Dirofilaria immitis, or heartworm, through antigen in sera is the primary means of diagnosing infections in dogs. In recent years, the practice of heat-treating serum prior to antigen testing has demonstrated improved detection of heartworm infection. While the practice of heat-treating serum has resulted in earlier detection and improved sensitivity for heartworm infections, it has been suggested that heat treatment may cause cross reactivity with A. reconditum and intestinal helminth infections of dogs. No studies have assessed the potential cross-reactivity of these parasites with heartworm tests before and after heat treatment using blood products and an appropriate gold standard reference.Detection of n=163) was used to evaluate a heartworm antigen-ELISA (DiroCHEK®) and potential cross-reactivity with common parasitic infections. The heartworm status and additional parasite infections were confirmed by necropsy and adult helminth species verified morphologically or by PCR, and feces evaluated by centrifugal fecal flotation.Canine sera . In the 105 dogs without heartworms, specificity remained unchanged at 100% both before and after heat treatment despite confirmed infections with A. reconditum, Ancylostoma caninum, Ancylostoma brasiliense, Trichuris vulpis, Toxocara canis, Dipylidium caninum, Spirometra mansonoides, Macracanthorynchus ingens, Cystoisospora sp., Giardia sp., and Sarcocystis sp.Intestinal parasites were confirmed in 140 of the dogs by necropsy, and 130 by fecal flotation. Acanthocheilonema reconditum, intestinal helminths, and protozoal parasites in dogs. These findings suggest that the use of heat treatment improves sensitivity of heartworm tests and is unlikely to cause false positive antigen results due to  Dirofilaria immitis antigen tests is in contrast to the cross-reactive nature of antibody tests historically used for D. immitis [Dirofilaria sp. [D. repens infected dogs [The use of antigen testing to aid in the diagnosis of canine heartworm infections has been a vital tool for veterinarians since 1985. Commercialized antigen tests, primarily based on monoclonal antibodies generated from adult heartworm antigen preparations following acid and heat purification , demonst immitis , 7 and mted dogs .D. immitis antigen [O. lupi [Prior to 1995, manufacturer protocols for many commercial heartworm antigen tests used chemical and/or low heat (60–70Cº) immune complex dissociation (ICD) steps. However recently heat ICD treatment of serum at an elevated temperature of 104 Cº has been shown to allow earlier detection and improved sensitivity for  antigen . No decr antigen , 11. Hea[O. lupi .Acanthocheilonema reconditum, Dirofilaria repens, Onchocerca lupi, Toxocara canis, Ancylostoma caninum, Ancylostoma brasiliense, Uncinaria stenocephala, Trichuris vulpis, Dipylidium caninum, Strongyloides stercoralis, Spirocerca lupi and Angiostrongylus vasorum [A. vasorum [S. lupi [Acanthocheilonema dracunculoides, induced a false antigen positive result using normal manufacturer protocols [Dirofilaria repens has recently been confirmed to cross-react with 3 heartworm tests following heat ICD in a small number of experimentally infected n=3 dogs [D. repens infections, have positive antigen results post-heat ICD [Studies evaluating commercial antigen test specificity have used blood products from dogs experimentally or naturally infected with  vasorum –19, 21.  vasorum –16, 19,  vasorum , and thr[S. lupi . In a rarotocols . Dirofilheat ICD .Acanthocheilonema reconditum causes false positives with heartworm tests [D. immitis infection or D. repens infection. Additionally, conclusions of studies using non-biologically relevant samples, such as saline soakings of intestinal helminths, to assess cross-reactivity with heartworm tests, are potentially misleading [A. reconditum or intestinal helminths with heartworm antigen tests, with or without heat ICD of naturally sourced canine serum, have ruled out occult D. immitis infection by necropsy.Several recent publications have added confusion to the literature by stating rm tests –27 miscirm tests , which csleading . CurrentA. reconditum, intestinal helminths and protozoan infections using a commercially available heartworm antigen test.In this study, using sera from 163 dogs, 100 of which were previously characterized based on heartworm composition (adults and microfilariae) and antigen results with and without heat ICD , we asseCryptosporidium verification) [From 2017 to 2020, 163 previously euthanized dogs were collected and necropsied at the University of Florida College of Veterinary Medicine. All dogs collected during this time period were included in this study. The abdominal and thoracic cavities were thoroughly inspected for parasites prior to and following removal of the heart, lungs, caudal esophagus, thoracic aorta, and gastrointestinal (GI) tract. The entire GI tract was opened and mucosa scraped and rinsed into a 355 µm sieve for parasite recovery. Helminths were rinsed in phosphate buffered saline (PBS) and stored in 70% ethanol. Feces removed from the colon was examined by centrifugal flotation using Sheather’s sugar (SG 1.26), and when necessary, by acid fast staining (i.e. ication) . Antigenication) .D. immitis (12S rDNA) and D. repens (12S rDNA), using single-plex reactions to increase sensitivity [A. reconditum (cox1) was performed as described [Whole blood from all dogs was examined for microfilariae by direct smear and Modified Knott’s technique (MKT). Microfilariae recovered from whole blood by MKT were identified morphometrically , 29, 30.sitivity . Specifiescribed  modifiedescribed .χ2 test was used to compare SE and SP before and after heat treatment of serum and for the calculation of two sided p-values, with P<0.05 considered statistically significant [The prevalence (PR), sensitivity (SE), specificity (SP), negative (NPV) and positive predictive values (PPV), and 95% confidence intervals (“exact” Clopper-Pearson) were calculated for antigen results using a diagnostic test 2x2 contingency table using a commercial statistical software . The McNemar paired nificant . The PPVnificant .D. immitis adult or immature adult worms was verified by necropsy. Viable heartworms were found in 58/163 (35.6%) dogs . A total of 105 dogs, without viable heartworms present at necropsy, were classified as non-infected. Microfilariae were detected in 38/163 blood samples by the MKT , 2 samples had microfilariae of A. reconditum only, and 12 with microfilariae of both D. immitis and A. reconditum. Microfilariae of all 8 MKT positive samples in heartworm non-infected dogs were identified as A. reconditum  (95% CI 64.7–88.7%) and 98.0% (50/51) (95% CI 89.6–99.9%) p=0.002. The observed increases in sensitivity were statistically significant. Altogether, 90.9% of heartworm infected dogs initially testing false antigen negative but post-heat ICD antigen positive were verified as occult, male or female only heartworm infections. All 105 dogs verified as heartworm negative at necropsy tested antigen negative, a specificity of 100%, (95% CI 96.6–100.0%) both before and after heat treatment (Table The sensitivity of the DiroCHEK® before and after heat treatment of sera for ns Table  was 69.0n=128), cestodes (n=72), and acanthocephalans (n=1). Helminths recovered by necropsy in heartworm infected dogs included A. caninum, A. brasiliense, T. vulpis, D. caninum, and Spirometra mansonoides. Helminths recovered by necropsy in heartworm non-infected dogs included A. caninium, A. brasiliense, T. vulpis, D. caninum and S. mansonoides. Additionally, GI helminths recovered only heartworm non-infected dogs