Clostridium perfringens type D epsilon toxin (ETX) produces severe, and frequently fatal, neurologic disease in ruminant livestock. The disorder is of worldwide distribution and, although vaccination has reduced its prevalence, ETX still causes substantial economic loss in livestock enterprises. The toxin is produced in the intestine as a relatively inactive prototoxin, which is subsequently fully enzymatically activated to ETX. When changed conditions in the intestinal milieu, particularly starch overload, favor rapid proliferation of this clostridial bacterium, large amounts of ETX can be elaborated. When sufficient toxin is absorbed from the intestine into the systemic circulation and reaches the brain, two neurologic syndromes can develop from this enterotoxemia. If the brain is exposed to large amounts of ETX, the lesions are fundamentally vasculocentric. The neurotoxin binds to microvascular endothelial receptors and other brain cells, the resulting damage causing increased vascular permeability and extravasation of plasma protein and abundant fluid into the brain parenchyma. While plasma protein, particularly albumin, pools largely perivascularly, the vasogenic edema becomes widely distributed in the brain, leading to a marked rise in intracranial pressure, coma, sometimes cerebellar herniation, and, eventually, often death. When smaller quantities of ETX are absorbed into the bloodstream, or livestock are partially immune, a more protracted clinical course ensues. The resulting brain injury is characterized by bilaterally symmetrical necrotic foci in certain selectively vulnerable neuroanatomic sites, termed focal symmetrical encephalomalacia. ETX has also been internationally listed as a potential bioterrorism agent. Although there are no confirmed human cases of ETX intoxication, the relatively wide species susceptibility to this toxin and its high toxicity mean it is likely that human populations would also be vulnerable to its neurotoxic actions. While the pathogenesis of ETX toxicity in the brain is incompletely understood, the putative mechanisms involved in neural lesion development are discussed. Clostridium perfringens, classified as A, B, C, D, E, F and G based on their elaboration of one or more of six principal exotoxins, namely, alpha (CPA), beta (CPB), epsilon (ETX), iota (ITX), enterotoxin (CPE) and necrotic enteritis B-like (Net-B)   1]. Typ. TypClosClostridium perfringens type D enterotoxemia is one of the most common clostridial diseases of sheep and goats, and is of worldwide distribution [ribution ,5,6. Sinribution ,8,9, andribution ,11. Howeribution , althougClostridium perfringens type D epsilon toxin commences with changes in the intestinal milieu that favor massive proliferation of this resident bacterium, resulting in the production of large quantities of ETX  produces immunologically distinct major exotoxins, some of which cause inflammatory lesions in the gastrointestinal tract and/or enterotoxemia. A range of other toxins, some of which enhance virulence, but are not used in the classification of Clostridium perfringens, is also recognized. The pattern of toxin production varies with each Clostridium perfringens type and determines the ensuing clinical syndrome, with sustained levels of clostridial exotoxin usually being required for the development of systemic clinical signs. Clostridial infections invariably need favorable predisposing conditions which, in the case of Clostridium perfringens type D, is an alteration to the intestinal microbiota caused mostly by an abrupt change to the feed type, namely, large amounts of grain or succulent pasture [Clostridial diseases can be divided into three categories, based on toxin activity and tissues affected. These are neurotoxic, histotoxic and enteric diseases. Neurotoxic clostridia, such as  pasture ,21,22,23Clostridium perfringens type D secretes a relatively inactive, ~33 kDa polypeptide, termed epsilon prototoxin (pETX). This prototoxin is proteolytically activated by intestinal enzymes, or other proteases such as lambda toxin produced by some strains of C. perfringens, to the active ~29 kDa ETX, by removing C-terminal amino acids [no acids ,25,26. Eno acids .+ levels, with a consequent influx of Na+ and Cl− into the cell, leading to cell necrosis [ETX is a member of the aerolysin family of pore-forming toxins, which oligomerize at the cell surface, form pores of varying size in the membrane and eventually cause cell lysis. One domain of ETX interacts with receptors on the host cell, while another domain possesses an amphipathic loop that inserts into membranes during pore formation. Once bound to a receptor, ETX uses lipid rafts and caveolins to oligomerize into a heptameric pre-pore on the host cell surface, then extends a β-hairpin loop into the membrane lipid bilayer to form an active pore. This pore formation results in a rapid decrease in cytoplasmic Knecrosis ,28.Clostridium perfringens type D in their small intestine, but bacterial numbers are generally small, in part due to peristalsis. Clinical disease does not occur unless the microbial balance in the gut milieu is disrupted. The small amounts of ETX normally present in the intestinal lumen appear to be relatively harmless [A small number of ruminants harbor harmless ,22,23. TWhen sheep and goats, and more particularly lambs, graze abundant lush pasture or young cereal crops, or after heavy grain feeding in feedlots, large quantities of undigested starch pass into the small intestine. Since it takes days or weeks for the rumen microflora to adapt to this sudden overload of ingested carbohydrates, the starch overload, which has given the disease the colloquial designation “overeating disease”, provides an excellent substrate for these saccharolytic clostridial bacteria, and they proliferate rapidly. Large amounts of ETX are produced. However, while the presence of excess starch in the small intestine encourages clostridial proliferation, there is experimental evidence that, in fact, it is a lack of glucose in this microenvironment, due to a failure to digest the starch overload, which appears to stimulate ETX production ,22,23.Type D enterotoxemia in sheep, and likely cattle, is a pure toxemia, with no bacterial invasion of tissues. Large amounts of ETX in the intestinal lumen appear to increase mucosal permeability, thus facilitating toxin absorption into the bloodstream . When aClostridium perfringens type D enterotoxemia has not yet been determined.The circulating toxin is distributed to a range of target tissues, including brain, lungs, liver, heart, kidneys and eye, where it binds to a receptor. This binding can be experimentally, albeit transiently, prevented via competitive inhibition by prior injection of pETX ,30,31,32Clostridium perfringens type D enterotoxemia of sheep spans a disease continuum, ranging from a peracute clinical course with sudden death at the severe end of the spectrum to a subacute or chronic intoxication with a more prolonged course. When large amounts of ETX are absorbed from the intestine, the disease can be rapidly fatal [ly fatal .Clostridium perfringens type D [Acute ETX neurotoxicity mainly affects young, rapidly growing lambs and goat kids, but sheep and goats of all ages may succumb. It was traditionally believed that newborn lambs and kid goats are not susceptible to type D enterotoxemia, probably because of the low level of trypsin activity in the intestine due to the trypsin inhibitory action of colostrum. However, recent evidence indicates that rare cases may occur in newborn lambs and goat kids colonized by lambda toxin-positive strains of s type D . In thess type D ,22,23.At autopsy, lamb carcasses are usually well-nourished. There is frequently excess, straw-colored pericardial fluid, which clots after being exposed to air, and forms fibrin strands. Pulmonary congestion and edema, as well as hemorrhage beneath the endocardium and epicardium of the left ventricle and other serosal membranes, are common macroscopic findings. Kidneys may be soft, friable and congested with type D enterotoxemia, sometimes referred to as “pulpy kidney disease”. However, since this gross appearance probably reflects accelerated autolysis, its diagnostic importance should not be overestimated, especially if the autopsy is delayed. There is usually scant evidence of enteritis, but segments of small intestine are sometimes distended with gas and the mucosa is hyperemic ,23,42,43C. perfringens by anaerobic bacteriologic culture [Hyperglycemia and glycosuria, the result of rapid mobilization of hepatic glycogen, are pertinent clinical chemistry diagnostic aids. However, they are not consistently present, especially if urine collection is delayed post-mortem, or etiologically specific, being found in the terminal stages of other, particularly hepatic, ruminant disorders. A diagnosis of type D enterotoxemia is also abetted by smears of the intestinal mucosa containing large numbers of proliferating, Gram-positive, rod-shaped bacteria, which then need to be positively identified as  culture .C. perfringens type D can sometimes be found in clinically normal sheep. These small quantities of ETX are, nevertheless, below the detection limit of most routine laboratory diagnostic tests used to detect ETX. A few techniques have been used to detect ETX in the intestinal contents of sheep and goats, including polyclonal capture enzyme-linked immunosorbent assay (ELISA), monoclonal capture ELISA, a mouse neutralization test, and counterimmunoelectrophoresis. However, the results of these tests have sometimes been inconsistent [The presence of a large quantity of ETX in the intestinal contents is confirmatory of ETX poisoning, although small amounts of toxin and numbers of nsistent ,46,47 anIn peracutely, acutely and subacutely intoxicated sheep, and to a lesser degree, goats, the brain microvascular endothelium forming the BBB appears to be the principal target of ETX neurotoxicity ,48,49,50These fine structural changes are also found in ETX-exposed pulmonary, hepatic, renal and retinal microvessels ,58,59,60In ETX-intoxicated goats, microvascular intramural accumulation of proteinaceous edema fluid is more often found in the brain than the perivascular lakes that are a common histologic feature in ovine cases. This is posited to be due to the lower doses of toxin absorbed from the intestine in goats and, consequently, less severe microvascular injury and BBB breakdown ,65.Clostridium perfringens cause acute cardiopulmonary lesions, of varying severity, in sheep. Death can occur very rapidly when these lesions are severe and, in addition to brain damage, cardiac dysfunction caused by ETX is a potential cause of mortality. Hydropericardium (with a high fibrin content) and pulmonary edema are the