our docking experiments revealed for all five investigatedTYRs that hydroxy groups in high-molecular-weight phenolic polymersare sterically blocked from accessing the active center due to thebulky structure of the polymer . Thus, while high-molecular-weight phenolic polymers have a significantimpact on the activities of hydrolytic enzymes, they will not be directlyaffected by increased TYR activity caused by the aeration of previouslyanoxic wetland soils.Following investigations focused on theinhibition of β-glucosidase by low-molecular-weight phenolicTYR substrates, the inhibition of hydrolytic enzymes (with β-glucosidaseas a model enzyme) by high-molecular-weight phenolic polymers, suchas lignin decomposition products (lignosulfonic acid) and humic acids,has been investigated. These compounds have previously been reportedto function as suitable representatives for phenolic polymers naturallyencountered within wetland ecosystems.via ultrafiltration. Thismethod has previously been shown to yield fractions with similar physicochemicalproperties , which are separate according to theirdegree of polymerization.87 For humic acidsand lignosulfonic acids, the relative quantities (in terms of mass)have been determined for the fraction <30 kDa and SinATYR (1 mg/L). As a negative control, humicacids and lignosulfonic acids (1 g/L each) have been incubated with p-coumaric acid (100 mg/L) but without SinATYR. The molecular weight distribution profiles of humic acids andlignosulfonic acids have been redetermined after 48 h and revealedsubstantial shifts in their degree of polymerization for samples withadded SinATYR . For humic acids and lignosulfonicacids, the molecular weight distribution shifted toward higher molecularweights, indicating an increased degree of polymerization. This effecthas been observed to a similar degree for the monophenolic substrate p-coumaric acids and for the corresponding diphenolic substratecaffeic acid  and has been morepronounced for humic acids, compared to lignosulfonic acids  has been investigatedfor the first time. In a natural wetland environment, low-molecular-weightphenolic compounds are present alongside high-molecular-weight phenolicpolymers. Consequently, quinones produced by TYRs (from low-molecular-weightphenolic TYR substrates) can not only react with proteins, as hasbeen demonstrated for β-glucosidase within this study, but alsoreact with high-molecular-weight phenolic polymers. To yield furtherinformation on the effects of increased TYR activity on carbon cyclingwithin wetland ecosystems, the interplay between low-molecular-weightphenolic compounds (which are accepted by TYRs as substrates) andhigh-molecular-weight phenolic polymers  in the presence of TYR activityhas been investigated. Therefore, humic acids and lignosulfonic acids(52 kDa), both of which represent complex mixtures of polymers, havebeen characterized in terms of their molecular weight distributionprofile by fractionation  SinATYR 4. In con SinATYR S8. For h SinATYR S8. For ho-quinones can interact with high-molecular-weightphenolic polymers in two main ways. They can either bind directlyto the polymer, thus adding their molecular weight to the mass ofthe polymer, or they perform redox exchange. o-quinoneshave been shown to react with thiol groups, the sulfur atom of thioethergroups, amino groups, and phenolic hydroxy groups, and, therefore, o-quinones have the potential to react with humic acidsand lignosulfonic acids .88 On the other hand, redox exchange leads to thereduction of a quinone (produced from a low-molecular-weight phenoliccompound by a TYR) to the corresponding diphenol, while the redox-exchange-partner gets oxidized to the corresponding quinone .88 This“secondary quinone” (formed via redoxexchange) can then undergo the aforementioned reactions, which canconsequently lead to the cross-linking of two polymer molecules, whichdrastically increases the molecular mass of the resulting molecule.Since the solubility of humic substances is negatively correlatedto their molecular weight,90 this process can resultin the precipitation of the cross-linked polymer, as has been observedin our study  and to originate fromdifferent geographic locations (Five TYR sequences previously identified withinthe genomes of bacteria indigenous to wetland ecosystemsocations 1.Escherichia coli) nucleotide sequencescoding for CanSTYR, CabSTYR, SinATYR, PseSTYR, and ChrSTYR (Table S1) were obtained from a commercialsupplier  and cloned into thepGEX-6P-SG expression vector91 adjacentto either a GST-tag  or a 10xHis-tag (CabSTYR and SinATYR) using the restriction endonuclease Esp3I via restriction enzyme recognitionsites introduced into the optimized sequence. The ORFs were sequenceverified in the forward and reverse directions using Sanger sequencingprovided by a commercial supplier .The codon-optimized (toward the codon usageof E.coli BL21(DE3) and purified using affinity chromatography. The successful expression of TYR enzymes was checkedusing SDS-PAGE  and ESI-MS . A detailed descriptionof the expression and purification procedure can be found in the Supporting Materials and Methods Section.All enzymes were heterologously expressed in Supporting Materials and Methods Section. 100 μg of recombinantly expressedand purified TYRs was mixed with 1 mM of one of the following phenolicsubstrates: caffeic acid, catechin, dopamine, epicatechin, ferulicacid, gallic acid, l-DOPA, p-coumaric acid, p-hydroxybenzoic acid, protocatechuic acid, syringic acid,tyramine, l-tyrosine, and vanillic acid . The activities toward the flavonoid substratesisorhamnetin, kaempferol, quercetin, and taxifolin, which do not yieldcolored reaction products and show poor solubility in water, wereassessed in a reaction mixture containing 10% DMSO supplemented with5 mM MBTH (3-methyl-2-benzothiazolinone hydrazine hydrochloride hydrate).Optimal SDS molarities  were added, and the pH of the reaction mixture was adjusted with50 mM MES (2-(N-morpholino) ethanesulfonic acid; CanSTYR and CabSTYR) or 50 mM Tris (tris(hydroxymethyl)aminomethane; SinATYR, PseSTYR, and ChrSTYR) to the pH optimum of the respective enzyme . The reactions were performed in atotal volume of 200 μL. The activity was assessed visually bya clear change in color within 24 h at 22 °C.The determinationof the SDS optima and pH optima of recombinantly expressed TYRs isdescribed in detail in the 92 or have been determined withinthe scope of this study .92 Adequate amounts of the TYR enzyme (Table S4) were mixed with a substrate in a 50mM buffer solution  supplemented with previously identified optimalSDS molarities .Maximum reaction rates were determined for seven to eight substratemolarities per substrate–enzyme combination in triplicate.Nonlinear curve fitting of the Michaelis–Menten equation towardthe measured reaction rates implemented in OriginPro 8 software wasused to calculate Km values and kcat values .Kinetic parameters 2 for enzp-coumaric acid, caffeic acid, catechin, lignosulfonic acids(18 and 52 kDa), and humic acids  in 50 mM Tris-HCl (pH7.0). SinATYR (1 mg/L) was added to the mixture forsamples with TYR activity. Samples solutions were incubated at 4 °Cand β-glucosidase activity has been determined after 24, 48,72, and 96 h.β-glucosidase was incubated with tyramine, d-glucopyranoside(Sigma-Aldrich) was used to measure β-glucosidase activity.Fluorescence measurements were performed on a TECAN infinite M200reader 77 in triplicate. Foreach measurement, 10 μL of sample solution was mixed with 5μL of 10 mM 4-methylumbelliferyl-β-d-glucopyranoside(dissolved in 100% 2-methoxyethanol). The solution was filled up to200 μL with H2O and adjusted to 50 mM Tris-HCl (pH7.0). As a control sample, β-glucosidase (1 mg/L) was incubatedat 4 °C in 50 mM Tris-HCl (pH 7.0) without additional phenolics.4-Methylumbelliferyl-β-via evaporation.Vivaspin ultrafiltration devices  and Microsept advance ultrafiltration devices  were used for the fractionation of phenolic polymers. Phenolicpolymers were separated by centrifuging at 4.000 g at 20 °C.Phenolic contents in the supernatant  and flow-through  were determined gravimetrically in triplicate after removingthe solvent (water)


This paper describes the dataset associated with the paper “Product-Specific Human Appropriation of Net Primary Production (HANPP) in US Counties” . This dataset comprises human appropriation of net primary production (HANPP) values for 3101 counties in the conterminous US for the years 1997, 2002, 2007, and 2012. For this dataset, HANPP is the carbon content of specific crop, timber, and livestock grazing products appropriated by humans in a county in a year. To calculate HANPP, raw agricultural data were downloaded from public databases such as USDA-National Agricultural Statistics Service Quick Stats and Cropland Data Layer, US Forest Service Timber Product Output, and NPP data from MODIS. These data were processed in Microsoft Excel using stoichiometry derived from established scientific literature. HANPP was partitioned by year, county, product, used and unused and above- and below-ground. This complete dataset is published in Mendeley Data and the methods used to compile them are included to make our research well documented, reproducible, and useful for future studies. Specifications Table1•These data are useful because they provide the first product-specific, county-level measurements of HANPP in the U.S., a valid indicator of human land use intensity and an ecological indicator that has advantages over ecological footprint due to its spatial specificity and empirical foundation in net primary production.•These data will be beneficial to researchers and land managers who want to measure HANPP on a large scale in relation to other environmental variables.•HANPP can be used in further experiments that look at the effect of human land use on biodiversity, how HANPP can be broken down into different subsets, or how HANPP relates to ecosystem service provision.