were T. canis and Macracanthorynchus ingens  were found, along with Giardia cysts  and included species of nematodes n=18, cestodAcanthocheilonema reconditum, intestinal helminths and protozoan parasites in this study. The specificity was unchanged at 100% both before and after heat treatment assessed using serum from 105 confirmed heartworm non-infected dogs. Given the high level of parasitism in these heartworm non-infected dogs  were detected post heat-ICD, once thought to be undetectable due to no or low antigen released. Alternatively, if intestinal parasites do release cross reactive antigens they may be limited to the local intestinal environment, and may not enter circulation. The results of this study, in agreement with the historical literature [No cross reactivity was seen between the commercially available DiroCHEK® heartworm assay and the sera from heartworm non-infected dogs naturally infected with s Tables , 3 this artworms /7 were dterature , 14, 16,A. reconditum post heat-ICD, agree with previous observations of previous studies using heat ICD in dogs where the heartworm status was unknown, only A. reconditum microfilariae were detected, and no antigen detected following post-heat ICD [A. reconditum microfilariae, initially tested “no antigen detected” but converted to post-heat ICD antigen positive [The findings of this study, no observed cross-reactivity by heat ICD , 35. Recpositive . Though A. reconditum could cause false positive antigen results on heartworm antigen tests [D. repens were not ruled out by necropsy, the accepted gold standard [Wolbachia and Filariid PCR [A. reconditum identified by MKT and PCR. Another recent case report assuming cross reactivity without knowing the true heartworm status involved a previously stray dog from D. immitis endemic Spain, imported into the Netherlands, and suspected of heartworm infection due to microfilariae, and a post-heat ICD antigen positive test [Acanthochilonema dracunculoides cross reacts with heartworm tests post-heat ICD based primarily on the post-heat ICD antigen results, PCR results, as well as echocardiography not visualizing heartworms in the trunk or pulmonary arteries, the latter a finding not uncommon in mild asymptomatic heartworm infected dogs [D. immitis infection, known to test false negative on antigen tests, and as demonstrated in this study, 90.9% of false negative results testing positive post-heat ICD were due to male or female only occult infections. Male heartworms may also be a possible scenario for the initial post-heat ICD antigen positive results and short period (~104 days) to “no antigen detected” post-heat ICD, particularly due to the low level of antigen released by male heartworms [A. dracunculoides does indeed cross react with heartworm tests post-heat ICD, and shown to react using standard protocols under extreme infection intensities [A. dracunculoides, and with occult D. immitis and Dirofilaria repens verified absent at necropsy, or experimental samples evaluated, similar to that recently reported for D. repens [The results reported in this study underscore the importance of using well-characterized samples by combining multiple diagnostic methods with necropsy to verify or rule out occult heartworm infections prior to assessing cross-reactivity of an organism with a diagnostic test. Previous studies have suggested that en tests –27 by mien tests , an inapstandard . Additioriid PCR , though riid PCR . In thisartworms . The obsartworms . While tensities  at this . repens .D. immitis in the thoracic cavity. One of these 4 dogs had no heartworms present in the cardiopulmonary system, only the thoracic cavity, and would have been presumably an unverifiable antigen positive by other diagnostic methods with the exception of potential eosinophilia on a CBC blood panel [While the specificity observed with the DiroCHEK® in this study was unchanged at 100% both pre-and post-heat ICD antigen testing of serum, it is important to consider the animal history and clinical factors when interpreting antigen results . Four ofod panel . In anotod panel . In thatD. repens in a shelter dog and cat from a Florida, USA shelter and unknown whether those cases were imported or were acquired locally, there is a need for a multiple diagnostic approach and increased awareness and surveillance particularly among sheltered animals [Given the recent reports of  animals . It has  animals . Importe animals .A. vasorum and S. lupi, both rarely reported and often asymptomatic, can be diagnosed using different means and therefore, differentiated from D. immits [A. vasorum is considered an emerging threat and is endemic in wild canids of Newfoundland and Nova Scotia, Canada [Spirocerca lupi is endemic in wild canid populations in the southeastern US and has been reported in bobcats, grey and red foxes, and coyotes in Florida, USA [D. repens was not identified by MKT or PCR testing of blood. Dracunculus insignisis, also not found in this study is suspected to cross-react with heartworm antigen tests [D. immitis. Since they were present in a dog with a known D. immitis infection, it is not determined whether or not their presence contributed to the positive heartworm antigen result.Parasitic infections in North America known to cross-react with heartworm tests include . immits , 42. In , Canada , 43 with, Canada . Spiroceida, USA . In thisen tests and shoulLimitations in this study include the use of the MKT, known to have a lower sensitivity for microfilariae versus a filtration concentration method, particularly when microfilaremia is very low . AlthougA. reconditum or the intestinal parasites evaluated here with or without the use of heat ICD of serum. When evaluating potential cross reactivity of diagnostic tests, ruling out occult D. immitis infections, intestinal, or tissue inhabiting parasites by necropsy combined with a multiple diagnostic approach is important. Additional surveillance is needed for helminths in domestic dog and wild canid populations by necropsy and/or other diagnostics. Further research to determine potential cross-reacting organisms is important to improve heartworm antigen tests, aid development of a definitive confirmation test, and to aid in differential diagnosis. Given the high sensitivity following heat treatment for detection of mature heartworm infections demonstrated here, and the high specificity observed in this study, the use of heat treatment may allow increased confidence for detection of mature heartworm infections in dogs without fear of compromising results due to some common, often concurrent, parasitic infections.The overall data presented here concludes that the DiroCHEK® heartworm antigen test is unlikely to cross-react with A. reconditum, intestinal helminths and protozoal parasites in dogs.The conclusions of this study suggest that the use of heat treatment improves sensitivity of heartworm tests without false positive antigen results due to