most common cardiopulmonary lesions, followed by hydrothorax, the latter being highly correlated with interstitial pulmonary edema. Endocardial and myocardial hemorrhage are also frequently found. Cardiomyocyte necrosis is common, especially in the region of the atrioventricular node, and this site predilection could lead to cardiac dysfunction and be responsible for marked clinical deterioration and even death [It has recently been confirmed  that onlen death .The primacy of microvascular damage in the pathogenesis of acute EXT neurotoxicity is supported by the fact that this toxin produces a rapid and dose-dependent cytotoxic effect on cerebral microvascular endothelial cells in vitro ,67. FurtHowever, although these vasculocentric histologic changes are found in acutely to subacutely ETX intoxicated ruminants, and support a diagnosis of type D enterotoxemia, it is not known why there is an inconsistency, in both frequency and neuroanatomic distribution, of vascular lesions between individual cases. It is possible that only a subset of cerebral blood vessels possesses endothelial receptors for ETX or, given that ETX neurotoxicity is expressed in a dose- and time-dependent manner, these factors could determine which microvessels in selected brain areas are exposed to sufficient toxin to sustain endothelial damage. In experimentally ETX-intoxicated laboratory rodents, the toxin has been shown to cause either selective or more widespread neural injury, depending on the dose of circulating ETX and the time interval between toxin administration and death. In general, higher doses of ETX tend to expand the distribution of brain lesions, although still favoring certain vulnerable areas, whereas lesions in sub-acutely intoxicated animals exposed to lower doses of ETX are smaller, tend to be well-circumscribed and are confined to fewer susceptible brain regions. Moreover, as the time from toxin administration to death increases, the distribution of brain lesions tends to become more widely distributed . AnotherIn acute ETX intoxication, the brain edema due to breakdown of the BBB is initially vasogenic. This microvascular incompetence permits abundant extravasation of plasma proteins and fluid into the extracellular space (ECS). The protein-rich fluid principally affects the white matter, extending from the gray matter along adjacent white matter tracts. White matter edema is facilitated by the freedom with which fluid can move through parallel fiber bundles, and this edema progression can result in tissue necrosis. However, excessive water accumulation and dissemination eventually become a mix of vasogenic  and cytotoxic (cellular) edema, the latter characterized by intracellular accumulation of water, mainly in astrocytes, and then in neurons .Cerebral edema is a severe, deleterious and frequently fatal result of ETX-induced microvascular injury. It is defined as an increase in brain volume due to an elevation of tissue water content and results from the passage of free water from the vascular compartment to an intracellular or extracellular site, or both. This definition does not differentiate between either location, although it has been argued that cytotoxic edema is not a true edema as it is intracellular rather than interstitial . Brain eIn acute ETX intoxication, there is widespread extravasation of plasma albumin , which hSheep that develop FSE lesions usually survive for 5 to 7 days, and sometimes up to 14 days, and this more chronic form of ETX intoxication is characterized by neurologic signs including blindness, aimless wandering, ataxia, bruxism, head pressing, nystagmus, posterior paresis, lateral recumbency, ophistotonus and paddling convulsions ,20,71.Lambs and older sheep with subacute or chronic ETX intoxication may show herniation of the cerebellar vermis through the foramen magnum (“cerebellar coning”) which, although highly suggestive of type D enterotoxemia, can also occur with other disorders causing raised intracranial pressure .In goats, both kids and adult animals may be affected, and acute, subacute and chronic forms of the disease are recognized ,73,74. TIn ETX-exposed sheep following a more protracted clinical course, bilaterally symmetrical foci of necrosis are sometimes found in certain brain regions of predilection, including the basal ganglia, thalamus, internal capsule, midbrain, medulla oblongata, and cerebellar peduncles , this neWhile FSE is pathognomonic for the more chronic manifestation of type D disease in sheep, the reason for the selective vulnerability of certain brain regions remains elusive. It could reflect the higher susceptibility to ETX of the neural parenchyma, or vasculature, in these areas. After gaining access to the brain by damaging the microvasculature, it is possible that ETX could have a differential cytotoxic action on specific neuroanatomic sites . HoweverStudies of ETX pathogenesis in laboratory rodents have shown that this toxin causes excessive release of glutamate from pre-synaptic, glutamate-rich sites in the hippocampus, leading to post-synaptic dendritic injury and pyramidal cell death. The toxin also binds to the soma and dendrites of glutamatergic cerebellar granule cells and damages dopaminergic areas of the brain ,82,83,84The development of focal necrotic lesions in only some brain regions in FSE conforms to the concept of selective vulnerability . In mostWhile a cogent explanation for selective vulnerability is lacking for most neuropathologic entities, these lesions can, nevertheless, be diagnostically useful and may provide some insight into pathogenetic mechanisms. In FSE, the malacia (necrosis) denotes a grossly observable softening of the brain, but the malacic process is not pathognomonic for this clostridial disease, being the final common pathway of a variety of toxic, vitamin and nutritional deficiency, and ischemic insults to the central nervous system in domestic animals. However, there is valuable diagnostic specificity in the neuroanatomic pattern of distribution of FSE lesions .Centaurea solstitialis) and pigs with selenium toxicoses [In addition to FSE, there are a few examples in domestic animals of well-circumscribed, bilaterally symmetrical, selective necrosis in specific gray matter areas of the brain and spinal cord, for example, in the Australian cattle dog, Alaskan Husky, Yorkshire Terrier and Angus and Simmental cattle . A similoxicoses . Sheep ioxicoses .While reasons for selective vulnerability in most neurologic disorders is not understood, there are a few neuropathologic examples in humans where the selective vulnerability of a particular brain region is striking and correlated with a known pathogenic factor. In carbon monoxide (CO) poisoning, the well-defined necrosis in the globus pallidus and substantia nigra is due to the high affinity of the CO molecule for these iron-rich areas of the brain. Moreover, in transient global ischemia, the copious release and accumulation of the neurotransmitter, glutamate, into the brain extracellular fluid overstimulates N-methyl-D-aspartate (NMDA) receptors in certain hippocampal neurons, leading to excitotoxic death . In ordeIn ruminant ETX intoxication cases, there is sometimes displacement of the cerebellar tonsils through the foramen magnum, termed cerebellar coning . While tIn terms of disease prevention measures, vaccination with an alum-adsorbed, ETX toxoid produces solid immunity in sheep and goats and has been effective in reducing the prevalence of this disease. In sheep, two doses of vaccine, administered 4–6 weeks apart, are generally protective for about 12 months; an annual booster is recommended. Lambs from vaccinated ewes are usually given their first immunization at 4–6 weeks of age. Since the immunity produced in vaccinated goats is lower, and of shorter duration, than that of sheep, a booster is recommended every 3–4 months in this species. Pregnant small ruminants are usually vaccinated 2–4 weeks prior to parturition in order to provide colostral immunity to lambs and goat kids . EpsilonC. perfringens overgrowth, with excessive ETX production, in the intestinal milieu [It is also advisable, especially with unvaccinated animals, to prevent unrestricted access of lambs to large amounts of starch-rich feed, a major predisposing nutritional factor to l milieu .In ruminant livestock acutely intoxicated with ETX, microvascular endothelial (BBB) damage appears to be the fundamental brain lesion, resulting in diffuse parenchymal edema, and a resulting marked and rapid rise in ICP. The finding at autopsy of severe pulmonary edema, with hydrothorax and ascites, abundant pericardial fluid which forms fibrin clots when the pericardial sac is opened, and subendocardial hemorrhages are diagnostically useful for a diagnosis of acute ovine and caprine ETX intoxication. Microscopically, a microangiopathy, with deposition of a protein-rich perivascular fluid, is found in most acute ovine case, but in less than 20% of caprine cases. Cerebellar coning, due to an uncompensated increase in ICP, is also regarded as highly diagnostically suggestive but not confirmatory of type D enterotoxemia. In ovine but not caprine cases following a more protracted clinical course, bilaterally symmetrical necrotic foci are sometimes found in certain selectively vulnerable neuroanatomic regions, a pattern not observed with the same distribution in any other ruminant neurologic disorder.While these neuropathologic changes are generally sufficient for the diagnostic pathologist to make an etiological diagnosis, much remains to be elucidated about the different events marking the journey of ETX from the intestinal lumen through the systemic circulation to the brain. It is also unclear why certain brain regions, and/or their microvasculature, appear to be selectively vulnerable to ETX-induced injury. Pathogenetic studies are important; although vaccination has reduced the occurrence of this disease in ruminants, clinical cases still occur too commonly, with concomitant economic loss. Moreover, ETX is a potential zoonosis and bioterrorism threat. Since a wide range of species are known to be susceptible to this potent neurotoxin, it is likely that humans are also at risk, particularly as ETX is the third most potent clostridial toxin after botulism and tetanus.ETX was classified by the Centers for Disease Control and Prevention in the United States as a select agent—that is, one determined to pose a potential threat to public health and safety—but it has now been removed from this list. However, ETX is still classified as a potential biological weapon in some countries and authorization is required for its laboratory use. There is currently no therapy or vaccine approved for use in humans if ETX were to be used in a nefarious manner .