•These production-oriented data on HANPP can be paired with data on HANPP consumption and trade to delineate a system of ecological interdependencies.•HANPP data can be used to describe ‘anthromes’ as human-oriented ecological categories in contrast to biomes.2Product-Specific Human Appropriation of Net Primary Production (HANPP) in US CountiesData in Brief article adds to the original research paper by including more details about the data collection, processing, and database structure.This dataset compiles measurements for 3101 counties in the conterminous U.S. and on a product-specific basis in five-year intervals. The article 3−2yr−1 and HANPP mass in kilotonnes C. There are separate folders for the different measurements of HANPP. Within each unit folder are five spreadsheet files that contain the total county HANPP, HANPP partitioned into aboveground and belowground, and HANPP partitioned into used and unused. Note that grazing HANPP is defined as entirely aboveground and entirely used. Note also that we did not calculate onsite HANPP densities for timber because the locations of harvest within a county are undetermined.This data repository contains the processed data for HANPP measurements. The HANPP data sets are organized into four folders representing the four years for which HANPP was calculated . Within each year are folders representing the different measurements of HANPP; county-wide HANPP and onsite HANPP density in gCm−2yr−1 or kilotonnes), and the year . Each row contains data for a single county. The following list describes the data included in each column:STATE: The state that the county is in.STATE_FIPS: The government Federal Information Processing Standard FIPS code for the state.STATE_COUNTY: The state and county, listed in the following form: ALABAMA_AUTUAGA. This value is unique and can be used as a primary key.COUNTY: The county name.COUNTY_FIPS: The government FIPS code for the county.YEAR: The year that HANPP is being calculated.COUNTY_AREA: The area of each county in meters squared.corn_grain: HANPP of corn grain.corn_silage: HANPP of corn silage.winter_wheat: HANPP of winter wheat.spring_wheat_durum: HANPP of durum spring wheat.spring_wheat_excluding_durum: HANPP of spring wheat other than durum.soybeans: HANPP of soybeans.hay_alfalfa: HANPP of alfalfa hay.cotton_pima: HANPP of pima cotton.cotton_upland: HANPP of cotton upland.sorghum: HANPP of sorghum.other_crops: Average HANPP of other crops that make up the remaining 23% of cropland, after the other top 10 crops.total_crops: HANPP of all crops, which is the sum of columns “corngrain” through “other_crops”.hardwood: HANPP of hardwood.softwood: HANPP of softwood.total_timber: HANPP of all timber, which is the sum of “hardwood” and “softwood”.blm_grazing: HANPP of grazing that takes place on Bureau of Land Management land.usfs_grazing: HANPP of grazing that takes place on US Forest Service land.private_land_grazing: HANPP of grazing that takes place on private land.total_grazing: HANPP of all grazing, which is the sum of “blm_grazing,” “usfs_grazing” and “private_land_grazing”.total_hanpp: The total HANPP for the county, which is the sum of “total_crops,” “total_timber” and “total_grazing.”File Organization: For each HANPP spreadsheet file, each filename includes the type of HANPP , the method of HANPP (county-wide or onsite), the units of HANPP , NPP, and NPP measurements) and “national_HANPP” . The “HANPP_NPP_NPPeco” folder contains two folders for county and national totals. The county totals folder contains three folders for the years 2002, 2007, and 2012 (there is no NPP data available for 1997). Within each of these year-based folders are two spreadsheet files for gCm−2yr−1 and tonnes.In the repository there are also Microsoft Excel spreadsheets that contain the formulas and methodology to calculate HANPP for crops and timber. These are called “HANPP_crop_calculator” and “HANPP_timber_calculator.” To utilize the timber HANPP calculator sheet, the user inputs timber harvest data at the county level in units of cubic feet for roundwood and mill residues, as well as all removals, for both hardwood and softwood. To utilize the crop HANPP calculator, the user inputs crop harvest data in the indicated units, which conform to USDA-NASS Quick Stats listings. The stoichiometry formulas in the spreadsheet will output HANPP measurements for specific products for HANPP total, aboveground, belowground, used, and unused in both gCmIn the repository there is also an R markdown file that contains code used to produce the HANPP bar charts that we included in the accompanying paper. This code can be modified to produce different county, state, or national HANPP bar charts. There is commentary in the document to help guide the user.4To calculate total HANPP for each county, we used a detailed, bottom-up approach. This was done by calculating three subsets of HANPP  on a product-specific basis and adding them together to get a total HANPP signature for each county. The Mendeley Data page includes crop and timber HANPP calculators that users can employ for years not included in the study.4.1We obtained crop yield data for each county for all four years  from USDA-National Agricultural Statistics Service Quick Stats HANPP (harvest) = (economic yield * dry fraction *carbon content) / (harvest index * % shoot)We developed crop-specific stoichiometry estimates from peer-reviewed, gray, and extension literature to estimate the different economic yield, dry fraction, carbon content, and harvest index percent shoot for each of the crops . Economi−2yr−1) is the national mean for minor crops. We also assumed this to be constant for 1997, 2002 and 2007 because Cropland Data Layer data only became available for the entire U.S. in 2008.In addition to calculating HANPP for the main crops, we also calculated HANPP of all minor crops aggregated together (we refer to them in the dataset as “Other Crops”). The Cropland Data Layer in 2012 was used with ArcGIS software to identify the acreage of minor crops in each county. However, data on yields and the stoichiometry of these numerous minor crops are not available. We therefore assumed that the on-site HANPP density of major crops  for each CONUS county in the years 1997, 2002, 2007 and 2012 were obtained from the U.S. Forest Service database Timber Product Output (TPO) −2yr−1. On-site densities could not be calculated because the data are not site-specific within the county and the U.S. Forest Service does not report the area harvested.This produced timber HANPP measures for hardwood and softwood for each county in each year in total metric kilotonnes of carbon and county-average density in gCm4.3−2yr−1 for each allotment in the most recent permit.Estimating HANPP from livestock grazing is less straightforward than crops or timber because grazing takes place on both public and private lands that have quite different data sources. For public lands, the USDA Forest Service (USFS) and the Bureau of Land Management (BLM) provide permits to ranchers in the form of animal-unit months (AUMs) appurtenant to allotments of land For private lands, we first quantified the NPP resources contained in grassland and pasture in each county. This was done by matching 30m pixels classified as grassland/pasture in the Cropland Data Layer with NPP data from Landsat in 2012. We then categorized counties into 20 USDA Land Resource Regions (LRR) that may vary in the percentage of NPP that is utilized by livestock. Within each LRR, counties were selected that had a population below 100,000 and lacked confined animal feedlot operations (CAFOs). We then quantified grazing demand by obtaining data on the number of beef cattle from the USDA-NASS Cattle Inventory for the years 1997, 2002, 2007, and 2012. Each beef cattle was assumed to require 12 AUMs or 1944 kgC per year from grazing. The total grazing demand for selected counties in each LRR was then compared to NPP resources in those counties to derive a percentage of NPP grazed as the measure of HANPP in each respective LRR . These c−2yr−1. Grazing HANPP was defined as entirely used and entirely above-ground.Total grazing HANPP in each county was divided by the area of grassland/pasture to obtain on-site density and by the area of the county to obtain county-wide HANPP density in gCm4.4−2yr−1. HANPP in kilotonnes was calculated by summing all HANPP totals, while HANPP in gCm−2yr−1 was calculated by summing all HANPP in gC but dividing by the total area of all counties in m2. These calculations were done using Microsoft Excel software.All crop, timber, and grazing HANPP was summed to get a measurement of total HANPP for the 3101 CONUS counties. This was then summed to get a measurement of national HANPP. National HANPP totals are in the units of total metric kilotonnes of carbon and densities are measured in gCm4.5−2yr−1 and aggregating 250m pixels for each county. NPP was calculated by subtracting total HANPP(harvest) from each county from NPP in terms of kilotonnes and county-wide density in gCm−2 yr−1.The net primary production of each county was calculated by downloading Moderate Resolution Image Spectroradiometer (MODIS) data Data in Brief. Neither human study participants, animals, nor social media platforms were involved in collecting the data presented here. All data used in the study are public domain.The authors have read and followed the ethical requirements for publication in Suman Paudel: Methodology, Software, Validation, Formal analysis, Investigation, Data curation, Visualization. Kaeli Mueller: Software, Validation, Formal analysis, Data curation, Writing – original draft, Visualization. Gustavo Ovando-Montejo: Methodology, Software, Validation, Formal analysis, Investigation, Data curation, Visualization. Lauren Tango: Methodology, Software, Validation, Formal analysis, Data curation. Richard Rushforth: Conceptualization, Methodology. Christopher Lant: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Writing – review & editing, Supervision, Project administration, Funding acquisition.