In ancient freshwater lakes, an abnormally large species diversity is observed. The mechanisms that generatedextremely high biodiversity in the ancient lakes have not been sufficiently studied and remain only partiallyknown. Sequences of environmental changes in highly complex ecosystems such as Lake Baikal, may induce sophisticatedcombinations of microevolutionary processes. These processes are likely to result in unusual “patterns” ofgenetic variability of species. The most unusual patterns include the ones when speciation is followed by incompletelineage sorting as well as mitochondrial or nuclear introgression. All these phenomena are diagnosed by comparingthe topologies of phylogenetic trees inferred from molecular markers of evolution located in mitochondria andnuclei. Mitochondrial and nuclear introgression is a particularly interesting and complex case, which is the process ofincorporating the gene alleles of one species into the gene pool of a sister species due to interspecific hybridization(introgressive hybridization). In many cases, existing methods for molecular phylogenetic analysis do not automaticallyallow the observed patterns of polymorphism to be explained and, therefore, cannot provide hypotheses thatwould explain the mechanisms which resulted to these patterns. Here we use adaptive dynamics models to studyneutral molecular evolution under various scenarios of interaction between sister species and the environment. Wepropose and justify a set of criteria for detecting how two evolutionary trees may differ, with a special focus on comparinga tree inferred from nuclear DNA to one from mitochondrial DNA. The criteria react to branching pattern andbranch lengths, including relative distances from ancestral lineages. Simulations show that the criteria allow fast andautomated detection of various types of introgression, secondary breaches of reproductive barriers, and incompletelineage sorting. Significant advances in gathering the data on genetic polymorphismsof all kinds of organisms have shed light on importantfeatures of the evolutionary process. For example, recenttechnique, allow one to study previously cryptic features ofthe mechanisms, generating and maintaining the diversity oflife. Among the most challenging foci of modern evolutionarystudies are the hyper-diverse and geographically constrainedancient freshwater lakes . These lakes are inhabited byrapidly evolving species assemblages generated by adaptiveradiation, mostly in sympatry, and ultimately responding tothe fast and powerful environmental challenges generated byglobal changes . Studies of speciation processes in ancient lakesrevealed numerous cases of presumably complicated evolutionaryhistories and therefore many unexpected patterns ofgenetic diversity. The most striking are the cases of dramaticdiscordance between the patterns resulting the studies ofmitochondrial and nuclear DNA described in .Although these phenomena are well and long known to existin many species, their explanations involve the assumptionof large-scale range shifts  and thus are hardly applicable to the situationsof sympatric or parapatric speciation responsible for the mostof the species diversification in relatively small and closed ecosystems.A systematic study of disagreements between mitochondrialand nuclear phylogenies requires a formal procedureautomating search for such cases, such as testing significanceof the disagreement and modelling of evolutionary scenarioslikely to cause generationof the discrepancies between nuclearand cytoplasmic phylogenies. Here we describe fast simultaneousanalysis of two tree topologies allowing one to detecta discordance, test its significance and diagnose its type. Wetest this approach on a set of trees resulting simulations ofevolutionary events and on the real-world data set on endemicto Lake Baikal gastropods of genus Baicalia.Individual based modeling. Individual-based models simulating evolution of two sisterspecies of diploid organisms possessing both mitochondrialand nuclear DNA markers were designed as described in. Differential responses of the sisterspecies to the same environmental challenges were modelledby pre-setting independent curves of environmental nichecapacity for the two sympatrically occurring species. Modelalso allows independent variation of gene flows between thespecies thus mimicking asymmetric or symmetric breach of the reproductive barrier during defined periods of time. Eachsimulation was succeeded by collection of certain amount of marker sequences from the same “individuals”, savedin separate files and maximum likelihood trees were inferredwith PhyML version 3.3.20180129 ,using the model of molecular evolution set to JC because ofthe simulation settings. Tree comparisons were performedwith custom Python scripts using ete2 library .Types of discordances between mitochondrial and nuclearmarkers. Discordance between nuclear and mitochondrialtrees should be declared when their branching patternsdiffer significantly so that one of them looks distorted stronglyif compared to the other. We test following kinds of phylogenydiscordances of two sister groups of organisms may occur:splitting into two species;introgression is when on the one tree all groups form separateclades, while on the other tree one group appears insidethe cluster formed by the other group. In other words, thecommon for one group allele becomes fully substituted withthe allele originated from the other group;inherited polymorphism (incomplete lineage sorting)is when due to resent breach of reproductive barrier bothgroups acquire alleles from the other ones. Discriminatingbetween different types of branching order discordances.In order to detect the cases of introgressions while comparingdata sets, we had developed the test, which was used thento optimize the processing of fairly large amounts of data.The test employs three values estimated from the phylogenies. The first value is the corrected mutational distancebetween the common ancestors of the two species (nodes LA and LB) and the most basal node CA (common ancestor of thetwo species). In the case of inherited polymorphism all threepoints in fact are the same point and thus all three distancesare equal to zero. Therefore if all three distances insignificantlydiffer from zero one may assume incomplete sorting ofancestral lineages. Obviously all three distances would havesignificant lengths if unaccomplished divergence of the twospecies took place. We can also find the distance between CAand L (d ) for two species and find their difference (D). Withinherited polymorphism and separation into two types, thedistances da and db will be approximately equal.The second value useful for detecting of introgression isthe sum of all distances from all representatives of the samespecies to the most recent common ancestral node of the tree. In spite of any accomplishments this value shouldbe approximately equal if molecular clock hypothesis holds. The latter condition may be tested separately. The significantlydifferent from zero value of the difference between EA andEb determined for one molecular marker, i. e. mitochondrialgene while remaining approximately equal for other marker(s)points at full introgression .http://etetoolkit.org/). Based on the topologyof the tree it calculates the coefficient of monophyly, P.It becomes 0 in case of a monophyletic clade and 1 in caseof polyphyly.The possibility of a dramatic violations of molecular clockhypothesis as well as numerous other reasons make it necessaryto support the hypothesis on the introgression independently.Here we propose to use for that purpose the testfor monophyly of the clades in question. We employed thesimplest form of the test as it is implemented in ete2 and ete3libraries for Python ; CA – closest common ancestor; L – furthestdescendant of the common ancestor indexed by the species;d – distance  from CA to L indexed by thespecies.It is important to note that the protocol (pipeline) describedhere makes it easy to test directly the statistical significance ofthe diagnosis. This may be done by applying it to the bootstrapreplicates of the original data set followed by odds ratio testto estimate the support to the conclusion.In the case of the full introgression all sequences of onetype appear as the ingroup(s) to the recipient type. The