The main objective of this study was to determine the impact of increased demand for peregrine falcons via breeding  on the genetic structure of the birds. In the analysis, 374 specimens from six countries were sampled in 2008–2019 (omitting 2009), and all the birds analyzed were released into the wild as part of the Polish reintroduction program. The assessment of genetic variation was based on a well-known panel of 10 microsatellite markers described for the species. We calculated a fixation index for the samples from each year, and based on this, we determined the level of inbreeding. We also performed an analysis using the Bayesian cluster method, assuming that 1–19 hypothetical populations would define the division that best fit the samples. The most probable division was into two groups; in the first group, the samples from individuals delivered in 2013 were most often segregated; moreover, in this year, a jump in inbreeding, expressed by the fixation index, was observed. The earliest evidence of falconry activity dates to around 5000 B.C. and comes from Tell Chuera, an archaeological site in northeastern Syria . HoweverFalco cherrug) in Bulgaria [Reintroduction of the peregrine falcon was possible, thanks to intensive breeding efforts  ReintrodBulgaria .Falco peregrinus peregrinus [In Poland, peregrine falcon reintroduction has been taking place since 1990 and focuses on restoring the tree-nesting ecotype population, with individuals from breeders located in Poland and Western European countries ,12. In 2regrinus .Nesje et al.  describeThe aim of this study was to assess how the genetic structure of individuals used in reintroduction efforts has changed over the years 2008–2019.n =127), the Czech Republic (n = 117), Germany (n = 79), Slovakia (n = 38), Denmark (n = 10) and the Netherlands (n = 3)—prior to the birds being released into the wild as part of a polish restitution program. Samples were obtained during the years: 2008 (n = 11), 2010 (n = 46), 2011 (n = 53), 2012 (n = 73), 2013 (n = 65), 2014 (n = 43), 2015 (n = 35), 2016 (n = 6), 2017 (n = 16), 2018 (n = 18) and 2019 (n = 8). All individuals, divided by country, were used for the first part of the analysis; then, 170 individuals from the 6 breeders that provided the most falcons for reintroduction were analyzed. Of these breeders, 1 was from Germany (breeder 1), 1 was from Slovakia (breeder 3), 2 were from the Czech Republic (breeder 2 and breeder 5) and 2 were from Poland (breeder 4 and breeder 6).Peripheral blood was collected from wing veins via needle puncture, and then, stored in 96% ethanol . Blood samples were collected from 374 captive peregrine falcons. The samples were obtained from 47 breeders from 6 different countries—Poland  in accordance with the manufacturer’s instructions.Falco peregrinus species [Microsatellite genotyping was performed for all samples using 10 markers for which primers have been described in the literature for  species ,16,17,18Based on the related literature, 10 markers were selected: NVHfp5 (labeled with VIC), NVHfp13 (labeled with 6-FAM), NVHfp46_1 (labeled with VIC), NVHfp54 (labeled with PET), NVHfp79_4 (labeled with 6-FAM), NVHfp82_2 (labeled with VIC), NVHfp86_2 (labeled with PET), NVHfp89 (labeled with 6-FAM), NVHfp92 (labeled with NED) and NVMfp107 (labeled with NED). These markers were characterized by the presence of high locus polymorphism, primer melting points and product length, which allowed the reactions to be carried out as one multiplex PCR (polymerase chain reaction) procedure. Reactions were performed as described by Puchała et al. . Marker Visualization of the PCR product was performed using an ABI3500 DNA analyzer. For this purpose, the reaction product was mixed with formamide loading and size standard.Allele sizes were assigned using GENEMAPPER 4.0 software, produced by Applied Biosystems Inc.Using GenAlEx v6.5  ,22 softwRelatedness and inbreeding analyses were performed using GenAlEx, and the fixation index for all analyzed samples and for groups which were analyzed in the second stage of the analysis were calculated. Data were visualized using Excel.The genetic distance between the populations of the 6 largest breeders was estimated using Nei’s method  This metSimulation of the most probable division of individuals into groups was performed using STRUCTURE v2.3.4 ,28,29,30Multilocus genotypes were obtained in 10 loci. A total of 347 out of 374 individuals were genotyped in all loci, with 25 in nine loci and 2 in eight loci. Among the individuals genotyped at all loci, 85 had a unique genotype. No null alleles were found. The number of alleles observed at the loci ranged from four to nine. In one population, the private allele was present in the genotypes of 4 out of 38 individuals originating from Slovakia. The fixation index among all samples was 0.409. The genetic variability parameters across all the samples are presented in For groups divided by year of collection, Nei’s genetic distance varied from 0.009 (for the 2010–2011 pair) to 0.830 (for the 2008–2018 pair) with an average value of 0.280.STRUCTURE analysis was performed for all the samples, divided by years. As presented in A fixation index (F) was used to determine the change in inbreeding over the years. The course of changes over the years is shown in Multilocus genotypes were obtained in nine loci (Marker NVHfp79-4 was excluded). A total of 163 out of 170 individuals were genotyped in all loci, 5 in no loci and 2 in seven loci, and 69 individuals had a unique genotype. No null alleles were found. The number of alleles observed at the locus ranged from four to nine. The fixation index among all samples was 0.409. The genetic variability factors across all samples are presented in On the phylogenetic tree, shown in For the analysis of individuals from all six breeders, as for the analysis of all samples, the K = 2 groups best fit the data. In In both the analysis of all samples  and the Compared to our previous study, which included both breeding and wild falcons , the numCompared to the Finnish wild population, where 145 individuals sampled over 5 years were analyzed , for sevCompared to the 32 (16 wild and 16 breeding) individuals from the Czech Republic [Based on the comparisons shown, it can be concluded that the captive falcon population shows signs of genetic diversity. The large standard deviation of the F-ratio seen in Breeders from Poland are on both branches . Birds fDespite the demonstrated genetic similarity between breeding pairs and their offspring for the selected breeders , it can In the case of the six breeders that have been supplying individuals most regularly over the years, a change in the genetic structure of the individuals supplied is also evident b. Additin = 5), Slovakia (n = 2), the Czech Republic (n = 4) and Poland (n = 5). Such different origins of the birds and the fact that these breeders, to our knowledge, do not cooperate with each other, may explain the decrease in inbreeding in 2017. In 2010, the year in which intensive restoration of the species began in Poland, the level of inbreeding, expressed by the fixation index, was relatively high (compared to, for example, the population analyzed by Mengoni et al. [The reason for the drop in inbreeding in 2017  may be ti et al. , in whici et al. . The sigi et al. , such a The change in the genetic structure of the samples studied is clearly visible in the structure charts  and occuThe idea of saving a wild species through breeding work is often criticized due to the apparently greater success of wild-to-wild translocations . In the The data we collected suggest an impact of increased demand for birds, associated with reintroduction, on the genetic structure of birds kept in the breeding facilities from which the birds originated. This is a factor that should be taken into account in reintroduction plans, especially when the planned activities are intensive and extend over time.


Tissue factor‐procoagulant activity (TF‐PCA) on cells is modified by multiple molecular mechanisms of encryption and decryption. The risk of thrombosis is higher for patients with a high tissue factor antigen level at registration as this enables patient’s blood more PCA‐high status before the onset of cancer‐associated thromboembolism (CAT). ELISA, including the Quantikine assay with validation as performed in our study, can contribute to more precise prediction of CAT. The authors have no conflict of interest.Dear Editor,We would like to address the concerns regarding tissue factor (TF) antigen assays recently raised by Mackman et al.® ELISA, which we used in our study, fails to measure plasma TF levels. The conclusion by Mackman et al.The authors conclude based on their prior work that the commercial ELISA system, Quantikine® may not detect all forms of TF‐EVs, it does not invalidate our measurements of pancreatic cancer‐derived TF‐EVs.Their study also found no measurable TF antigen in plasma samples from ovarian cancer patients, whereas TF‐procoagulant activity (TF‐PCA) on isolated extracellular vesicles (EVs) from these plasmas was detectable.® kit because of the strong proportional relationship between TF antigen levels in nondenatured cell lysate evaluated by this ELISA and those evaluated by western blotting using the TF 10H10 antibody and also TF mRNA levels evaluated by RT‐PCR (figures 1, S1, and S3 in Ref. [® ELISA kit, we evaluated TF antigen levels in plasma of healthy volunteers and pancreatic cancer patients. We found that higher TF antigen level significantly correlated with lower fibrinogen level, shorter prothrombin time, and higher D‐dimer level,We carefully selected the ELISA kit in our study,Mackman's research group recommends TF‐PCA assay in evaluation of TF level in blood samples because it sensitively detects TF‐EVs increased in response to cell stimulation with LPS.TF‐PCA on cells is modified by multiple molecular mechanisms of encryption and decryption.In summary, accuracy of ELISA, including Quantikine, in detection of plasma TF may vary between cancer types. However, we believe that ELISA including the Quantikine assay with validation as performed in our study can contribute to more precise prediction of CAT.