Antimicrobial resistance (AMR) is a global health threat with 1.27 million and 4.95 million deaths attributable to and associated with bacterial AMR, respectively, in 2019. Our aim is to estimate the vaccine avertable bacterial AMR burden based on existing and future vaccines at the regional and global levels by pathogen and infectious syndromes.We developed a static proportional impact model to estimate the vaccination impact on 15 bacterial pathogens in terms of reduction in age-specific AMR burden estimates for 2019 from the Global Research on Antimicrobial Resistance project in direct proportion to efficacy, coverage, target population for protection, and duration of protection of existing and future vaccines.Mycobacterium tuberculosis and Streptococcus pneumoniae by pathogen. In the baseline scenario for vaccination of primary age groups against 15 pathogens, we estimated vaccine-avertable AMR burden of 0.51 (95% UI 0.49–0.54) million deaths and 28 (27–29) million disability-adjusted life-years (DALYs) associated with bacterial AMR, and 0.15 (0.14–0.17) million deaths and 7.6 (7.1–8.0) million DALYs attributable to AMR globally in 2019. In the high-potential scenario for vaccination of additional age groups against seven pathogens, we estimated vaccine-avertable AMR burden of an additional 1.2 (1.18–1.23) million deaths and 37 (36–39) million DALYs associated with AMR, and 0.33 (0.32–0.34) million deaths and 10 (9.8–11) million DALYs attributable to AMR globally in 2019.The AMR burden avertable by vaccination in 2019 was highest for the WHO Africa and South-East Asia regions, for lower respiratory infections, tuberculosis, and bloodstream infections by infectious syndromes, and for Increased coverage of existing vaccines and development of new vaccines are effective means to reduce AMR, and this evidence should inform the full value of vaccine assessments. Haemophilus influenzae type b, rotavirus, Streptococcus pneumoniae, Salmonella typhi and influenza on antimicrobial resistance (AMR) in specific settings.There is some evidence on the impact of vaccines against To our knowledge, this is the first study to estimate attributable and associated bacterial AMR burden avertable by vaccination against 15 bacterial pathogens for a combined set of existing and new vaccines in the pipeline by pathogen, infectious syndrome and region.Mycobacterium tuberculosis and Streptococcus pneumoniae by pathogen.The AMR burden avertable by vaccination in 2019 was highest for the WHO Africa and South-East Asia regions, for lower respiratory infections, tuberculosis and bloodstream infections by infectious syndromes, and for Our model-based projections facilitate evidence-based decision-making for scaling up of existing vaccines to regions in most need with higher AMR burden and prioritise development of new vaccines with high potential for lowering AMR burden by pathogen, infectious syndrome and region.Our study contributes to the WHO-led value attribution framework for vaccines against AMR, and specifically to the criterion focused on vaccine averted AMR health burden.Since the discovery of penicillin in 1928, antimicrobials have been used to treat bacteria, fungi, parasites and viruses, saving countless lives.3Vaccination, when used in conjunction with other preventive measures, has the potential to significantly reduce AMR transmission through several pathways.6The Global Research on Antimicrobial Resistance (GRAM) project estimated the deaths and disability-adjusted life-years (DALYs) attributable to and associated with resistance by replacing all drug-resistant infections with susceptible infection or no infection, respectively. It estimated that 1.27 (95% UI 0.91–1.7) million deaths and 47.9 (35–64) million DALYs were attributable to bacterial AMR and 4.95 (3.6–6.6) million deaths and 192 (146–248) million DALYs were associated with bacterial AMR in 2019.To address this evidence gap, our aim is to analyse the findings from the GRAM project and estimate the vaccine-avertable bacterial AMR burden based on the profiles of existing and future vaccines by pathogen and infectious syndromes at the regional and global levels in 2019. Such pan-pathogen analyses using standardised approaches are critical to inform vaccine development, funding, introduction and use. They also inform the WHO-led value attribution framework for vaccines against AMR,Neisseria gonorrhoeae include only morbidity and no mortality.We used the bacterial AMR burden estimates from the GRAM project which provided data for age-specific deaths and DALYs associated with and attributable to AMR by pathogen, infectious syndrome and region for 2019.Two sets of estimates are presented—burden attributable to AMR, that is, deaths and DALYs that could be averted if all drug-resistant infections would be replaced by drug-sensitive infections; and burden associated with AMR, that is deaths and DALYs that could be averted if all drug-resistant infections would be replaced by no infections. As vaccines prevent drug-resistant and drug-susceptible burden, we infer that the associated AMR burden is the appropriate metric for measuring the impact of vaccination on AMR burden.—Acinetobacter baumannii, Enterococcus faecium, Escherichia coli, Group A Streptococcus, Haemophilus influenzae, Klebsiella pneumoniae, Mycobacterium tuberculosis, Neisseria gonorrhoeae, non-typhoidal Salmonella, Pseudomonas aeruginosa, Salmonella paratyphi, Salmonella typhi, Shigella spp, Staphylococcus aureus and Streptococcus pneumoniae. We selected pathogens that are part of the WHO evaluation of the value of vaccines in preventing AMR. We used vaccine profiles  for two scenarios—baseline scenario (for 15 pathogens) for primary vaccination of specific age groups, and high-potential scenario (for a subset of 7 pathogens) that includes additional age groups at risk of infection based on expert opinion.A. baumannii, E. faecium, Extraintestinal Pathogenic E. coli (ExPEC), K. pneumoniae , P. aeruginosa and S. aureus. For S. pneumoniae, we explored the high-potential scenario by administering a vaccine to an elderly population with the highest disease burden.Vaccine profiles with the corresponding product characteristics for efficacy and duration of protection for vaccine-derived immunity, and coverage and target population for the baseline and high-potential scenarios are described in We conducted a Monte Carlo simulation of 400 runs (sufficient for results to converge) to propagate the uncertainty in the AMR burden, vaccine efficacy and coverage through the model simulations to estimate the uncertainty in our projected outcomes of vaccination impact. We provide summary estimates in terms of vaccine-avertable deaths and DALYs attributable to and associated with AMR by region, infectious syndrome and pathogen with 95% UIs for the baseline and high-potential scenarios. Additional details on the modelling process, scenarios and uncertainty analysis are provided in 10.1136/bmjgh-2022-011341.supp1Supplementary dataWe analysed anonymised secondary data in our study. The data analysed originated from the GRAM project, and they were analysed in aggregate. As a result, it was not appropriate or possible to involve patients or the public in the design, or conduct, or reporting, or dissemination plans of our research. The public will benefit from the findings of our study as our model-based projections facilitate evidence-based decision-making for scaling up of existing vaccines to regions in most need with higher AMR burden and prioritise development of new vaccines with high potential for lowering AMR burden by pathogen, infectious syndrome and region.https://github.com/vaccine-impact/vaccine_amr and Dryad open data publishing platform.17We conducted our analysis using the R (version 4.2.3) programming language for statistical computing,At the global level in 2019 for the baseline scenario, we estimated that vaccines against the 15 pathogens  could avert 0.51 (95% UI 0.49–0.54) million deaths and 28 (27–29) million DALYs associated with AMR, and 0.15 (0.14–0.17) million deaths and 7.6 (7.1–8.0) million DALYs attributable to AMR. In the high-potential scenario, we estimated that vaccines against a subset of 7 pathogens could avert an additional 1.2 (1.18–1.23) million deaths and 37 (36–39) million DALYs associated with AMR, and 0.33 (0.32–0.34) million deaths and 10 (9.8–11) million DALYs attributable to AMR globally in 2019.S. pneumoniae at 2019 coverage levels averted 44 (37–52) thousand deaths and 3.8 (3.3–4.5) million DALYs associated with AMR in 2019. By reaching the WHO recommended coverage level of 90% globally, 59 (50–69) thousand deaths and 5.1 (4.5–5.9) million DALYs associated with AMR could have been averted in 2019. Expanding the coverage to elderly populations would increase the vaccination impact to avert 71 (63–81) thousand deaths. We estimated that vaccination against H. influenzae at 2019 coverage levels averted 11 (9.7–13) thousand deaths