othermarker yields monophyletic clusters consisting of a singletype of sequences. The four cases above may be diagnosedby measuring the distances between the common ancestors ofsequences as they appear on the tree and comparing them todistances from the common ancestors to the common root incombination with the monophyleticity test for the both sistergroups. Indeed, the inner branch between the common ancestorsof two groups passes the node defined by the outgroup inall cases except for complete introgression. In the latter casethe common ancestor of the recipient group is connected tothe outgroup via the common ancestor of the donor group. Inthis case the donor group is always polyphyletic, while therecipient may be both monophyletic or polyphyletic if theintrogression occurred more then, once or multiple donor’slineages were involved. Mutual introgression from the recentbi-directional hybridization generates the latter pattern in bothdirections. In this case each type of sequences appears in theother cluster as an ingroup.Introgression differs from incomplete lineage sorting becausein former case common ancestors of the invader (donated)lineages would be significantly younger then the commonancestor of the recipient. In contrast, incomplete sortingof ancestral lineages results in the absence of separate clustersfor the two groups in case of one of the markers. The simplestcause of the latter may be the insufficient rate of molecularevolution of the marker affected.In all cases the internal branching order must be robustlysupported, and the branches crucial for the diagnosis must havesignificant lengths. Therefore testing the diagnosis significancesimply turns into testing the significance of the sum of branchlengths between common ancestors of the groups (species) and the most ancestral node of the tree. There are several methodsof estimating the confidence limits of the branch length.Additional feature helping to diagnose the distortions of treetopology is the monophyleticity of the groups. In the case ofthe lack of any discordance, the mutual correspondence musthold between the groups and clades on the tree.If the tree is rooted by outgroup  CAA and CAB labelled with colored dotsdesignate the common ancestors of the respective groups, Ois their common ancestor as it is defined by the outgroup)linked to the common ancestor of species A and B with theircommon ancestors designated as it is shown on the samefigure, the relative position of the three nodes will distinguish between full introgressions if taken together with the resultsof testing the groups for monophyleticity. All possible outcomesare summarized in Table 1 (L is the distance betweenthe common ancestor of a groups to the common ancestor ofthe two groups).Baicalia sequences. Sampling locations of Baicalia specimensare specified in . GenomicDNA was extracted from muscle tissue using a modifiedmethod described earlier . Gene fragmentsof mitochondrial cytochrome c oxidase subunit 1 (COI) wereamplified using primers L1490 (5ʹ-GGTCAACAAATCATAAAGATATTGG-3ʹ) and H2198 (5ʹ-TAAACTTCAGGGTGACCAAAAAATCA-3ʹ) ; fragments of the internaltranscribed spacer (ITS1) were amplified using primersKp-2F (5ʹ-AAAAAGCTTCCGTAGGTGAACCTGCG- 3ʹ)and 5.8SR (5ʹ-AGCTTGGTGCGTTCTTCATCGA-3ʹ) . An average of 1–3 μL of DNA extracted wasamplified in a 25 μL reaction using BioMaster HS-Taq PCRKit  under the conditions recommendedby the manufacturer. The conditions for amplification, electrophoresisand amplicon purification are those in T.E. Peretolchinaet al. (2007). GenBank accession numbers ofITS1 sequences: FJ598711, FJ598712, FJ598715–FJ598723,FJ598727–FJ598732, FJ598735–FJ598741, FJ598743–FJ598745, FJ598748–FJ598760, FJ598762–FJ598771,FJ598832–FJ598848; of COI sequences: Z92995 , HQ113269–HQ113278, DQ436384–DQ436443,GU22640–GU22649, KT885116, FJ749133.Software availability. All custom software used in thiswork is available from https://github.com/dysh/MRDR.We have used several example phylogenies resulted fromcomputer experiments where various scenarios were usedto generate different patterns of disagreements between maternallyinherited mitochondrial and nuclear DNA of diploid organisms. The simulations involved differential responsesof sister species to the changes of environmental capacityaccompanied by periods of reproductive isolation breaches.Each simulation produced two sets of “DNA sequences” used for tree inferences subsequentlycompared to each other using the procedure described here.All kinds of disagreements between the trees were obtainedin course of these simulations and successfully classifiedusing the distances between ancestral roots and tests formotophyleticity (Table 2). An example of full mitochondrialintrogression is shown on Fig. 3 (circles designate commonancestors. The trees are rooted with the starting sequence usedin the simulation).Experimental data example: genus Baicalia. Gastropodgenus Baicalia belonging to the subfamily Baicaliinae endemicto Lake Baikal consists of five species diverged from thecommon ancestor relatively recently in confines of the lake. Species of this genus differ morphologicallyand ecologically. The most remarkable ecological differencebetween them is their substrate – dependent matingbehavior. Both nuclear and mitochondrial markers have beenused in phylogenetic inferences involving the species of Baicalia. Dramatic discrepancies between phylogeniesinferred from several nuclear markers and mitochondrialmarkersare reported to be typical for the whole group and forBaicaliain particular . Here we have reproduced phylogenetic inferencesusing the data set consisting of two markers of different inheritancemode belonging to three Baicalia species: B. carinata,B. dybowskiana and B. turriformis from . Two separate phylogenies have been obtained for thenuclear gene ITS1 and mitochondrial COI. Maximum likelihoodtrees obtained differed from each other .The two species, B. dybowskiana and B. turriformis, appearas monophyletic separate clades in a phylogeny inferred from nuclear sequences. According to the tree inferred from thesequences of mitochondrial origin both B. dybowskiana andB. turriformis become in-groups to B. carinata. However, theirclusters are separated from each other. This example illustratesthat discrepancies between phylogenies may be relative andcomplicated if traced in more than two groups: while thereare clear indications of full mitochondrial introgressionsfrom B. carinata to B. turriformis and to B. dybowskiana, ifthe representatives of latter two species are subjected to phylogeneticinference in the absence of B. carinata there is nodiscrepancy between the trees inferred from different markers.Interestingly, the two introgressions were not simultaneousand occurred with a large time gap.We present here a simple and fast procedure allowing oneto distinguish automatically between contrasting patterns ofdisagreement between trees inferred for the same groups oforganisms using different sets of data. In this communicationwe concentrate on the comparison between trees inferredfrom DNA sequences of mitochondrial and nuclear origin.First, we define different kinds of disagreements between thetree topologies  andpropose the set of criteria, which may be estimated from aphylogeny. The approach is based on the estimating distancesbetween common ancestors of groups defined externally, forexample, by species identity of their members. Two trees are used as the input, the groups are tested if they are monophyleticand then set of distances between clusters is measured. At thispoint it is possible to test for the statistical significance of thedistortions and their diagnoses using any appropriate approachsuch as various kinds of bootstrapping.This procedure is required for any modeling efforts aimingat the elucidation of ecological circumstances favoring differenttypes of disagreements between trees inferred for thesame organisms. 