White blood cell count to mean platelet volume ratio (WMR) and neutrophil-to-platelet ratio (NPR) have been demonstrated as prognostic inflammatory biomarkers of the acute coronary syndrome. We aimed to evaluate the prognostic value of WMR and NPR among myocardial infarction with nonobstructive coronary arteries (MINOCA) patients.  A total of 274 MINOCA patients were enrolled. Baseline clinical data, blood cell panel, and biochemical parameters were evaluated. The patients were classified according to the medians of WMR and NPR. The primary endpoint of the present study was major adverse cardiovascular events (MACE). Multivariable Cox regression analysis was used to assess the effect of independent variables of WMR and NPR on the dependent variable (MACE). P = 0.004 and P = 0.002, respectively). The combined high WMR and high NPR showed a significantly higher rate of MACE (long-rank P = 0.001). Cox regression analysis showed that the combined high WMR and high NPR were independent predictors of long-term MACE with the highest hazard ratio .  The median values of WMR and NPR were 701 and 0.03, respectively. During the median follow-up of 28 months, a total of 58 incidences of MACE occurred. The MACE rate was more frequent in high WMR and high NPR patients. In Kaplan-Meier analysis, the incidence of MACE was higher in WMR>701 and NPR>0.03 (long-rank  High WMR and NPR separately or in combination were correlated with increased risk of MACE among MINOCA patients, suggesting WMR and NPR may assist as a reliable inflammatory marker in risk prediction of MINOCA patients. Globally, ischemic heart disease remains a major cause of death, and its prevalence constantly increases . Around Inflammatory cells (leukocytes) and platelets play a crucial role in the pathogenesis and progression of atherosclerosis and AMI, producing intense inflammatory reactions –14. WhitTherefore, the present study aimed to explore the association and prognostic values of inflammatory biomarkers (WMR and NPR) for the first time in acute settings of patients with MINOCA.This was a single-centered retrospective observational study that enrolled patients with AMI who underwent invasive coronary angiography (CAG), with new-onset chest pain with an ECG presentation of STEMI and NSTEMI at the cardiology department of Shanghai Tenth People's Hospital , during the period from 2014 to 2019.MINOCA was diagnosed if a patient fulfilled the AMI criteria in the absence of obstructive coronary arteries  as recommended by the recent American Heart Association statement that introduces an updated definition incorporating the 4th universal MI definition, which by agreement excludes Takotsubo syndrome and myocarditis from the eventual diagnosis of MINOCA . PatientThe present study was in accordance with the Declaration of Helsinki and was approved by the hospital's ethical review board , and the informed consent was signed by each participant.The baseline and demographic clinical data  were collected from hospital records during admission. In addition, all cases underwent the invasive CAG, an echocardiographic assessment, and a serial electrocardiogram on hospital entry. Comorbidities such as diabetes mellitus, dyslipidemia, and hypertension were diagnosed as previously known, receiving specific therapy or fulfilling the well-established diagnostic protocols on workup during admission. All patients were initially managed with secondary preventative treatments recommended by the latest guidelines.Total WBC, neutrophils, lymphocytes, platelets, mean platelet volume (MPV), and other hematologic parameters were measured by an automated complete blood cell count. The present study used the full blood cell panel with the highest WBC count during the first 48 hours of admission to calculate the WMR (as the ratio of WBC count to MPV), NPR (as the ratio of neutrophils to platelets count), PLR (as the ratio of platelets to lymphocytes count), and NLR (as the ratio of neutrophils to lymphocytes count) from the same blood sample test report. The other laboratory parameters were measured and decided according to standard protocols.The median follow-up duration for this study was 28 months. Follow-up information was achieved through telephone calls to the patients or their relatives, checking computerized medical records and clinical notes obtained by expert cardiologists to assess the patients' medical status and outcome events or the 1st documented outcome case at the hospital. The primary endpoint of the present study was major adverse cardiovascular events (MACE), including (1) cardiovascular death ; (2) heart failure ; (3) nonx2) and Fisher's exact tests. The study population data analysis was classified according to the combination of WMR and NPR median values. The correlation between WMR and NPR was analyzed using the Spearman correlation coefficient. To determine the effect of independent variables of WMR and NPR on the dependent variable (MACE), Cox proportional regression models were constructed to obtain hazard ratio (HR). Multivariable Cox regression models were adjusted for traditional cardiovascular causative factors that are known to be associated with increased risk of MACE for MINOCA patients, such as age, sex, smoking, hypertension, diabetes mellitus, hyperlipidemia, atrial fibrillation, left ventricular systolic ejection fraction (LVEF), and the degree of coronary stenosis. To estimate the MACE-free survival, a Kaplan-Meier analysis was performed, and the log-rank test was used to determine the dissimilarity between groups. The receiver operating characteristic (ROC) graph was used to evaluate the significance of NPR and WMR in predicting the clinical outcomes. We also performed interaction term analysis to investigate the independent risk associated with WMR and NPR in specific patient subgroups . Statistical analyses performed were 2-sided; a P < 0.05 is considered significant.All analyses in this study used SPSS (v.22), and the figures were framed by GraphPad software . The mean ± standard deviation (SD) was used for numerical variables, and percentages were used for categorical data. Comparisons between groups of continuous variables were assessed by analysis of variance (ANOVA), while the categorical variables were expressed as the number of participants with percentage (%) and compared by the chi-square (n = 21) and loss of follow-up (n = 17). Finally, 274 MINOCA patients were enrolled in the final analysis of the present study  (n = 88), either low WMR or low NPR group (n = 77), and low WMR and low NPR group (n = 109). Heart rate was higher in the high WMR and high NPR group than in other groups, while the level of LVEF was lower. Systolic blood pressure was higher in the low WMR and NPR groups than in the other groups. Angiographic data showed that the prevalence of mild coronary artery stenosis (<50% stenosis) was higher in either the low WMR or low NPR group when compared with other groups. There were no significant differences in age, gender, hypertension, diabetes, atrial fibrillation, hyperlipidemia, and smoking history among the three groups.A total of 3472 AMI patients were consecutively selected, of whom 312 patients (9.0%) satisfied the diagnostic criteria of MINOCA. Of these, 38 patients were excluded due to missing WMR and NPR data (nt study . The mea< 0.001) . The parP = 0.001   (P = 0.003) and NPR  have moderate significant prediction of MACE.During the median follow-up duration of 28 months, 58 incidences of MACE were recorded, in which in the low WMR and low NPR group, 14 MACE occurred , in the either low WMR or low NPR group, 14 MACE occurred , and in the high WMR and high NPR group, 30 MACE occurred . The MACE rate was significantly higher in patients with high WMR and NPR. The rates of MACE in high WMR and high NPR vs. either low WMR or low NPR vs. low WMR and low NPR groups were 34.1% vs. 18.2% vs. 12.8%, respectively,  = 0.001 . The Kap Figures . Notably= 0.001) . The ROCP = 0.011). In model B, NPR>0.03 was an independent predictor of MACE outcome . In model C, the combined high WMR and high NPR were independent predictors of MACE events with the highest hazard ratio .To determine whether a correlation existed between WMR and NPR with MACE, we constructed three Cox hazard models . Model AP for interaction >0.05)  was performed, which demonstrated that there were no interactions between WMR>701, NPR>0.03, and clinically related variables  Tables  and 5. TThe current study was the first to demonstrate the association between WMR and NPR in predicting long-term outcomes in MINOCA patients. The main findings in the present study were as follows: (1) high WMR and high NPR were associated with worse long-term outcomes with the highest incidence of MACE; (2) WMR and NPR, both separately and in combination, are independent predictors of long-term outcomes among MINOCA populations.Despite MINOCA's high prevalence and poor clinical outcomes, the management of these patients remained controversial mainly due to unclear etiology, risk factors, and limited evidence-based clinical studies. The critical management of MINOCA is based on identifying the etiologic risk factors and final diagnosis , 6, 29. A recent clinical study showed that inflammatory markers such as NLR, NPR, PLR, and C-reactive protein at admission were significantly higher in the MINOCA population . AnotherTotal WBC counts and platelet counts have previously been implicated in the development of cardiovascular disease , 11. In On the other hand, platelets have been indicated to participate in atherosclerotic plaque development, extension, and atherothrombosis, which play a critical role in ACS events through interaction with leukocytes , 48, andTaken together, the findings of the present investigation are of significant clinical interest, which suggests that both WMR and NPR calculated from WBC subtype counts are readily and widely available inflammatory markers. The elevated levels of WBC, MPV, neutrophils, and platelets in acute MINOCA are indicative of a specific inflammatory pattern. The application of WMR and NPR may enhance clinical reasoning about the necessity of performing comprehensive risk stratification to reduce the MACE rate in MINOCA patients with high WMR and NPR. Large-scale prospective investigations are required to confirm our results and identify the impact of inflammatory biomarkers in the medical decisions in MINOCA populations.This study's findings need to be interpreted with a few limitations. First, it was an observationally retrospective, single-center, small sample size study; therefore, the data's generalizability is limited. Second, other inflammatory cytokines and oxidative stress markers were not available. Third, we used spot laboratory complete blood cell counts with the highest WBC count rather than blood test panel at a time interval. In addition, although we adjusted for potential confounding factors, we cannot eliminate unmeasured residual confounders. A prospective, multicenter study with time-interval blood cell counts, assessment of plaque burden, and evaluation of other inflammatory biomarkers is needed in future studies.High WMR and high NPR separately or in combination were correlated with increased risk of MACE among MINOCA patients, suggesting WMR and NPR may assist as a reliable inflammatory marker in risk prediction of MINOCA patients.