and 0.98 (0.85–1.2) million DALYs associated with AMR in 2019. At 90% coverage globally, 13 (11–15) thousand deaths and 1.1 (0.96–1.3) million DALYs associated with AMR could have been averted. We estimated that wider introduction and scale-up of vaccination against S. typhi could have averted 34 (26–44) thousand deaths and 2.8 (2.2–3.6) million DALYs associated with AMR in 2019.M. tuberculosis that meets WHO’s PPC criteria of 80% efficacy, given to infants, with lifelong immunity or boosting, would have averted 0.12 (0.11–0.13) million deaths and 4.5 (4.1–5.0) million DALYs associated with AMR. An improved vaccine against S. pneumoniae (70% efficacy against bloodstream infections (BSI), meningitis and other bacterial central nervous system infections, 50% efficacy against lower respiratory infection (LRI) and all related infections in the thorax, given to 90% of infants at 6 weeks of life) would have a relatively highest impact by averting 99 (86–115) thousand deaths and 8.6 (7.5–10) million DALYs associated with AMR in 2019. An M72-like vaccine against M. tuberculosis given to adolescents and older populations with lifelong immunity or boosting would avert 71 (64–78) thousand deaths and 2.6 (2.3–2.8) million DALYs associated with AMR. A vaccine against all disease presentations of K. pneumoniae infection given to infants and elderly populations would avert 64 (59–72) thousand deaths and 3.7 (3.3–4.1) million DALYs associated with AMR.For pathogens with hypothetical vaccine profiles (developed by experts or provided in PPCs), we estimated that a vaccine against E. coli—non-diarrhogenic could avert 0.39 (0.37–0.40) million deaths and 13 (12–13) million DALYs associated with AMR in 2019. Vaccination of at-risk individuals against K. pneumoniae could avert 0.32 (0.31–0.34) million deaths and 14 (13–15) million DALYs associated with AMR, and vaccination against S. aureus could avert 0.32 (0.31–0.33) million deaths and 11 (10–11) million DALYs associated with AMR.In the high-potential scenario see , we estiS. pneumoniae, S. aureus and K. pneumoniae account for most of the vaccine avertable AMR burden associated with LRIs. K. pneumoniae, A. baumannii and E. coli account for most of the vaccine avertable AMR burden associated with BSIs.For each infectious syndrome, we stratified the vaccine avertable AMR burden for deaths and DALYs by pathogen in the baseline scenario, as shown in M. tuberculosis and S. pneumoniae by pathogen.We estimated vaccine avertable disease burden attributable to and associated with AMR for existing and new vaccines in the pipeline by pathogen, infectious syndrome and region based on the most recent, comprehensive estimates of the global burden of AMR. The AMR burden avertable by vaccination in 2019 was highest for the WHO Africa and South-East Asia regions, for LRIs, TB and BSIs by infectious syndromes, and for H. influenzae type b (Hib) and typhoid conjugate vaccine (TCV) on reducing AMR burden attributable to and associated with S. pneumoniae, H. influenzae and Salmonella typhi, respectively. We highlight the critical need to scale up existing vaccines to high and equitable immunisation coverage, and the acceleration of TCV introductions in high burden countries. Also, we show that vaccines can contribute towards preventing a significant proportion of the AMR burden for pathogens which have vaccines in late-stage clinical development with clear attributes or published PPCs or TPPs, such as for ExPEC and M. tuberculosis. Novel regulatory and policy mechanisms should be developed to accelerate the approval and use of these vaccines to prevent AMR. Based on the estimated high vaccine avertable burden associated with AMR for K. pneumoniae, S. aureus and A. baumannii, we urgently call for studies to enhance biological understanding and improve the feasibility of developing vaccines for these pathogens. For the remaining pathogens that have vaccine candidates in the early stages of clinical development or no vaccines in the pipeline, we recommend investing in vaccine development to resolve biological challenges as well as feasibility in terms of product development, market access and product implementation.Our estimates show the impact of existing vaccines for pneumococcal conjugate vaccine, Our analysis included a baseline and high-potential scenarios. In the baseline scenario, we model vaccine delivery based on known vaccine attributes, including a defined target age group that has been immunised with a vaccine in the past, during clinical trials or identified in vaccine TPPs. In contrast, the high-potential scenario makes no assumptions about vaccine delivery and target age group and shows the highest probable vaccine impact, should there be a policy recommendation and feasibility of delivery to all who would benefit from a vaccine. We recognise that the high-potential scenario includes multiple challenges that need overcoming such as immunisation of adults and the elderly, timely immunisation to prevent nosocomial infections, vaccine efficacy in patients who are immunocompromised and with comorbidities, vaccine demand and financing.H. influenzae type b (Hib), rotavirus, pneumococcal, typhoid and influenza vaccines have been directly associated with reduction of resistance, antibiotic use and related clinical complications,et al modelled the global burden of drug-resistant TB avertable by a future TB vaccine.26Pan-pathogen analyses with standardised methodologies are critical to inform vaccine funding and development and should be followed up with detailed vaccine-specific analyses, considering pathogen biology and transmission, and accounting for varied disease burden patterns across the spatial and temporal scales. Enterobacter spp, Group B Streptococcus, E. feacalis, Proteus spp, Citrobacter spp and Morganella spp were excluded. However, inclusion of these pathogens appears unlikely to significantly affect our overall inferences considering that the included 15 pathogens are responsible for the majority of the AMR burden. Third, while our analysis was based on the estimates generated by the GRAM project, which represents the most comprehensive estimates of bacterial AMR burden to date, limited input data to the GRAM project especially from low-income and middle-income countries is a significant data gap that necessitates newer surveillance data and platforms to inform the updates, validity and confidence in the estimates of the GRAM project. In particular, estimates from the GRAM project for TB do not include TB associated with HIV. Fourth, we did not consider the impact of viral vaccines on reducing the AMR drivers of antibiotic misuse and overuse.29Our study has limitations. First, since we included the direct effect of vaccination but excluded indirect effect and transmission dynamics of AMR pathogens, our vaccine impact estimates on averted AMR burden are conservative. Second, our analysis focused on 15 bacterial pathogens and additional pathogens included in the GRAM project such as The value of vaccines in preventing AMR should be systematically considered in the decision-making process during scale-up of existing vaccines and introduction of new vaccines. Vaccines should be explicitly incorporated as tools to combat AMR into National Action Plans on AMR10.1136/bmjgh-2022-011341.supp2Supplementary data


We agree with the author of the letter regarding our paper that researchers should followthe science rules, and, in this way, it is important to mention that all authors havedecades of experience on the theme cryopreservation and are doing good science ; 2019,aThe author of the letter regarding our paper states that the prototype Tvitri-4 is a“homemade” medium, but this assertion does not correspond to reality. Tvitri-4 wasdeveloped by the biotech Invitra, a spin-out of University of São Paulo, foundedin 2013 and that initiated its main laboratory activities in 2016 supported by twoFAPESP research grants for Innovative Research in Small Business (PIPE) ,a RHAE Researcher in Company grant from Brazilian Programme on Human Resources forStrategic Areas/RHAE/CNPq , and a Productivity Scholarship inTechnological Development and Innovative Extension CNPq . Besides, allsteps of the Tvitri-4 medium development were performed in a controlled environment in alaboratory with state-of-the-art equipment and standard operational procedures.According to the references cited in our paper, there is not a defined standard for theminimum or maximum number of oocytes to be vitrified per straw. In addition, there is nosuch indication in the study published by In the article from Regarding the Tvitri-4 formulation, it is surprising that the author of the letter on ourpaper is not familiar with the process of intelectual property protection. The prototypeTvitri-4, developed by Invitra, is protected by an industrial secret, a modality ofintellectual property (IP) rights on confidential information which may be sold orlicensed. This was the recommendation of the innovation agents and the IP legal officethat advises us. Moreover, Invitra in partnership with the University of SãoPaulo is co-owner of an invention patent filed in 2019 intitled “Vitrification and/orwarming composition, process for improving cell cryopreservation, and use of thevitrifying and/or warming composition, 2019, Brazil. BR1020190136979”(It also should be noted that in ongoing studies by our research group focused onsupplementing vitrification media with fatty acids and L-carnitine, the Tvitri-4 mediumhas also shown excellent performance compared to the Irvine Scientific vitrificationmedium as a control, regarding the oocyte membrane lipids profile and IVF outcomes(