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Escherichia (E.) coli EPEC, Shigella (Sh.) sonnei, Salmonella (S.) typhimurium, Klebsiella (K.) pneumoniae and Clostridioides (C.) difficile were determined. Investigated products were a yeast mono strain probiotic containing Saccharomyces (Sac.) boulardii, bacterial mono strain probiotics containing either Lactobacillus (L.) rhamnosus GG or L. reuteri DSM 17938, a multi strain probiotic containing three L. rhamnosus strains , and a multi strain synbiotic containing nine different probiotic bacterial strains and the prebiotic fructooligosaccharides (FOS). Inhibition of pathogens was moderate by Sac. boulardii and L. rhamnosus GG, medium by L. reuteri DSM 17938 and the L. rhamnosus E/N, Oxy, Pen mixture and strong by the multi strain synbiotic. Head-to-head in-vitro pathogen growth inhibition experiments can be used to differentiate products from different categories containing probiotic microorganisms and can support the selection process of products for further clinical evaluation.A variety of activities potentially contribute to the beneficial effects of probiotic bacteria observed in humans. Among these is a direct inhibition of the growth of pathogenic bacteria in the gut. The present study characterizes head-to-head the in-vitro pathogen growth inhibition of clinically relevant infectious bacterial strains by different types of probiotics and a synbiotic. In-vitro growth inhibition of  The invention of antibiotic therapy some 100 years ago was a major step forward in medical practice, allowing medical practitioners to manage otherwise deadly infections caused by pathogenic bacteria. However, in the recent past, the rapid increase of bacterial antibiotic resistance has become a pressing problem of global healthcare [C. difficile. Pathogenic bacteria are omnipresent in the human gut. However, as long as the gut microbiota is well balanced and diverse, no pathogen overgrowth will take place [Every orally taken antibiotic is leading to alterations of the gut microbiota, in some patients with symptoms , in others without symptoms . While ake place . Disturbke place .E. coli EPEC, Sh. sonnei, S. typhimurium, K. pneumoniae and C. difficile by representatives of different categories of products containing probiotic microorganisms are studied. The evaluated probiotics are the clinically well-established mono strain probiotics containing either Sac. boulardii [L. rhamnosus GG [L. reuteri DSM 17938 [L. rhamnosus strains , the other being a complex multi strain synbiotic containing six lactobacilli , three bifidobacteria strains (Bifidobacterium (B.) bifidum, B. longum, B. lactis) and the prebiotic FOS.Today, a huge variety of products containing probiotic microorganisms are available. These products can be categorized according to their composition: (i) yeast and bacterial products, (ii) mono strain and multi strain products or (iii) probiotic and synbiotic products. In the present study, the inhibitions of five bacterial pathogens, oulardii , L. rhamnosus GG  or L. reMost published in-vitro growth inhibition studies focused on the characterization of only one pathogenic bacterium and one or a limited number of products containing probiotic microorganisms. The present study evaluated the antagonistic activity of a range of products in head-to-head in-vitro growth inhibition experiments with a number of different bacterial pathogens, which can support the selection of products for future, more in-depth investigations.Sac. boulardii probiotic Enterol®  contains in each capsule 4.5 × 109 colony forming units (CFU) of the Sac. boulardii strain CNCM I-745. Dicoflor®  contains 6 × 109 CFU of L. rhamnosus GG ATCC© 53103 per capsule. BioGaia®,  contains 108 CFU L. reuteri DSM 17938 per 5 drops. Lakcid®  contains a total of 2 × 109 CFUs as mixture of the L. rhamnosus strains E/N (40%), Oxy (20%) and Pen (40%), [® Baby  is a freeze-dried powder. Each sachet contains a total of 109 CFUs as a mixture of equal CFU amounts of L. acidophilus LA-14; L. casei R0215; L. paracasei Lpc-3; L. plantarum Lp-115; L. rhamnosus GG, L. salivarius Ls-33, B. lactis Bl-04, B. bifidum R0071, B. longum R0175 and 1.43 g of the prebiotic ingredient FOS.The yeast E. coli EPEC , Sh. sonnei ATCC© 9290TM , S. enterica subsp. enterica serovar typhimurium ATCC© 14280TM  and K. pneumoniae subsp. pneumoniae ATCC© 700603TM  the respective pathogen was inoculated on Columbia agar with 5% sheep blood  and incubated at 37 °C under aerobic conditions for 24 h [Sac. boulardii, L. rhamnosus GG, L. reuteri DSM 17938, the multi strain L. rhamnosus E/N, Oxy, Pen and the complex multi strain synbiotic) each containing 106 CFU were inoculated on MRS agar  and incubated for 48 h in the presence of 5% CO2 [For the in-vitro pathogen inhibition experiments with f 5% CO2 . After tC. difficile ATCC© 9689TM , the pathogen was cultivated under anaerobic conditions at 35–37 °C for 24–48 h on Schaedler agar  [6 CFU of the five evaluated products were inoculated on MRS agar and incubated for 48 h in the presence of 5% CO2. 10 mm diameter bars were transferred to a Mueller–Hinton agar with 5% horse blood and 20 mg/L NAD  and incubated under anaerobic conditions for 24 h.For the in-vitro pathogen inhibition studies with Germany) . SuspensFor testing a potential pathogen growth inhibitory effect of FOS, 100 μL of a solution containing 14.3 mg/mL FOS  was applied to a 10 mm filter disk that was then administered to respective pathogen testing plates. The multi strain synbiotic was tested on the same plates as a positive control.p-value < 0.01 was achieved.At the end of the incubation, measurements of inhibition zones around the tested colonies were taken from the outer edge of the colonies to the outer edge of the clear zones. Each test was performed in triplicate and the arithmetic means of the radii measuring from the edges of the colonies to the edges of the clear zones were calculated as well as the standard deviations SD . Independent T-test statistical analyses of datasets were conducted with GraphPad Prism software version 8.2 , Datasets were considered as significantly different when a In-vitro growth of all five tested pathogenic bacteria were inhibited by all evaluated products. However, the extent of in-vitro growth inhibitions by the products varied significantly for all tested pathogens .E. coli EPEC and K. pneumoniae were similar. The antagonistic effects of the tested products ranked from weak to strong inhibition as follows: L. rhamnosus GG < Sac. boulardii < L. reuteri DSM 17938 = multi strain probiotic L. rhamnosus E/N, Oxy, Pen < complex multi strain synbiotic. The inhibitory patterns found for Sh. sonnei and S. typhimurium an inhibition ranking from weak to strong of Sac. boulardii = L. rhamnosus GG < L. reuteri DSM 17938 = multi strain probiotic L. rhamnosus E/N, Oxy, Pen < complex multi strain synbiotic was found. While there was a significant difference  between the inhibitions of Sh. sonnei by Sac. boulardii or L. rhamnosus GG and the L. reuteri DSM 17938 probiotic or the multi strain probiotic L. rhamnosus E/N, Oxy, Pen, the difference of the inhibitions by these two groups of probiotics of S. typhimurium was not significant .For the inhibition of C. difficile by the tested products resembled that of Sh. sonnei and S. typhimurium. The weakest inhibitions were found for