Plasmodium vivax (PvCSP) plays a critical role in parasite biology. It has been extensively studied as a leading vivax-malaria-vaccine candidate. In this study, the genetic polymorphism and natural selection of pvcsp in P. vivax isolates collected from the Central Highlands, Vietnam were analyzed to understand the genetic structure of the parasite circulating in the endemic area and to provide baseline information for effective vaccine development based on the protein. Only two major alleles, VK210 and VK247, were detected in Vietnamese pvcsp, with VK247 being the predominant one. The N-terminal and C-terminal regions of Vietnamese VK210 and VK247 variants showed a low genetic diversity. Amino acid substitutions, insertions of a single amino acid or octapeptide (ANKKAEDA in VK210 and ANKKAGDA in VK247), and tetrapeptide repeat motifs (GGNA) were the main factors generating genetic diversity in the two regions of the Vietnamese VK210 and VK247 variants. Interestingly, these two regions of Vietnamese pvcsp displayed a unique natural selection pressure distinct from global pvcsp, particularly with the neighboring Southeast Asian pvcsp population. Meanwhile, the central repeat region (CRR) in both the VK210 and VK247 variants showed a high degree of polymorphic characters, caused by varying numbers, types, and combinations of peptide repeat motifs (PRMs) in Vietnamese pvcsp. Highly complicated polymorphic patterns of the CRR were also detected in global pvcsp. These results expand our understanding of the genetic structure of Vietnamese pvcsp and the population dynamics of P. vivax in the Central Highlands, Vietnam.The circumsporozoite surface protein of  Plasmodium, including P. falciparum, P. vivax, P. malariae, P. ovale, and P. knowlesi, transmitted by Anopheles mosquitoes , C[n = 41) , India ((n = 41) , Mexico [n = 19) , Colombi(n = 30) , Vanuatu[n = 21) , and Vie 5.10.00 . Polymorpvcsp from 69 Vietnamese P. vivax isolates successfully yielded 116 pvcsp amplicons with different sizes ranging from 0.3 kb to 0.8 kb. Multi-clonal infections, presumed to have at least two different sizes of amplicons in an isolate, were detected in 43 isolates. The mean multiplicity of infection was 1.68. Among the 43 samples with multiclonal infections, 4 samples were multiple infections with different allele types of VK210 and VK247. Sequence analysis of these 116 pvcsp amplicons revealed only two allelic variants of Vietnamese pvcsp, VK210, and VK247, but not the P. vivax-like variant (n = 78). The VK210 variants accounted for the remaining 32.8% (n = 38). A comparative analysis of the global distribution of these two major pvcsp variants revealed that the proportions of these two major pvcsp variants differed by country. However, geographical relevance was not observed (PCR amplification of  variant . The VK2observed . The VK2pvcsp was highly conserved (91KLKQP95). Twenty-eight (73.7%) sequences belonged to H2, while the remaining ten (26.3%) sequences were classified into H1. For the VK247 variants, three distinct haplotypes, H1, H2, and H3, were detected (97DGAGNQPG104) at the end of the N-terminal region. Polymorphic patterns of the N-terminal region of global pvcsp were also analyzed by combining Vietnamese pvcsp sequences and currently available pvcsp sequences in the GenBank database. As reported previously [pvcsp population displayed a low level of polymorphic characters in the N-terminal region. In the VK210 variants, insertion of an alanine at the end of the C-terminus was the most notable variation, albeit several rare amino acid substitutions were detected in the Indian VK210 variants (97DGAGNQPG104) was found in only the VK247 variants from Vietnam and Myanmar. Despite these sequence variations and insertion/deletion in the global VK210 and VK247 variants, the R1 motif was well conserved in both variants of global pvcsp.The N-terminal non-repeat region of Vietnamese onserved . The VK2onserved a. Haplotdetected b. H1, wheviously , the glovariants a. Meanwhvariants b. Amino pvcsp displayed high levels of size in polymorphisms and sequence variations in both the VK210 and VK247 variants. In the VK210 variants, nine species of PRMs, including GDRADGQPA, GDRAAGQPA, GGRADGQPA, GNGAGGQAA, GDGAAVQPA, GDGADGQPA, GDGAAGQPA, GNRAAGQPA, and GDRAAGQAA, were basic building blocks constructing the CRR (n = 8). Most haplotypes started with GDRADGQPA and terminated with GNGAGGQAA. The CRR of the Vietnamese VK247 variants displayed more complex patterns than that of VK210. A total of 12 distinct types of PRMs were detected. They constructed 44 distinguished haplotypes (H1–H44) with different copy numbers and combinations of PRMs (n = 6). Most haplotypes started with ANGAGNQPG and ended with the same PRM. Only H5 and H43 started with different PRMs, which were ANGAGNRPG and ANGAGNQLG, respectively. Meanwhile, three haplotypes  were terminated with ANGAGNQSG or ANGASNQPG. The 8 PRMs, ANGAGNRPG, ASGAGNQPG, ANGAGNQPR, ANEAGNQPG, ANGASNQPG, ANGAGNQLG, TNGAGNQPG, and ANRAGNQPG, identified in the Vietnamese VK247 variants were novel ones that were not previously reported.The CRR region of Vietnamese  the CRR . Two PRM of PRMs . ANGAGNQpvcsp showed highly polymorphic patterns. A total of 98 types of PRMs were found in the global VK210 variants, some of which were uniquely identified in a particular country , overall haplotype diversity (Hd), and nucleotide diversity (π) were 0.102, 0.102 ± 0.065, and 0.0024 ± 0.0016, respectively (p > 0.1). Fu and Li’s D and F values were positive for both the N- and C-terminal regions of Vietnamese VK210. The N-terminal region of Vietnamese VK210 showed similar or slightly higher values of K, Hd, and π than that of the global VK210 variants, except for the Indian VK210 variants. The N-terminal region of the VK210 variants showed different trends in natural selection by country. The dN–dS values were negative for the VK210 variants from Vietnam, India, and Brazil, whereas such values for the VK210 variants originating from Myanmar, Cambodia, and South Korea were positive, suggesting a positive natural selection. For the VK210 variants from Iran, Mexico, Sudan, and Vanuatu, these values were neutral (0) as only one haplotype was identified from each country. Tajima’s D values for the N-terminal region of the VK210 variants from Vietnam, Myanmar, Cambodia, India, South Korea, and Brazil were negative, suggesting a purifying selection in the region. The C-terminal region of the global VK210 variants also showed nucleotide diversity and patterns of natural selection. The Vietnamese VK210 variants displayed the greatest nucleotide diversity. This might be attributed to the octapeptide deletion (97DGAGNQPG104) observed in the Vietnamese VK210 variants. Meanwhile, no nucleotide diversity (π = 0) was found in the VK210 variants from Cambodia, Iran, Brazil, Mexico, and Vanuatu. The C-terminal region of global VK210 was also under neutral or purifying selection considering that its Tajima’s D values were 0 or negative. However, the C-terminal region of Vietnamese VK210 had a positive Tajima’s D value, suggesting a balancing selection.The nucleotide diversity and natural selection of N- and C-terminal non-repeat regions in Vietnamese and global ectively . These vSimilar to the VK210 variants, substantial levels of nucleotide diversity and natural selection were also found in the Vietnamese VK247 variants . The K, P. vivax in the Central Highlands is also becoming a major concern in current efforts to control and eliminate malaria in the endemic areas [Plasmodium populations in the endemic areas will provide fundamental data for evaluating and improving efforts to control and eliminate malaria in Vietnam. However, limited information on the genetic make-up or structure of Vietnamese malaria parasites, both for P. falciparum and P. vivax, is currently available [pvcsp in Vietnamese P. vivax isolates collected in Central Highlands, Vietnam were analyzed to understand the genetic structure of the parasite and to provide expanded information in designing a vivax malaria vaccine based on PvCSP, a leading vaccine candidate.The malaria burden in Vietnam has remarkably reduced in the recent years. However, the Central Highlands remains a major malaria transmission area . The incic areas . Understvailable ,38,39,40pvcsp, namely VK210 and VK247, were identified in the Vietnamese pvcsp population analyzed in this study. Interestingly, the VK247 allele was a dominant allele accounting for 67% in Vietnam. This does not coincide with the general trend that VK210 alleles are prevalent in other neighboring Southeast Asian countries, including Cambodia, Myanmar, and Thailand [pvcsp alleles in Vietnam is not apparent. The population of mosquito vectors has been proposed as a potential factor affecting the distribution of pvcsp alleles in endemic areas [Anopheline fauna showed a greater species diversity than that of other places in the world [Anopheles species have been identified as vectors transmitting malaria in Asia-Pacific region [Anopheles species have been recorded and 16 of them, such as An. Dirus, An. Minimus, An. Harrisoni, and An. Epiroticus, have been recognized as vector species transmitting malaria [Anopheles mosquitoes in Vietnam is necessary to understand the differences in the adaptability of each allelic variant to different mosquito vectors that affect the prevalence of VK247 in Vietnam.Only two allelic variants of Thailand ,26,41. Tic areas . In Southe world . At leasc region . In Viet malaria ,46. Furtpvcsp [97DGAGNQPG104). Similar to other previously reported VK247 variants [pvcsp were relatively well-conserved without a significant regional or continental difference in the global pvcsp population. Notably, the R1 motif acting in the invasion of the malaria parasite into host cells [pvcsp, suggesting that this motif might be an attractive target for designing a PvCSP-based vaccine [Consistent with previous studies on the genetic diversity of global pvcsp ,26,47,48pvcsp , althougvariants , few amist cells  was tigh vaccine . Analysepvscp. Similar patterns in the genetic complexity of the