Myocardial bridging (MB) is a congenital coronary anomaly in which a segment of a coronary artery, most frequently the left anterior descending artery, deviates from its epicardial route by passing through the myocardium. The advent of cardiac computed tomography angiography (CCTA), equipped with its multiplane and three-dimensional functionalities, has notably enhanced the ability to identify MBs. Furthermore, novel post-processing methods have recently emerged to extract functional insights from anatomical evaluations. MB is generally considered a benign entity with very good survival rates; however, there is an increasing volume of evidence that certain MB characteristics may be associated with cardiovascular morbidity. This review is intended to depict the diagnostic and prognostic role of CCTA in the MB context. Myocardial bridging (MB) is an anatomical entity whereby a portion of the epicardial coronary artery takes an intramyocardial route. The layer of muscle that covers the artery is known as a “myocardial bridge,” and the artery that traverses through the myocardium is labeled as a “tunneled artery” .This phenomenon was first mentioned in 1737 by Reyman in autopsy series , but GeiMB can occur in any of the coronary arteries, but the most common artery affected is the left anterior descending artery , with its mid segment being the most frequent site of bridging 46.7%), followed by the distal LAD [6.7%, folThe prevalence of MB varies widely depending on the diagnostic modality applied. In autopsy series, the prevalence varies from 20% to 85% , which iIn most patients, MB is an incidental finding associated with an excellent survival rate of 97% at 5 years . HoweverIn the MB setting, CCTA allows for anatomical visualization of both the coronary artery lumen and the myocardium and, more recently, the functional assessment for evaluating the clinical relevance of MB . This review is intended to illustrate the feasibility and potential value of CCTA in MB.CCTA images for the detection of MB are obtained using the standard protocol ,22.The target coronary attenuation value is 350–400 Hounsfield units (HU), achieved through an iodinated contrast medium volume of 245–370 mgI/kg administered intravenously into an antecubital vein at a flow rate of 4–5 mL/s for 10–20 s. A 50–60 mL saline flush is used at the same injection rate as the contrast material. A bolus-tracking method is employed with monitoring placed in the ascending aorta. A tube potential of 100 peak kilovoltage (kVp) is applied in patients with body weight ≤ 100 kg or BMI < 30 kg/mq and an automated current adjustment mode is utilized. The scan range covers the entire heart, starting from the tracheal bifurcation to the diaphragm with craniocaudal scan direction during the middle inspiratory breath-hold.A high-pitch acquisition can be considered in patients with stable sinus rhythm and a heart rate of less than 60 bpm and a body weight ≤100 kg (very low radiation dose); a prospective ECG-gated acquisition is performed in situations with a regular rhythm, where high-pitch or high-volume scans are not available or contraindicated; retrospective ECG-gated acquisition should be considered in patients with an irregular rhythm or high heart rate. With the introduction of the 320-slice multi-slice CT, scanning of the entire heart has become possible in a single heartbeat with a very low dose of radiation . If the heartbeat is greater than 65 bpm, intravenous β-blockers are given before CCTA; furthermore, after ruling out hypotension, a commonly used regimen of 400–800 μg sublingual nitroglycerin is administered 5 min before the exam.The image reconstructions are done using 10 phases in 10% of each RR interval. Images are reconstructed with a slice thickness of <1 mm, a slice increment of 50% of the slice width, an image matrix of 512 × 512, and a field of view (FOV) of 200–250 mm. A diastolic and systolic dataset with the best image quality is selected. Images are restructured using multiple post-processing methods. Transverse source images, multiplanar reformations (MPR), curved multiplanar reformations, and maximum intensity projection images are used for evaluating the intramyocardial course. Whole-heart volume-rendered images of the heart are also reconstructed and rotated to best display the coronary arterial anatomy. CCTA has significantly improved the anatomical characterization of MB. Its strength lies in its high spatial resolution. In terms of anatomy, LAD-MBs can exhibit variations in depth, categorized as superficial (>1 to 2 mm), deep (≥2 mm), and very deep (≥5 mm)  Figure . The degCCTA can describe muscle bridging as being partial or complete, depending on the extent to which the coronary artery segment with bridging is surrounded by myocardium. A partial MB is one in which >75% but <100% of the coronary artery is encased by myocardium, and a complete bridge is one that is entirely encased by myocardium [Third, MB is classifiable into short (<25 mm) or long (≥25 mm) according to the length of the tunneled segment .The length of the encasement appears to impact the dynamic compression of both the trapped LAD segment and the septal branches originating from or near the affected LAD segment . One repSince both the length and depth of the MB are closely related to systolic compression, their synergy may generate contractile force exerted by the MB. To assess the mass volume of the MB muscle, the value of MB depth (in millimetres) multiplied by MB length (in millimetres) is defined as the MB muscle index (MMI) .The major anatomical MB classifications are schematically outlined in When conducting CCTA, it is crucial to consider any additional significant anatomical characteristics of the LAD-MB, such as the concurrent number of arteries or tunneled segments, as well as the degree of systolic diameter reduction or kinking.MBs that develop into symptomatic disease often occur in a complex interplay also related to left ventricle hypertrophy and the presence of coronary atherosclerosis . Even in this field, CCTA can be of great help for anatomical evaluation. Regarding left ventricular hypertrophy, information on left ventricular mass is readily available for analysis without additional testing  and is dFurthermore, the development of atherosclerotic coronary disease has been associated with MB. The systolic compression of the tunneled artery produces retrograde flow proximal to the MB . This reNonetheless, these altered biomechanical forces might also be accountable for certain non-atherosclerotic complications linked to the MB. It is plausible that the elevated compressive stress exerted by the MB onto the tunneled artery could induce intimal damage, potentially evolving into dissection .Another frequently observed characteristic of MB is its relatively low occurrence of plaque within the tunneled artery segment. This phenomenon can be elucidated through several observations: Firstly, MB leads to the separation of the coronary artery from the perivascular adipose tissue in the epicardium, which is tied to proinflammatory signaling pathways . SecondlDistally, due to increased endothelial shear stress, decreased blood flow and volume, atherosclerosis is underdeveloped and a decrease in luminal diameter (secondary coronary hypoplasia) is often noticeable.CCTA can not only serve as a valuable non-invasive test for the anatomical definition of a MB, but can also provide insight into the potential hemodynamic significance of the bridge in patients with recurrent symptoms of typical angina without obstructive coronary artery disease. Categorized by the degree of lumen constriction during the systolic phase in CCTA, individuals with MB can be sorted into three categories: those lacking systolic compression and without luminal narrowing, those with <50% systolic compression, and those with systolic compression leading to ≥50% luminal stenosis. Nonetheless, it has been acknowledged that dynamic compression is not solely linked to the systolic phase; it also continues into the mid-to-late diastole .Moreover, a patient undergoing CCTA is usually administered a combination of beta-blockers, calcium antagonists, and/or ivabradine along with nitroglycerin prior to the scan. This step is necessary to enhance