Sac. boulardii and L. rhamnosus GG. Inhibitions by L. reuteri DSM 17938 and the multi strain probiotic L. rhamnosus E/N, Oxy, Pen were intermediate and the best inhibitory effect was found for the complex multi strain synbiotic.The inhibition of the gram-positive E. coli EPEC. FOS also had no inhibitory effect on the growth of the other four tested pathogens (data not shown).As shown in A large number of studies have been published which are characterizing the in-vitro pathogen growth effects of individual probiotic microorganisms. There are also studies which compare the growth inhibitory effects of individual strains in head-to-head experiments. Less data are available for the in-vitro growth inhibitory effect of individual multi-strain products. To our knowledge there is no study published which compares the in-vitro pathogen growth inhibitory effects of probiotic products from different categories  head-to-head. The present study compares the in-vitro pathogen growth inhibitory properties of products containing probiotic microorganisms commonly used by physicians in Poland. The in-vitro pathogen growth inhibition experiments of the present study are not aiming to identify the underlying mechanisms of growth inhibition or to identify the causes of potential synergistic effects of probiotic bacteria in the multi-strain products, but to focus primarily on establishing a ranking of efficacy in this one particular experimental set-up. Inhibition of pathogen growth is only one potential effect exhibited by probiotic bacteria in the gut and other activities  are also potential contributors to their efficacy. However, in-vitro pathogen growth inhibition testing is a helpful measure that can be used to support the selection of products for further investigation, e.g., in clinical trials.For the in-vitro growth inhibition testing, five bacterial pathogens were selected which physicians encounter in their day-to-day practice. All five selected bacterial pathogens represent major health care concerns. As outlined in the following, infections with these pathogens are not always requiring antibiotic therapy. In addition, the increasing rate of developing antibiotic resistance in these bacteria has stimulated the interest in probiotics as prophylactic, alternative or adjuvant therapy for infections caused by them.Enteropathogenic E. coli EPEC is a major cause of infant diarrhea [E.coli against antibiotics [E. coli infections [E. coli growth antagonism by the yeast Sac. boulardii [E. coli have been described for a number of mono strain bacterial probiotics, among them L. rhamnosus GG [L. reuteri DSM 17938 [diarrhea . As longibiotics , probiotfections . In-vitroulardii . In contnosus GG , L. reutSM 17938  and multSM 17938 ,18,19.Shigella infections are a major public health problem in areas of poor sanitation with especially high incidence, morbidity and mortality in children [Shigella bacteria can already cause clinical disease. Most patients recover from Shigellosis without antibiotic treatment within 5−7 days. Various antimicrobial agents are effective in the treatment of severe cases of Shigellosis, however, a globally emerging antibiotic resistance is observed [Sac. boulardii has been shown to interfere with Shigella pathogenesis [Sh. sonnei by Sac. boulardii has not been published. Inhibition of in-vitro growth of Sh. sonnei has been demonstrated for L. rhamnosus GG [L. reuteri [lactobacilli [Sh. sonnei have not been demonstrated.children . Shigellobserved . Consequogenesis , howevernosus GG , for L.  reuteri  and a nuobacilli . For theS. typhimurium. Antibiotic therapy is not advised for most of the patients with S. typhimurium infection, as the infection is often self-limiting and antibiotic resistance of the bacterium is on the rise [Sac. boulardii has potential in the prevention and treatment of infections with S. typhimurium [S. typhimurium by Sac. boulardii with that of bacterial probiotics. In-vitro growth inhibition of S. typhimurium has been shown for a number of individual lactobacilli strains, [lactobacilli strains with growth inhibitory potential against S. typhimurium [S. typhimurium was more strongly inhibited by a multi strain synbiotic (different from that investigated in the present study) when compared with the in-vitro growth inhibition caused by its individual bacterial strains [Most food-borne bacterial gastroenteritis is caused by the rise . Therefothe rise , non-antthe rise . There ihimurium . Howeverstrains, ,29,30. Thimurium ,31. Our K. pneumoniae is responsible for an alarming increase in hospital infections, especially in intensive care units. More and more antibiotics have lost their efficacy against K. pneumoniae, and strains of K. pneumoniae have emerged that are resistant against most of the presently available antibiotics [K. pneumoniae has emerged. Few studies have investigated the effect of probiotics on the in-vitro growth of K. pneumoniae [ibiotics ,33. Conseumoniae ,35,36, hC. difficile infection (CDI), the exposure to antibiotics is the most important risk factor [C. difficile, production of toxins and disease development. Characteristic symptoms are diarrhea and abdominal pain. In severe cases, the CDI can result in a life-threatening pseudomembranous colitis. Recurrence of CDI is a not uncommon observation. First step in the treatment of CDI is discontinuing the therapy with the inciting antibiotic as soon as possible. Depending on severity of the CDI, three antibiotics can be considered for therapy: metronidazole, vancomycin and fidaxomicin [For an initial k factor . In thisaxomicin . In addiaxomicin .C. difficile by a number of mono strain probiotics, among them L. rhamnosus, and bifidobacteria has been described [C. difficile by representatives from different categories of products containing probiotics.In-vitro growth inhibition of escribed ,41. To oSac. boulardii and L. rhamnosus GG, intermediate for L. reuteri DSM 17938 and the L. rhamnosus E/N, Oxy, Pen mixture and strongest inhibition is found for the multi strain synbiotic. This finding is in line with the hypothesis that multi strain probiotics exhibit superior growth inhibitory effects towards pathogenic bacteria because they are combining a broader range of independent antibacterial activities, some of which might even act synergistically [The products evaluated in the present study can be categorized by the following criteria: (i) yeast/bacteria, (ii) mono strain/multi strain and (iii) probiotic/synbiotic (containing probiotic and prebiotic components). While the in-vitro pathogen growth inhibitory effects vary among the tested products, the inhibitory effects seem not to be pathogen-dependent. Weakest pathogen inhibition is observed for stically ,43. HoweAn obvious limitation of the present study is that it is using only one experimental set-up to establish an in-vitro efficacy ranking among the evaluated products. No efforts have been undertaken to investigate the underlying mechanisms of the observed pathogen inhibitions or the underlying mechanisms of potential synergies in the multi strain products. Dedicated human clinical studies will be necessary to evaluate if these in-vitro findings will translate into clinical benefits for patients.In-vitro growth inhibition of a variety of pathogens is helpful to differentiate products and product categories containing probiotic microorganisms. Based on the results of the present study, multi strain probiotics should be preferred in case a strong in-vitro growth inhibition of a broad range of pathogens is desired. Our study results can support the product selection for future clinical investigations.