CRR were also identified in the global VK210 and VK247 variants. Up to now, highly various PRMs, which probably originated from a common ancestor [pvcsp. New point mutations in PRMs might generate novel PRMs, which can further increase the genetic diversity of the CRR and contribute to the appearance of unique haplotypes in global pvcsp [pvcsp. Due to high levels of size polymorphisms in the CRR caused by varying numbers and arrangements of diverse PRMs, we could not conduct a direct analysis of the natural selection of the CRR in Vietnamese and global pvcsp. However, evidence supporting natural selection in the CRR has been previously reported [The CRR of both the Vietnamese VK210 and VK247 variants showed high levels of genetic polymorphisms. Different copy numbers and arrangements of diverse species of PRMs induced genetic diversity of the CRR in Vietnamese ancestor , have beal pvcsp . A recomal pvcsp  might alreported ,50,52.P. vivax isolates collected at different geographical sites in the endemic areas are necessary to gain an in-depth knowledge on the genetic structure of the parasite. Further analyses of genetic make-up of other polymorphic markers are also necessary.The limitation of this study was the small size of samples. Considering malaria is mainly prevalent in the Central Highlands, Vietnam, studies with larger numbers of pvcsp in the global P. vivax population can be a hurdle when developing a universal vaccine based on PvCSP. Like other global pvcsp, Vietnamese pvcsp also shows polymorphic characters throughout the gene, which is attributed to amino acid changes, insertions/deletions, and varying numbers of repeating PRMs. However, the Vietnamese pvcsp population displays a different evolutionary force from global pvcsp, especially with the neighboring Southeast Asian pvcsp population. The VK247 variants were prevalent. Substantial levels of genetic diversity and natural selection detected in Vietnamese pvcsp suggest that an adequate P. vivax population size remains to generate or maintain the genetic diversity of the parasite in Vietnam, despite a recent remarkable decline in malaria incidences. Moreover, substantial levels of multiplicity of infection and potential asymptomatic infections in the Central Highlands [P. vivax in the endemic area. The results of this study could expand our knowledge of the genetic structure of the Vietnamese P. vivax population and the global pvcsp population. Genetic polymorphic characters of global pvcsp also suggests that further analysis of genetic diversity in global pvcsp is essential to provide fundamental information for the development of an effective vaccine based on this antigen.PvCSP is one of the most intensively studied vaccine candidate antigens. However, the genetic diversity of ighlands  have emp


The growing concentration of the population in urban areas presents great challenges for sustainability. Within this process, mobility emerges as one of the main generators of externalities that hinder the achievement of the Sustainable Development Goals. The transition of cities towards innovations in sustainable mobility requires progress in different dimensions, whose interaction requires research. Likewise, it is necessary to establish whether the experiences developed between cities with different contexts can be extrapolated. Therefore, the purpose of this study was to identify how the conditions that determine a city’s readiness to implement urban mobility innovations could be combined. For this, qualitative comparative analysis was applied to a model developed using the multi-level perspective, analyzing 60 cities from different geographical areas and with a different gross domestic product per capita. The R package Set Methods was used. The explanation of the readiness of cities to implement mobility innovations is different to the explanation of the readiness negation. While readiness is explained by two solutions, in which only regime elements appear, the negation of readiness is explained by five possible solutions, showing the interaction between the landscape and regimen elements and enacting the negation of innovations as a necessary condition. The cluster analysis shows us that the results can be extrapolated between cities with different contexts. A global increase in the population living in cities has taken place, leading to projections such as 70% living in major urban centers by 2050 . This isThe greater the concern for the SDGs, the greater the attention should be paid to the events that take place in cities . Such isTo explain the processes and dynamics manifested in cities, as well as the interactions between cities and their context, urban transformation recognizes radical innovations as determinants in achieving social and environmental improvements ,19,23,24A city’s smartness is measured based on the factors that determine its level of readiness for transformation and change ,27,28. TThe smart city is the result of a transformation process followed by many cities around the world aiming to increase their efficiency, facilitate the participation of their citizens, and reduce the environmental impact of their activities . AlthougMass urbanization and resource scarcity are global phenomena for which urban transformation towards a smart city is the most viable solution . SustainThe purpose of this article was to identify how the conditions that determine the level of readiness among cities for implementing urban mobility innovations could be combined. To achieve this, we developed a model supported by the MLP, since it has been used to explain the implementation and adoption of innovative mobility solutions ,35,36,37The study of environmental transformations acting across different levels commonly employs the MLP of sustainable transitions in socio-technical systems ,42. A soThe landscape—the macro level—consists of deep structural trends and social or physical conditions—exogenous environment—that shape systems ,33,42,43This paper proposed a model based on the MLP to study the readiness of cities to undertake transitions towards mobility innovations, using components at three levels: landscape, regimen and niche . The firAmong the landscape conditions is the city’s level of innovation. An urban transition must recognize the relevance of innovation, experimentation and initiatives developed, as well as the processes that are specific to them . Cities Regime conditions include infrastructure, market attractiveness, system efficiency and social impact. Infrastructure comprises durable assets that define the physical configuration of mobility systems . InfrastAt the niche level, the readiness of a city to implement mobility innovations was used. The transformative capacity of a city is a comprehensive and holistic framework for identifying the relevant contextual conditions to advance changes towards sustainability . TurningSocio-technical transition (STT) recognizes that cities are complex, adaptive, dynamic and open systems in constant evolution ,47. SustProposition 1.Landscape and regime conditions are combined to explain the readiness of cities in order to implement mobility innovations.Each city drives its transformation processes and development levels in different directions according to its contextual conditions, needs and resources . In factProposition 2.The combination of landscape and regime conditions that explain the readiness of cities to implement mobility innovations differs between the contexts in which the city is located as measured by GPD per capita.The development of this work was based on the indicators presented in the Urban Mobility Readiness Index 2021 for a sample of 60 cities from different regions. Geographically diverse cites, ranging from sprawling megacities to more compact cities or to fast-developing metropolises, were examined. They present distinct mobility challenges and display varied solutions that they are pursuing. To rank the cities, the Index employs five dimensions that are the result of 57 key performance indicators that identify cities that excel in meeting their future mobility needs . The OliUrban sustainability is a complex system affected by economic, social and environmental dimensions, with interactions between them ,65. TherQCA is a technique that integrates elements of qualitative and quantitative analysis. It uses Boolean logic and set analysis to identify combinations of conditions that explain the presence of a result. Initially, analysis was focused on crisp variables—those that can only take the value of 0 or 1—hence its name csQCA (crisp set QCA). Fuzzy set QCA (fsQCA) is a variant of QCA that allows the calibrated values to oscillate between 0 and 1, so it can identify both differences in kind and differences in degree. fsQCA is an interesting complement to regression analysis as it focuses on the analysis of effects from a different perspective. While regression methods find the net effect of the variables, fsQCA offers a set of combinations—called recipes or complex causal patterns—that explain the result ,71. The The first step in the analysis was to calibrate the ratings received by the components of the landscape, regime and niche dimensions using the 95th and 5th percentiles for complete inclusion or exclusion and the mean for the crossover point . To ensuIn order to explain how the different conditions of the MLP interact in the suitability for implementing mobility innovations, the two-step protocol was applied. This is because, between landscape and regime conditions, a distinction can be made between remote (landscape) and proximate (regime) factors. This protocol establishes a first step corresponding to a necessity analysis for the remote conditions, and a sufficiency analysis for the proximate conditions and those that have been found to be necessary . RStudioWithin the first step of the two-step protocol, the necessary conditions were analyzed. A condition is necessary for the outcome if whenever the outcome is present, the condition is also present . As a reEnhanced standard analysis was applied to establish sufficient conditions. A condition is sufficient if its presence explains the appearance of the result. A consistency of 0.85 and one case per conjunction were used to create the truth table. No contradictory simplifying assumptions were identified, so only implausible counterfactuals had to be eliminated. All atomic regime conditions that were necessary from the necessity analysis were taken as directional expectations. For OVE, these are INF , SIM , MAT  and SEF . For ~OVE, they were as follows: ~INF  and ~SEF . 