image quality by extending the diastolic phase and promoting coronary vasodilation. Nevertheless, these medications have the potential to mask the clinical importance of MBs, thereby constituting a constraint in the study of MBs through CCTA. Employing dobutamine and/or exercise to induce the physiological state in which MBs are most likely to elicit symptoms thus poses a notable technical obstacle. Nonetheless, last-generation CT scanners have recently been used to perform CCTA stress perfusion. In more recent times, novel post-processing methods have emerged to extract functional insights from the anatomical evaluation conducted through CCTA.Two of these techniques include the Transluminal Attenuation Gradient (TAG) and the CCTA-derived Fractional Flow Reserve (FFR) .The former entails the linear regression coefficient between luminal attenuation and the axial distance from the coronary ostium ,37. TAG is characterized as the transluminal contrast intensity gradient in CCTA from the proximal to the distal regions of vessels. For each targeted vessel, cross-sectional images are reconstructed perpendicular to its centerline. Hounsfield units (HU) determine the TAG and are measured at 5-mm intervals, ranging from the ostium to the point where the lumen area falls below 2 mmq. This attenuation gradient is established by assessing the HU change over a 10-mm span of the coronary artery. It is defined using the linear regression coefficient between the intraluminal attenuation in HU and the distance from the vessel ostium in millimeters.The TAG demonstrates a direct linear relationship with the degree of intracoronary transluminal stenosis . ImportaThe CCTA-derived FFR involves a computational fluid dynamics simulation that utilizes adenosine-induced hyperemia in conjunction with CCTA imaging. It relies on the attenuation observed within the coronary artery, taking into account the vessel lumen’s morphology and various other parameters ,41. CCTAΔCCTA-FFR in the systolic phase has shown the highest sensitivity and negative predictive value whereas ΔCCTA-FFR in the diastolic phase has the highest specificity among all parameters for diagnosing MB-related ischemia . Abnormal CCTA-FFR values (defined as ≤0.80) have a prognostic role with a positive association with the symptoms of typical angina .The role of CCTA-FFR has also been studied in predicting proximal plaque formation associated with LAD-MB using DML approaches . While tCCTA offers numerous advantages over invasive and other non-invasive diagnostic techniques when it comes to identifying, assessing the anatomic structure, and gauging the functional characteristics of MBs.Its effectiveness lies in its high spatial resolution, contributing to an improved detection rate of MBs compared to coronary angiography ,46.CCTA facilitates precise scrutiny of the vessel wall and lumen, along with the surrounding myocardium and all adjacent structures, rendered in three dimensions. This capability permits the analysis of the artery’s trajectory and categorization of LAD-MBs based on their depth and length.These anatomical assessments carry noteworthy clinical, prognostic, and therapeutic implications.Notably, the deeper variant is more frequently linked to symptoms and adverse cardiac events ,48. MB tFurthermore, CCTA could assist in guiding the selection of treatment options. Medication with beta-blockers or calcium antagonists is a possible strategy. For patients resistant to medication, superficial and shorter-length MBs might be more effectively addressed through PCI. Conversely, very deep (≥5 mm) or long (≥25 mm) MBs are likely to benefit from surgical intervention .Complications linked to coronary stenting encompass issues such as perforation, fracture, restenosis, and thrombosis, which have imposed limitations on its utilization . SurgicaIn cases of substantial MB length (≥25 mm), significant depth (≥5 mm), proximal plaques, or incomplete decompression of the bridged coronary segment during diastole, coronary artery bypass grafting (CABG) is preferred over myotomy . NeverthA novel procedure has been devised that involves utilizing a segment of the LIMA to connect the proximal and distal portions of the MB . This inCCTA allows for accurate surgical pre-planning by identifying the arterial course and therefore avoiding complications such as perforation of the right ventricle wall during attempts to isolate the intramuscular artery ,56 Figu.Although invasive coronary angiography with IVUS, dobutamine stress diastolic FFR, and instantaneous wave-free ratio (iFR) methods can reliably provide an anatomical and functional assessment of the MB ,58,59,60Specifically, CCTA has proven comparable to IVUS in determining MB length and location, the coverage of a MB by CCTA correlates well with the MB thickness by IVUS, and CCTA can give an indication of the hemodynamic significance of a MB .Noninvasive imaging techniques offer potential for detecting MB and exploring myocardial ischemia in patients with MB, given that this patient group tends to be younger, exhibits fewer cardiovascular risk factors, and thus has a lower pre-test probability of obstructive coronary artery disease .CCTA holds a significant role among noninvasive imaging methods within this context. CCTA may be preferred over other imaging techniques for MB identification in patients with low to intermediate risk of atherosclerotic coronary artery disease with recurrent symptoms of typical angina in order to consider dynamic ischemia from a MB in the differential diagnosis. Furthermore, CCTA may be selected for the diagnosis of MB in adults with hypertrophic cardiomyopathy because it is a noninvasive diagnostic imaging method with excellent spatial resolution. Lastly, CCTA may be of choice in athletes with exercise-induced ECG changes in whom intense physical activity increases the sympathetic tone leading to tachycardia and increased myocardial contractility, thus facilitating ischemia due to MB.Echocardiogram contributes to the functional evaluation of MB patients, highlighting the presence of “branch steal” by revealing a localized abnormality in septal wall motion during end-systole to early-diastole, accompanied by apical sparing. This phenomenon is referred to as “septal buckling with apical sparing” . FurtherWithin the domain of noninvasive diagnostic techniques, cardiovascular magnetic resonance imaging (CMRI), single-photon emission computed tomography (SPECT), and positron emission tomography (PET) facilitate the quantification of myocardial blood flow both at rest and during induced hyperemia. These methods offer potential avenues for assessing inducible ischemia and coronary microvascular dysfunction in MB patients .The significant drawback of CCTA is its relatively modest temporal resolution; early and mid-systolic CCTA datasets have mostly inadequate image quality due to severe motion artifacts caused by myocardial contraction. Nonetheless, suitable image quality of coronary arteries could be acquired using new generation scanners, allowing for feasible dynamic evaluation of MB.The use of CCTA is ultimately limited by radiation and contrast exposure, however, there are now novel scanning techniques and protocols that dramatically reduce the radiation and contrast injection dosing during scanning.Modern imaging technology has enabled a comprehensive assessment of both coronary anatomy and function.Accurate detection of anatomical characteristics and hemodynamic consequences of MB is essential to evaluate its clinical significance. Noninvasive CCTA has been widely used to detect MB in vivo. Currently, there are no clinical guidelines for the diagnostic evaluation of MB, but CCTA has the prospective to play a leading role in the MB setting, having demonstrated high diagnostic performance even to detect functional ischemia in addition to an exact description and classification of anatomical features. Machine learning has created great potential to advance CCTA scanning, by reducing exposure to radiation and harnessing the power of artificial intelligence. The integration of artificial intelligence algorithms into CCTA systems will enable physicians to provide better images, who will in turn provide better treatments based on more accurate diagnosis.MB is a common congenital anomaly encountered frequently in clinical practice. For quite some time, MB was regarded as an incidental discovery. Yet, an increasing volume of evidence points to its connection with symptoms and its potential to result in unfavorable cardiac events.Thanks to advances in CCTA technology, we now have a powerful noninvasive method to evaluate the location, depth, and length of a MB and any concomitant atherosclerotic changes. Furthermore, functional assessment of MB with CCTA is proving of great interest. Future development of CCTA approaches dedicated to the evaluation of MB is warranted for considering CCTA the tool of choice of the diagnostic and prognostic evaluation of MB.