Allergic asthma is the most common phenotype of the pathology, having an early-onset in childhood and producing a Th2-driven airways remodeling process that leads to symptoms and pathophysiological changes. The avoidance of aeroallergen exposure in early life has been shown to prevent asthma, but without repeated success and with the underlying preventive mechanisms at the beginning of asthma far to be fully recognized. In the present study, we aimed to evaluate if neonatal LPS-induced boost in epithelial host defenses contribute to prevent OVA-induced asthma in adult mice. To this, we focused on the response of bronchiolar club cells (CC), which are highly specialized in maintaining the epithelial homeostasis in the lung. In these cells, neonatal LPS administration increased the expression of TLR4 and TNFα, as well as the immunodulatory/antiallergic proteins: club cell secretory protein (CCSP) and surfactant protein D (SP-D). LPS also prevented mucous metaplasia of club cells and reduced the epidermal growth factor receptor (EGFR)-dependent mucin overproduction, with mice displaying normal breathing patterns after OVA challenge. Furthermore, the overexpression of the epithelial Th2-related molecule TSLP was blunted, and normal TSLP and IL-4 levels were found in the bronchoalveolar lavage. A lower eosinophilia was detected in LPS-pretreated mice, along with an increase in phagocytes and regulatory cells (CD4+CD25+FOXP3+ and CD4+IL-10+), together with higher levels of IL-12 and TNFα. In conclusion, our study demonstrates stable asthma-preventive epithelial effects promoted by neonatal LPS stimulation, leading to the presence of regulatory cells in the lung. These anti-allergic dynamic mechanisms would be overlaid in the epithelium, favored by an adequate epidemiological environment, during the development of asthma. Asthma is a heterogeneous disease with diverse underlying processes and many clinical expressions. The most common phenotype is allergic asthma, which has an early-onset in childhood and it is associated with a familiar history of allergic diseases. Histologically, it is characterized by chronic airway inflammation, with activated mast cells and an increased number of eosinophils, T cells, natural killer T cells, and CD4+ Th2 cells that release interleukin IL-4, IL-13, and IL-5. Additionally, IgE-secreting B cells are induced during the asthmatic process https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,  20 Apr 2020Responses to Academic Editor:1. Airway challenge in puberty and adulthood? Is this an appropriate statement for mice?http://dx.doi.org/10.1016/j.lfs.2015.10.025) Following bibliography and usual terminology, both terms are appropriate for age determination of laboratory mice  provided by protocols.io as submission-guidelines requirements.http://dx.doi.org/10.17504/protocols.io.bdwwi7fe >141Experimental design > http://dx.doi.org/10.17504/protocols.io.bdw9i7h6 >175Immunohistochemical analysis of lung tissue >http://dx.doi.org/10.17504/protocols.io.bdxzi7p6 >199Immunofluorescence > http://dx.doi.org/10.17504/protocols.io.bdx3i7qn >212Flow cytometry >http://dx.doi.org/10.17504/protocols.io.bbyhipt6 > 241Dot Blot Analysis > http://dx.doi.org/10.17504/protocols.io.bdx5i7q6 > 268RNA isolation and gene expression analysis > http://dx.doi.org/10.17504/protocols.io.bbymipu6> 273Clinical score assessment of the degree of respiratory distress >4. IFNg, PCR, and many others are standard abbreviations for the journal. No full definition is needed. TSLP ROS, PE, and PerCP probably too. Please correct. Note, that upon their first mentioning in the manuscript text, which is in the abstract, no full definition is provided.The abbreviations were added to the abstract and we revised the manuscript to keep them a minimum.AttachmentResponse to editor.docxSubmitted filename: Click here for additional data file. 23 Apr 2020Neonatal endotoxin stimulation is associated with a long-term bronchiolar epithelial expression of innate immune and anti-allergic markers that attenuates the allergic responsePONE-D-19-32052R2Dear Dr. Luciana Noemi Garcia,We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact With kind regards,Svetlana P. ChapovalAcademic EditorPLOS ONE 28 Apr 2020PONE-D-19-32052R2 Neonatal endotoxin stimulation is associated with a long-term bronchiolar epithelial expression of innate immune and anti-allergic markers that attenuates the allergic response Dear Dr. García:I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. 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Nodularia douglasiae  from Lake Kasumigaura, Japan, and inferred the Unioninae phylogeny using complete mitochondrial genome sequences. The complete F-type mitochondrial genome  contains 13 protein-coding genes, 2 rRNA genes, and 22 tRNA genes. Molecular phylogenetic analyses using complete F-type mitochondrial genomes from 15 Unioninae species including N. douglasiae from China and Korea were performed. This study should be basic data to investigate the genetic diversity of freshwater mussel N. douglasiae.We have sequenced the female type (F-type) mitochondrial genome of  The freshwater mussels, the family Unionidae, are benthic bivalves that inhabit rivers and lakes widely. In the last decade, the number of Unionidae has been steadily decreasing. Globally, there is a concern about the deterioration of the habitat of the mussel bivalves  inhabit not only in Japan but also in China, Korea, and eastern Russia  complete mitochondrial genome of N. douglasiae collected at Lake Kasumigaura, Japan, and molecular phylogenetic analyses were performed using complete F-type mitochondrial genomes of 15 Unioninae species including N. douglasiae from China and Korea.In Japan, 18 Unionidae species have been reported  was collected at Lake Kasumigaura, Japan . Genomic DNA was extracted from the mantle using DNeasy Blood and Tissue Kit . Libraries for next-generation sequencing were prepared using TruSeq DNA PCR-Free Library Preparation Kit , Nextera XT DNA Library Preparation Kit (Illumina), and KAPA HyperPlus Kit . The nucleotide sequence was determined by 301 bp × 2 pair-end sequence using MiSeq (Illumina). Trimming and de novo sequence assembly were performed using CLC GenomicsWorkbench (ver 11.0.1). Local BLAST search using complete mitochondrial genome of N. douglasiae from Korea (NC_040162) identified the complete mitochondrial genome of N. douglasiae from Lake Kasumigaura, Japan . The results of BLAST search at NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi) showed that the mitochondrial genome is the F-type. MITOS . The tree also showed that N. douglasiae from Japan (LC496352) is the sister to the clade of N. douglasiae from China (NC_026111) and Korea (MF314443), indicating the Japanese population diverged from the Eurasian group before the divergence of Chinese and Korean populations. This research would provide valuable information for investigating the genetic diversity of local populations of N. douglasiae.Molecular phylogenetic analysis was performed using the complete mitochondrial genomes of 15 Unioninae species. The sequences were aligned by ClustalW and a molecular phylogenetic tree was constructed by maximum-likelihood method (ML) using MEGA X (Kumar et al.