2 [The enhanced intermediate solution for OVE results in SIM + INF*MAT*SEF -> OVE. Thus, the solution is composed of two terms, in which the different regime-level conditions considered in the model are presented. Both terms have high parameters, SIM  and INF*MAT*SEF . Consistency—also called inclusion score—is the ratio of cases that, presenting the conditions of the solution, explain the result under analysis. In fsQCA, the consistency of the solution shows a similar role as the test ratios. Coverage is the ratio of cases that, presenting the results, are explained by the solution. The coverage of a solution measures the ability of the recipe to explain all of the observations and can be assimilated to R2 . Coverag2 . For exaIn the case of ~OVE, the enhanced intermediate solution is ~INN*~INF*MAT + ~INN*~INF*~SEF + ~INN*~SIM*MAT + ~INN*MAT*~SEF + ~INF*~SIM*MAT*~SEF -> ~OVE . The solThe graphical representation  of the sTo establish the robustness of the results and increase the credibility of the conclusions, existing tests were performed ,75. The In the table above , we obseTo establish whether the solutions obtained are generalizable across cities with a different GPD per capita, cluster analysis was performed to indicate whether the solution obtained for the pooled data using QCA was appropriate for each sub-population’s data. Unlike other techniques, in SetMethods, cluster is a diagnostic tool for clustered data. In other words, it is not intended to establish data grouping, but rather it is based on previous data grouping. In our case, we start from the allocation that is made of the cities under analysis in the index used, which groups cities based on their GDP in the following groups: high, upper-mid, lower-mid, and low . In thisIn the table above , we obset-test and the overall goodness of the regression model with R2 [To establish the individual net effects, a multiple linear regression was performed, in which OVE was explained on the basis of INF, SIM, MAT, SEF and INNThe significance of the regression coefficients evaluated with Student’s  with R2 . This reUrban mobility has great implications for the health and sustainability of cities and the population that resides in them. This situation deserves our attention for two main reasons ,7,54: thWe begin by analyzing the role of INN as a landscape condition. The literature has argued that increasing innovation can help cities to prepare for the transformation into a smart city by fostering cutting-edge technologies and ideas, stimulating business growth and generating employment opportunities ,49. HoweSince cities are systems with a constant evolution which must be guided by the SDG ,42,55, iIn the explanation of OVE, in line with , the relIn the case of OVE, the second solution is that in which INF, MAT and SEF are presented together. The transition processes towards sustainability can be shown through different paths that overlap each other . This reIf we explain ~OVE, ~INN is a necessary condition. Although from the results obtained for OVE, we cannot affirm that INN is among the factors that determine readiness for OVE ,49, we cIt seems striking that MAT, depending on the conditions with which it is combined, can determine both OVE and ~OVE. Thus, it appears that MAT is linked, to a greater extent, with the ability to attract a creative population or one with some economic strengths , rather Likewise, although it has been proposed in previous studies that INN and INF are the determinants of urban innovation, in our case, this is not verified. What is found is that ~INN and ~INF occur simultaneously to explain ~OVE. In the present work, based on previous works ,3,27, weCluster analysis shows us the possibility of explaining OVE and ~OVE independently of the context in which it takes place. However, this does not mean that there is only one way for cities to be ready to implement mobility innovations, as there are two options for explaining OVE. A similar situation arises for the explanation of ~OVE, where the values achieved show the suitability of analyzing all cities under analysis as a whole. The fact that the most enriched urban environments are not more proactive in investing in smart city projects can be explained by the fact that, as a result of this level, they are no longer interested in developing new projects .Finally, when analyzing the isolated effect of the conditions, the impact is similar, and there are notable differences when analyzing the joint effects of the conditions. In fact, INN, which obtains the lowest weight when establishing the isolated effects, comes to act as a necessary condition. Thus, the suitability of combining QCA and regression analysis is confirmed ,72. AddiThis paper has been developed with the aim of identifying how the conditions that determine the level of readiness among cities for adopting urban mobility innovations could be combined, responding to the request of the literature ,28. ThisThe first gap was identifying the combinations of factors that determine the level of readiness of cities to implement sustainable mobility innovations. The results show such combinations of factors for both OVE and ~OVE. Thanks to conjunctural causation, we have seen how, except in the case of the SIM effect for OVE generation, the conditions combine at least in groups of three to explain the phenomena under study. Thus, we can state, in line with , how difAs a second research gap, we have analyzed in different contexts the conditions that determine the implementation of mobility innovations in the smart city domain. Firstly, because all cities can be grouped together in the explanation of OVE and ~OVE, it does not seem that the context plays a determining role in the way in which the conditions determining their emergence combine. However, this does not mean that there is a single formula common to all cities. As we have already indicated, there are various combinations of factors that explain the phenomena under study in this paper.Just as mobility has not been established in the same way all over the world , the reaAmong the limitations of this work, it is shown how modifying the inclusion in the truth table would have altered the results obtained. Additionally, if we look at the case-oriented robustness parameters, the presence of shaky and possible cases is shown.


Tursiops aduncus, and the pantropical spotted dolphin, Stenella attenuata, are protected marine mammals in Thailand; however, knowledge regarding the populations of both species in Thai seas is minimal. We aimed to reveal the genetic diversity and population structure of two species, T. aduncus, and S. attenuata, based on inter-simple sequence repeats (ISSRs).The Indo-Pacific bottlenose dolphin, T. aduncus (n = 30) and S. attenuata (n = 23) found along Thai Andaman Sea coasts from 1998 to 2018 were used in this study. A total of 17 and 16 ISSR primers that produced clear and polymorphic bands were selected for T. aduncus and S. attenuata, respectively.Samples of stranded T. aduncus and S. attenuata were 93.750% and 92.857%, respectively. Phylogenetic dendrograms indicated that the population of each species was clustered into three groups. This outcome was consistent with the genetic population structure, as both suggested three genetic clusters (DK = 3). Genetic diversity analysis revealed that the average Shannon’s information index (I) was 1.926 ± 0.066 for T. aduncus and 1.714 ± 0.090 for S. attenuata, which indicate a high level of genetic variation. Further, low fixation index (F) values were observed for T. aduncus and S. attenuata at −0.231 ± 0.024 and −0.312 ± 0.042, respectively, suggesting that inbreeding is unlikely to have occurred for both species over the past decades.The highest percentages of polymorphic bands for At least three genetic clusters of both species were found in the Thai Andaman Sea, and the diversity indices of each species indicated that these species are not at a critical level for extinction. However, monitoring their population status should be prioritized to observe any future changes in the level of diversity. Tursiops aduncus, and the pantropical spotted dolphin, Stenella attenuata, have been categorized as rare marine animals in Thailand. However, T. aduncus has been recognized as a common species found along the coasts of the Thai Andaman Sea and the Gulf of Thailand [S. attenuata is an oceanic species with a pantropical distribution in all oceans between 40°N and 40°S. Moreover, this species has been observed in marine waters along both coasts of Thailand [T. aduncus was listed as a near-threatened species [S. attenuata was of the least concern [T. aduncus and 90 S. attenuata individuals were reported to inhabit the Thai Andaman Sea [Tursiops spp. and S. attenuata have been affected by various practices in the fishing industry in several regions of the world. Examples of these species include the offshore population of the common bottlenose dolphin Tursiops truncatus, which occurs in the Pilbara trawl fishery area in the waters of Northwest Australia [T. aduncus, which is often caught using gillnet fisheries in the waters of Japan and Pakistan [S. attenuata, which is negatively impacted by the tuna purse-seine fishery in the waters of the eastern tropical Pacific Ocean [T. aduncus and S. attenuata.All cetacean species inhabiting Thai seas, including two odontocetes identified as the Indo-Pacific bottlenose dolphin, Thailand . This coThailand , 3. MeanThailand , 2, 4. A concern . Further concern ; howeveraman Sea . All maraman Sea , 9. The aman Sea , 11. Fisaman Sea –14. Tursustralia ; T. adunPakistan , 17; andic Ocean , 19. In ic Ocean . HoweverDelphinus delphis) in Australian waters [Orcaella brevirostris) inhabiting the Gulf of Thailand [T. aduncus and S. attenuata inhabiting Thai seas. Consequently, the conservation status of the local population of these species in Thai seas cannot be evaluated. Thus, inadequate studies on local animal populations and inaccurate use of the conservation status could lead to ineffective management practices and may accelerate the extinction of local populations, as observed in the cases of the Antillean Manatee Trichechus manatus manatus, a subspecies occurring in Suriname [O. brevirostris, inhabiting the Songkhla Lake [Identification of management units is essential in developing conservation strategies for cetaceans, as most of these species are associated with extremely high mobility in marine environments, where geographical barriers are usually undetectable. Currently, genetic information is used to establish relevant management units for cetacean species in several regions of the world. In addition, this information is used in genetic population studies that have used nuclear and mitochondrial DNA markers for short-beaked common dolphins  using skin tissue samples from deceased dolphins.To monitor alterations in population diversity that may occur in the future and to collect additional information on the conservation biology of This study was approved by the Animal Use Committee of the Faculty of Veterinary Medicine, Chiang Mai University, Thailand, in 2020 (S14/2563).This study was conducted from June 2021 to February 2022 at the Faculty of Veterinary Medicine, Chiang Mai University, Thailand.T. aduncus and 23 deceased S. attenuata dolphins, which were stranded along the Thai coasts from 1998–2018, were provided by the tissue bank of the Phuket Marine Biological Center, Phuket, Thailand 4SO4, 50 mM Tris-HCl, 1.75 mM MgCl2, and 0.01% Triton™ X-100, New Jersey, USA), 0.2 mM dNTP , 0.2 mM ISSR primer, 1 U Taq DNA polymerase (Vivantis), and 10 ng/ml of the DNA sample. The PCR amplifications were performed in PTC-200 using a DNA Engine Thermal Cycler  under the following conditions: Pre-denaturation at 95°C for 5 min; 40 cycles comprising a denaturation step at 95°C for 30 s, annealing step at 55°C for 45 s, and extension step at 72°C for 1 min; and a final extension step at 72°C for 10 min. The PCR products were stained using RedSafe™ nucleic acid staining solution . They were separated using 2% agarose gel  electrophoresis through a PowerPac 200 (Bio-Rad) containing 1× Tris-acetate-ethylenediaminetetraacetate buffer at 120 V for 30 min. Finally, the PCR products were visualized under ultraviolet light using a GelMax 125 Imager .A total of 34 ISSR primers from the University of British Columbia  were initially screened to yield DNA fingerprints. Overall, 17 and 16 primers that produced reproducible bands were selected for the ISSR amplification of The ISSR amplification results of the DNA samples from each species were analyzed separately. The bands or loci that were clearly observed and unambiguous among the ISSR amplified fragments were scored for the presence (1) or absence (0) of bands. A binary matrix was generated to determine the level of polymorphism in each primer, which was represented by the percentage of polymorphic bands calculated using the formula established by Ng and Tan  as follohttps://darwin.cirad.fr/) [https://itol.embl.de) such as clustering analysis. The groups obtained from this process were then used for genetic diversity and differentiation in population analysis.Phylogenetic dendrograms were then created using the DARWIN software version 6.0 (rad.fr/) , 28 and https://biology-assets.anu.edu.au/GenAlEx/Welcome.html) [Genetic diversity parameters, including Shannon’s information index (I), observed heterozygosity (Ho), expected heterozygosity (He), and the fixation index (F), were determined using the GENALEX program, version 6.5 (me.html) . Furtherhttps://taylor0.biology.ucla.edu/structureHarvester/) [All samples of each species were analyzed using distance and model-based clustering methods to reveal population affinity and structure. The program STRUCTURE version 2.3.430   was usedvester/)  to identvester/) .T. aduncus were obtained using 17 ISSR primers (185 were polymorphic), whereas 160 DNA fragments of S. attenuata were obtained using 16 ISSR primers (135 were polymorphic) (T. aduncus were 93.570% (UBC861) and 37.500% (UBC807), respectively  . The higectively . In addi 25.000% . PhylogeT. aduncus indicated higher degrees of genetic variation in the a3 group than in the other groups . In the overall analysis, the Ho, He, and F of T. aduncus were 1.000, 0.817, and −0.231, respectively . In contrast, the greatest distance was observed between the a1 and a3 groups (0.103) , and the most distant relationship was observed between the b1 and b3 groups (0.305)  of r groups . Similarectively . Meanwhiectively . For T.  (0.103) . For S.  (0.305) .T. aduncus had a dominant genetic cluster (S. attenuata represented two dominant genetic clusters (The results from three independent runs indicated that the population structure of both species comprised three genetic clusters (ΔK = 3). The a1 and a2 groups of  cluster . The b2 clusters .T. aduncus and S. attenuata inhabiting Thai seas. To the best of our knowledge, this is the first study to analyze the available skin tissue samples of two stranded species of dolphins along the coasts of Thailand. Three genetic clusters of both species were detected using population structure analysis. Although the diversity indices of each species revealed high genetic variation, further genetic studies using on-field or biopsy samples are needed to obtain the exact location of each population.The results of this study provide genetic information on the population structure and genetic diversity of T. aduncus and S. attenuata. The average percentages of the polymorphic bands for T. aduncus and S. attenuata were high at 85.58% and 81.48%, respectively. These values were higher than those of several land mammals, such as cattle, goats, sheep, and Asian elephants, which were included in the previous studies [Limnonectes [In this study, we found that the UBC primer set 9 produced DNA fingerprints that could be used to investigate the genetic diversity of  studies , 35. The studies  and rhub studies , as well studies , dugong  studies , grass c studies , porpois studies , and Limnonectes .T. aduncus exhibited greater genetic relatedness to each other than to Group a2. This may be due to the fact that Groups a1 and a3 are likely to have a common ancestor in their recent evolutionary history. Similarly, according to S. attenuata, Groups b1 and b3 were more genetically related to each other than Group b2. Only one sample of S. attenuata obtained from the Gulf of Thailand (END194) was included in the population structure analysis, which revealed that the sample had not been separated from the other samples obtained from the Thai Andaman Sea and was determined to belong to Group b2. These results suggest a connection between S. attenuata populations inhabiting the Thai Andaman Sea and those inhabiting the Gulf of Thailand, as this oceanic species is also found around the Malay Peninsula, including the Malacca straits and in the waters of Singapore [According to the results of our study, the population structures of both species indicate that at least three genetic clusters of dolphin populations live along the Andaman coast. The Groups a1 and a3 of ingapore , 42. ImpT. aduncus inhabiting the Thai Andaman Sea that were observed in our study differed from those of the previous studies conducted by Natoli et al. [et al. [T. aduncus in regions around Mayotte and adjacent shallow reef banks had at least two genetic clusters. Further, S. attenuata population inhabiting the Thai seas was categorized into three genetic clusters. This outcome is similar to that of the three genetic clusters of S. attenuata reported in a study conducted in Hawaiian waters by Courbis et al. [The numbers of genetic clusters for dolphin populations differ in various regions because differences in marine environmental conditions can influence the diversification of each population. These conditions include water depth, ocean currents, sea surface temperatures, and the recycling of biological materials –45. The i et al. , which r [et al.  found ths et al. .A negative F value for both dolphin species in our study indicated high heterozygosity levels; thus, inbreeding is unlikely to be a problem for these dolphin populations. Both populations are unlikely to become extinct in the Thai Andaman Sea within a short period. Although the genetic diversity indices obtained in this study indicated a high level of variability, further genetic studies should be performed by including more samples. These studies should also include on-field or biopsy samples because using carcass-based studies to analyze population genetic structure can underestimate the genetic diversity in populations . ImportaT. aduncus and S. attenuata populations in the Thai Andaman Sea. At least three genetic clusters of both species were found, and the diversity indices of each species indicated that they are not at a critical level for extinction. However, due to the inadequate information on these populations and without proper monitoring plans, the abundance and genetic variability of these marine mammals may be significantly reduced in the near future. Thus, future studies are needed to address the existing knowledge gaps and to establish conservation plans for these populations.This study showed that ISSR can be used to successfully determine population structures and analyze the genetic diversity of PP: Conducted the study, analyzed the data, and wrote the manuscript. AP: Conducted the study and analyzed the data. KB: Supervised the laboratory work and wrote the manuscript. PK and KK: Provided the samples and relevant data. KN: Designed the study, analyzed the data, and wrote and finalized the manuscript. All authors have read and approved the final manuscript.