While HA is present naturally in periodontal tissues, its molecular weight can vary widely in vivo. The objective of this study was to directly compare the biological reactions of periodontal ligament cells to four distinct molecular weights of hyaluronic acid (HA).Immortalized human periodontal ligament cells (PDL-hTERT) were cultured for 21 days in culture medium alone (control) or enriched with osteogenic supplements (OS group). Other 4 experimental groups were cultured in OS medium with the addition of HA with different molecular weights . The cell morphology was examined daily. WST1 assays were performed to evaluate metabolic activity. Von Kossa staining and calcium deposition assay were used to analyze osteogenic differentiation and mineralization.Cell morphology remained unaltered in all groups. Cells stimulated with OS alone or with the addition of hyaluronan showed all the typical microscopic appearance of osteogenic differentiation. Metabolic activity increased in all groups over time. Hyaluronan stimulated greater metabolic activity than the control group, with LMW HA and MMW HA showing the most significant increase. All groups showed mineral deposits and calcium deposition after 21 days of stimulation.Our results suggest that hyaluronan can promote metabolic activity and mineralization of PDL-hTERT cells, with LMW HA being the most effective.These results shed light on how the various molecular weight fractions of HA promote tissue regeneration and repair, as well as help to identify an optimal molecular weight range for this application in periodontal tissues. Periodontitis is a chronic inflammatory disease that affects the supporting structures of teeth, including the periodontal ligament and alveolar bone . The extThis non-sulfated glycosaminoglycan HA in its native form is present as a long linear polysaccharide having a high-molecular weight >1000kDa (HMW HA) , 6. ThreHA has been found to be present naturally in periodontal tissues, gingival connective tissues as well as gingival epithelium , 9 produThe content of HA in human gingival epithelium has been calculated to be circa 5.58 μg/g dry wt., while in human gingival connective tissue circa 126.4 μg/g dry wt. [Several studies have suggested an important role for hyaluronan in maintaining periodontal tissue homeostasis . MoreoveIn case of inflammation, hyaluronidases, the activity of reactive oxygen and nitrogen species, etc. induce the HA into smaller fragments of different MW , 20. In The amount of hyaluronan, specifically the low-molecular weight HA (LMW HA) present in the gingival crevicular fluid (GCF) has been seen to be higher in patients affected by periodontitis, even after periodontal therapy .These apparently different involvements of HA, support the suggestion of Wu et al, that studying HA in knee osteoarthritis, suggested that HA of different MW present peculiar features and they should be not considered as a single group .Even having evidenced the presence of fractions with a different molecular weight HA, the present available literature is scarce in comparing their effects in the periodontal tissues.Understanding the effects of different molecular weight fractions of HA on PDL cells is important for elucidating the mechanisms involved in periodontal tissue maintenance and repair. By investigating these effects, we can also gain insight into the optimal molecular weight range for promoting tissue regeneration and repair.Thus, the aim of this study was to directly compare and better understand the biological reactions of PDL-hTERT cells to four distinct molecular weights of hyaluronan.Human immortalized periodontal ligament cells (PDL-hTERT) were established as previously described . PDL-hTEA control group of PDL-hTERTs was cultured in DMEM enriched as described above, without any hyaluronan supplements. Another group was constituted of PDL-hTERTs cultured in osteogenic differentiation medium (OS), which was DMEM with osteogenic supplement: 10% FBS, 1% Pen-Strep, 100nM Dexamethasone, 10mM β-glycerol phosphate and 50μM L-ascorbic acid .th and the 29th passage were used. PDL-hTERT were cultured in 1 ml medium, at 2x 104 cells/well in 12-well plates for 3 weeks.Hyaluronan (HA) with different molecular weights was purchased from R&D Systems, Minneapolis, MN, USA. The four groups further used for the experiments were as follows: High Molecular Weight Hyaluronan (HMW), Medium Molecular Weight Hyaluronan (MMW), Low Molecular Weight Hyaluronan (LMW), and Ultra Low Molecular Weight Hyaluronan (ULMW). HMW hyaluronan is provided by the company in a molecular mass range of >950kDa. The specific lot used had a mass of 1510kDa. MMW hyaluronan presents a molecular mass range of 75–350kDa. The lot used had a mass of 229kDa. LMW hyaluronan has a molecular mass range of 15–40kDa. The specific lot used had a mass of 37kDa. ULMW hyaluronan is in a molecular mass range of 4–8kDa. The specific lot used had a mass of 4.6kDa. HA was reconstituted as per manufacturer´s indications in phosphate buffered saline and sterilized by filtration with 0.2-μm syringe filters . The stimulating concentration of 100μg/ml was further used in the experimental groups, and it was determined based on previous studies , 26. PDLThe cell morphology was examined daily, and photographs were taken using a phase contrast microscope . For each studied group, the experiments were performed in triplicates and repeated three times.4 cells/well) and cultured for 7, 14, or 21 days, exposed to the above-mentioned HA solutions . The groups with medium alone and OS medium were also cultured under the same conditions. Medium change with 0.5 ml per well was performed every 2 days. At each experiment day, WST-1  was added according to manufacturer’s protocols in a 1:10 final dilution, incubated for 1 hour (5% CO2 and 37°C). The medium of each well was transferred into two wells of a 96-well plate, with a volume of 100 μl per well. The optical density of the samples was measured through a multi-detection microplate reader . Data analyses were performed with Magellan™ Software (TECAN). Absorbance readings were recorded at 450nm, with a wavelength correction at 650nm. The experiments were performed in triplicates and repeated three times.Water-soluble tetrazolium-1 (WST-1) assays were performed. For this assay, PDL-hTERTs were seeded in 24-well plates , pyrogallol acid solution , sodium hydroxide solution  and May-Grünwald solution . Afterwards, pictures were taken in standardized conditions for all groups.th passage were plated at a concentration of 2x 104 cells/well in 12-well plates and divided into 6 groups as above: control; +OS; +OS+ HMW, +OS+ MMW, +OS+ LMW and +OS+ ULMW. The cells were stimulated for 21 days and then harvested with QuantiChromTM Calcium Assay Kit  following manufacturer´s protocol. The calcium quantification was done with a microplate reader . The measurements were performed at 612 nm and the calculations of calcium deposition in mg/dl were done establishing a standard curve as per manufacturer´s indications.PDL-hTERTs in the 28U test and with Games-Howell test for WST-1. The p-value threshold was set at 0.05. If not otherwise indicated, all data are given as mean ± standard deviation (±SD).Statistical analyses were performed using SPSS software . Within each group, data were tested for normality with the Kolmogorov-Smirnov and Shapiro-Wilk tests. Homogeneity of variances between groups was tested with Levene test. Since data were not normally distributed, differences between study groups in calcium deposition for each time point were calculated with the Mann-Whitney The single cell morphology remained unaltered in all groups, which presented an elongated spindle shaped form Fig. . The celp> 0.05). The maximum increase in cell activity within the observed 21 days was seen in the cells stimulated with LMW and MMW. While the cells stimulated with MMW showed a higher difference in their metabolic activity from day 7 to day 14, this was more the case between day 14 and 21 for the cells stimulated with LMW. Moreover, it can be observed that the groups stimulated with HMW, MMW and ULMW hyaluronan presented at day 14 significantly higher metabolic activity than the control group.Considering the changes within the single study groups overtime, in all six groups, the cells´ activity increased with time, so that the highest value was measured for all groups on the 21st day. The osteogenic and the control groups increased equally within the observed time intervals and reached comparable values  between HMW group and ULMW can also be observed at day 7, which, however, could no longer be detected at day 14. At day 21, all groups stimulated with hyaluronic acid presented higher values than the control, but also higher than the OS group, but this reached significance just for LMW and MMW in the comparison with the control.A significant difference (We next assessed the capacity of PDL-hTERT cells stimulated with different molecular weight hyaluronan to differentiate into osteoblasts capable of forming a mineralized extracellular matrix. Mineral deposits by PDL-hTERTs were identified by von Kossa staining technique in all groups after 21 days of stimulation. Photographs of representative cultures are shown in Fig. The mineralization was also quantified in parallel experiments by calcium deposition assay Fig. . SpectroThe current study aimed to determine the influence of fragments of hyaluronic acid with varying molecular weights on periodontal ligament cells. This influence was evaluated first on the morphological, phenotypical expression of PDL-hTERTs, and then on the capacity of these cells to differentiate into osteoblasts, as described by Docheva et al. . The PDLFurthermore, these fibroblasts are the main cells occurring in the periodontal crevicular space, performing several functions in periodontal regeneration and homeostasis , 28. ConRegarding microscopic appearance, osteogenically stimulated PDL-hTERTs as well as those additionally treated with any of the fragments of HA showed an ongoing osteogenic differentiation at day 14. Contrarily to the control, all treated groups exhibited oval-shaped, cell-free areas that are typical of the beginning of mineralization/osteogenic differentiation. No differences were seen to be induced in cell morphology by HA, compare to OS alone. This has also been demonstrated by Kawano et al. for HMW hyaluronic acid of more than 2000kDa . HoweverAnalyzing metabolic activity, we observed a significant increase in PDL-hTERTs proliferation treated with MMW HA, already at day 7, until the end of the observation period, after 21 days, which is greater than the control and any hyaluronan with different MW. Similar results were found for the proliferation rate of skin keratinocytes by Ghazi et al., who hypothesized that MMW HA is a more effective therapeutic approach than native HA for promoting wound healing . In lineA recent study conducted on hamster ovary cells demonstrated that the application of sodium hyaluronate with a molecular weight > 1300kDa  inhibitsThe effect of LMW HA has been analyzed in 3D culture models and it has been demonstrated to enhance osteogenesis and bone matrix formation . This isIn contrast, ULMW HA in our study appeared to have no effect on cell morphology or mineralization potential of PDL-hTERTs. However, it induced a greater metabolic activity in PDL-hTERTs at the beginning of their osteogenic differentiation, at day 7 and 14.In a study by Ariyoshi et al. , HA withIt is worth considering that the biological effects HA can be cell type or tissue specific, as suggested by Cowman et al. . TherefoA limitation of this study is that it is performed with immortalized PDL cells isolated from one donor and, therefore, might not be representative of a general population. Another limitation of our study is that being an in vitro study, it lacks the possible mutual influence between HA fragments and the surrounding tissues, as these interactions influence the growth microenvironment of the cells regulating the HA response .In the light of our results, the molecular weight of the local HA could be used for its potential predictive value in amplifying periodontal regenerative healing. In terms of the current results particularly HA fragments with medium molecular weight are promising candidates to further delineate the regenerative potential of glycosaminoglycans, specifically HA.Also, the concentration of HA fragments might influence the obtained cell effects. Thus, further studies might consider covering a wider range for HA concentrations. It might be also interesting to investigate combined effects of HA with different molecular weights, as a bio-synergistic impact of two distinct molecular weights of HA was also proposed by Rosaming et al. .Overall, our in vitro findings suggest that the molecular weight of HA may play a key role in regulating the mineralization of periodontal tissues, and further investigation is needed to improve the understanding of the underlying biological mechanisms involved.


The MGDrivE (MGDrivE 1 and MGDrivE 2) modeling framework provides a flexible and expansive environment for testing the efficacy of novel gene-drive constructs for the control of mosquito-borne diseases. However, the existing model framework did not previously support several features necessary to simulate some types of intervention strategies. Namely, current MGDrivE versions do not permit modeling of small molecule inducible systems for controlling gene expression in gene drive designs or the inheritance patterns of self-eliminating gene drive mechanisms.Here, we demonstrate a new MGDrivE 2 module that permits the simulation of gene drive strategies incorporating small molecule-inducible systems and self-eliminating gene drive mechanisms. Additionally, we also implemented novel sparsity-aware sampling algorithms for improved computational efficiency in MGDrivE 2 and supplied an analysis and plotting function applicable to the outputs of MGDrivE 1 and MGDrivE 2. Mosquito-borne diseases, such as malaria, dengue, and Zika, remain a significant public health burden throughout the world , 2. TradCRISPR-based gene drives (GDs) offer a potentially sustainable approach to vector control via biasing the inheritance of a desired gene in a target population –6. A sucAs GD designs become more complex and field trials for wide-scale application are discussed, understanding and predicting how these designs behave under real-world conditions is of increasing importance . Such moThe MGDrivE software suite (MGDrivE 1 and MGDrivE 2) represent the state-of-the-art in gene drive modeling. MGDrivE 1  was uniqIn this paper, we build on the existing strengths of the MGDrivE 2 software suite by developing additional functionality to simulate new gene-drive constructs and transgenic tools for mosquito-borne disease control. More specifically, additional capabilities for modeling the performance of five novel gene drive designs are included, as well as improved analysis and plotting routines applicable to both the MGDrivE 1 and 2 software. Finally, we upgraded internal functionality for improved computational performance and software maintenance , and calculating simple statistics. Higher-level analysis and complex visualization functionality is provided by the excellent Python package MoNeT-MGDrivE , a gene We simplify the plotting and analysis process for bench researchers by implementing basic MoNeT-MGDrivE functionality within the MGDrivE suite, making tools a researcher needs readily accessible. New functions take the same analyzed output as MoNeT-MGDrivE but output files in a ggplot2  friendlyMGDrivE 2 represents models as stochastic Petri nets (SPN), separating the model specification from the simulation routines . This seStochastic simulations in MGDrivE 2 are simulated as competing Poisson processes, one for each transition in the system. Because models are described using SPN for mathematical specification, the intensity of a transition must have at least as many tokens in its input states as the weight of the input arcs. For example, the mortality transition for adult mosquitoes requires at least one adult mosquito in its input state for the intensity to be zero, or, in the language of SPN, for that transition to be enabled. Checking if all input states have at least the required number of tokens can be time consuming during large simulations. Therefore, we implemented first-order sparsity-aware samplers which only need to check that the first input state has a non-zero number of tokens. If the first input state has zero tokens, computation of the intensity can be skipped entirely, because all input states must have at least the minimum number of tokens to be enabled. This enhancement was applied to all samplers supplied with MGDrivE 2 and achieved ~ 5x runtime improvement when generating Fig. Developing extensions for MGDrivE 2 required a deep understanding of the original model and its implementation. To reduce the burden of future extensions, the software documentation was improved in three ways:New Documentation and ExamplesAll novel inheritance designs come with significant documentation, explaining how each design works, links to relevant publications, and describing how parameters impact inheritance. Additionally, we provide a new vignette with simple examples of disease-induced mortality and a self-elimination mechanism.Improvement of Existing DocumentationExisting documentation was augmented with additional examples, data structure clarifications for function input or return, spelling corrections, and links between functions of similar functionality or purpose.Documentation of Internal UpgradesSignificant internal improvements were made to support stability and maintenance of the codebase. Parameter checks were standardized, alleviating errors encountered near extreme ranges of parameter values, code duplication was substantially reduced, and several internal structures were simplified, reducing the computational complexity and maintenance efforts.We extend the excellent MGDRivE 2 software packages with the addition of novel scientific functionality, 5 new inheritance patterns based on a recent biodegradable design, and significant computational and software stability improvements. The addition of small-molecule sprays expands the utility of MGDrivE 2 and supports the use of inducible systems. Analysis, visualization, and computational improvements allow researchers to rapidly iterate on designs and parameterization, evaluating innovative ideas for their efficacy prior to expensive cage or field trials. Finally, internal improvements simplify upgrades and additions to the software suite, laying the foundation for future endeavors.While we provide significant enhancements of the MGDrivE 2 software suite, there are still limitations inherent to our work:Use of MGDrivE 2 as a foundation inherits any limitations thereof, such as computational limitations of the R language .Inheritance patterns are faithful mechanistic representations of our current understanding, which may change with experience, species, or biological experimentation.Sparsity-aware samplers rely on sparsity and provide no benefit in densely populated simulations. Additionally, they only apply to the SPN framework of MGDRivE 2 and not to the decoupled samplers.For compatibility with MGDrivE 1, analysis functions only work on entomological outputs and not epidemiological ones. For more complex analysis, we recommend using the MoNeT-MGDrivE library.


With the introduction of several novel medications, including angiotensin receptor neprilysin inhibitors and sodium-glucose cotransporter 2 inhibitors, in addition to conventional beta-blockers and mineralocorticoid receptor antagonists, mortality and morbidity in patients with heart failure improved significantly . ModulatThe presence of pulmonary congestion impairs patients’ quality of life, particularly in those with frailty, sarcopenia, and malnutrition . ResiduaOne of the challenges in the management of pulmonary congestion is the lack of a gold standard to accurately quantify the degree of fluid volume . PhysicaRemote dielectric sensing (ReDS) is a recently introduced technology that uses electromagnetic energy to non-invasively and rapidly quantify the amount of lung fluid without the need for expert technique . Its cliI am not denying the usefulness of these conventional methods. Each modality that includes a ReDS system has advantages and limitations. We can choose appropriate modalities, sometimes in combination, according to each clinical situation. Chest echocardiography is a good practical tool for assessing severe pulmonary congestion. However, it cannot accurately quantify the degree of mild congestion. Moreover, chest X-ray is an essential tool for assessing pulmonary congestion. It can visualize the thoracic anatomy and help us to assess other thoracic diseases. On the contrary, it cannot accurately quantify the degree of mild congestion. They may not be appropriate to adjust the dose of diuretics in patients with mild congestion. Plasma B-type natriuretic peptide is, needless to say, an essential method for estimating intra-cardiac pressure. However, there are several confounding factors, including obesity and renal dysfunction. Furthermore, an elevated B-type natriuretic peptide does not necessarily indicate the existence of pulmonary congestion, e.g., in patients with low cardiac output syndrome. The ReDS system can quantify the lung fluid. However, as detailed below, it cannot distinguish pulmonary congestion from other lung diseases that accompany lung fluid retention. This technology is more useful for screening out pulmonary congestion or tracking the degree of pulmonary congestion. The clinical impact of ReDS-guided management remains the next concern. Several prospective studies are underway. For example, we are currently comparing clinical outcomes between those who receive ReDS-guided congestion management by diuretic dose adjustment and those who receive diuretic adjustment without ReDS measurements. Another concern is the distribution of congestion. Pulmonary congestion is not necessarily associated with systemic congestion, which also has an independent prognostic impact . EstimatAnother concern is how to intervene against pulmonary congestion. Traditionally, we adjust the dose of loop diuretics. However, a higher dose of loop diuretics is associated with worse clinical outcomes, due to intravascular hypovolemia, the stimulation of the renin–angiotensin system, and the worsening of renal function . The useAs discussed above, the ReDS system is not a perfect tool for assessing pulmonary congestion. The ReDS system is non-invasive and does not require expert techniques. On the contrary, it cannot distinguish pulmonary congestion from other similar conditions, including pneumonia, lung cancer, and acute respiratory distress syndrome. The ReDS system is generally useful for screening or follow-up of mild congestion. Further intensive tests are highly recommended in patients with high ReDS values.