The progression of the nonalcoholic fatty liver disease to nonalcoholic steatohepatitis (NASH) is multifactorial, and there is still a lack of approved medications for its treatment. The study aimed to evaluate the impact of combined treatment with Pentoxifylline and Metformin on biochemical parameters in patients with Nash. Setting: Outpatient hepatology clinic.A prospective trial was conducted. The first cohort included patients with biopsy-proven Nash, while the second cohort consisted of patients with biopsy-confirmed NAFLD. Blood tests were checked at baseline and every three months. Pentoxifylline at a dosage of 400 mg t.i.d. and Metformin at the dosage of 500 mg t.i.d. were introduced for six months in Nash group. The impact of the treatment was assessed based on biochemical results after combined treatment with low-cost medications.All 33 Nash patients completed 24 weeks of treatment. We observed significant improvement (p<0.05) of median values after treatment for the following parameters: serum uric acid levels decreased by 51.0 mmol/L, calcium decreased for 0.27 mmoL/L, magnesium showed an increase of 0.11 mmoL/L. Insulin resistance improved as a reduction of HOMA - IR by 1.3 was detected. A significant decrease of median in liver enzymes, alanine aminotransferase, aspartate aminotransferase and gamma-glutamyltransferase by 24.0 U/L, 9.1 U/L, 10.8 U/L respectively, was noted.Pentoxifylline and Metformin may provide possible treatment option in Nash. Some new potential benefit of the therapy in improving liver function whilst decreasing cardiovascular risk was perceived. The epidemic extent of nonalcoholic fatty liver disease (NAFLD) reaching up to 30% of the general population, has become the most common chronic liver disease worldwide The progression of NAFLD is multifactorial, and there are currently no FDA-approved medications for the treatment of NASH Pentoxifylline (PTX), a phosphodiesterase inhibitor, although commonly used for peripheral vascular disease, is one of the antioxidants considered for NASH treatment. Besides its antioxidant activity and inhibition of proinflammatory cytokine production , it exerts pleiotropic pharmacological effects Acknowledging the complexity of the disease, it seems improbable that a single pharmacologic therapy will meet the requirements of the majority of this specific patient population. Hence, our study aimed to evaluate the efficacy of combination therapy of PTX and MET by monitoring biochemical parameters of patients with biopsy-diagnosed NASH.British Liver Trust) and were counseled on other lifestyle modifications.Data were collected prospectively from adult patients who attended a routine examination at the Clinic for Gastroenterology and Hepatology. Physical examination, abdominal ultrasound, laboratory testing, and liver biopsy were available for all patients. The first cohort included patients with biopsy-proven NASH. The second cohort consisted of age and gender-matched pairs with biopsy-confirmed NAFLD. Initially, all patients were instructed to follow the specific diet , amiodarone, methotrexate, tamoxifen, corticosteroids in the previous six months as well as patients with a history of excess alcohol ingestion  were also excluded from the study. Furthermore, this study dismissed patients with significant systemic or major illnesses  that might influence the treatment results.The abdominal ultrasound was performed using a Toshiba core vision with Doppler duplex convex probe 3.5 MHz. Corresponding to previous studies following criteria were applied: the diffuse hyperechoic echotexture, hepatorenal echo contrast in reference to the cortex of the right kidney, vascular blurring, and deep-echo attenuation Biopsies were performed for diagnostic purposes, during regular follow-up. Menghini liver biopsy needle was used. Histological criteria for steatohepatitis included: macrovesicular steatosis, hepatocellular injury, parenchymal and portal inflammation, fibrosis stage.2).All patients fasted overnight before physicians saw them in the clinic. We determined their height and weight, and body mass index (BMI) expressed as a weight (kg) divided by the square of the height . The analysis of hematological parameters were conducted immediately upon sampling, serum was separated by centrifugation, and analysis were performed. Biochemical parameters  and triglyceride concentrations (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), high sensitivity Creactive protein (hs-CRP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyltransferase (GGT), serum uric acid levels (SUA), calcium, magnesium, vitamin B12, folate and homocysteine) were measured employing routine methods . Additionally, insulin resistance was assessed by the homeostasis model assessment (HOMA-IR) Blood samples were collected under standard conditions between 7 and 8 am after 12h overnight fast. KPTX at a dosage of 400 mg t.i.d. and MET at the dosage of 500 mg t.i.d. were introduced for six months in NASH cohort.p value of less than 0.05 was considered statistically significant. The sample size was estimated based on Rosner calculation Descriptive statistic was applied to provide basic characteristics of the study population. The one-sample Kolmogorov-Smirnov test was performed to determine if the data showed normal distribution. Mann Whitney test or T-test was used to compare parameters between cohorts. Measured parameters were further analyzed by Wilcoxon Signed Ranks Test in the treatment group to detect potential differences in data before and after medication use. A The study was conducted in accordance with Guidelines for Good Clinical Practice, the Declaration of Helsinki, and local laws and regulations. The protocol was approved by the joint Research and Ethics Committee of the Clinical Centre of Serbia, Belgrade, filed under number 262/2. Written informed consent was obtained from all the participants in the study.Patients in the NASH cohort and NAFLD cohort were similar in regards to their anthropometric and ultrasonographic characteristics but showed differences in some biochemical parameters . We inclPatients reported the following adverse drug reactions (ADRs) during the first month of treatment: nausea (11%) and abdominal discomfort (14.9%). All ADRs were transient and mild; thus all patients included in the treatment group completed their 24 weeks medication regimen.Most NASH patients lost weight, 3.1± 1.6 kg on average in observed six months period.We detected significantly different values after three and six months when compared to the baseline parameters before treatment in NASH cohort for the following parameters: SUA, ALT, AST, GGT, calcium, magnesium, and HOMA-IR, as presented in In our study with given six months of NASH therapy, median SUA levels decreased by 51.0 µmol/L; serum calcium decreased by 0.27 mmoL/L, magne-sium showed an increase of 0.11 mmol/L. We detected a decrease in median values of ALT, AST and GGT by 24.0 U/L, 9.1 U/L, 10.8 U/L, respectively. Insulin resistance improved as a reduction of HOMA -IR by 1.3 was observed. Other monitored parameters  showed no statistically significant difference .When the comparison of parameters between two cohorts, the NASH treatment group, and the NAFLD group were applied after the study period, we detected no significant difference for SUA, calcium, AST, and GGT.However, magnesium, ALT, and HOMA-IR showed significant difference among observed cohorts after six months. In more details, ALT was still elevated in NASH group compared to NAFLD group (47.9 U/L vs. 37.7 U/L), magnesium reached the reference range in NASH treatment group but was lower than control group (0.77 mmoL/L vs. 0.90 mmoL/L), while HOMA-IR showed a decrease by 1.1 in NASH cohort when compared to NAFLD cohort .It is previously demonstrated that PTX and MET have a positive effect when used as monotherapy in NASH outcomes There is a growing body of evidence implying that increased SUA has a role in NAFLD Interestingly, a reduction of SUA levels was detected after our drug intervention. This observation may be explained by PTX effect on minimizing liver damage while reducing SUA levels as suggested in lately published research Notably, NASH patients had lower levels of magnesium at baseline similar to data from newly published research Moreover, a Korean population study by Shin et al. As in previously published studies on patients on MET therapy Although in use for more than 50 years, MET and PTX's new beneficial properties are still being discovered. PTX is a methylxanthine, like caffeine, for which recent evidence indicates that increased caffeine intake may decrease liver fibrosis While the individual treatment of NASH including patient-tailored lifestyle based on pharmacogenomics expects to tackle the core of the disease Treatment with PTX and MET of patients with NASH was well tolerated, with no clinically relevant side effects observed during our study. The results from this study imply some new potential benefit of PTX-MET treatment in improving liver function whilst decreasing cardiovascular risk in NASH patients.Author Contributions: Conceptualization, D.C., S.VK., and M.C.; Methodology, D.C., V.D., and S.VK.; Formal Analysis, M.C. and S.VK.; Investigation, M.C., and N.B. Resources, B.O., V.D., and B.M.; Writing-Original Draft Preparation, M.C.; Writing-Review & Editing, M.C., D.C., S.VK. and B.M.; Supervision, S.VK. and D.C.Funding: This research received no external funding.The authors stated that they have no conflicts of interest regarding the publication of this article.


Streptocarpus ionanthus (Gesneriaceae) comprise nine herbaceous subspecies, endemic to Kenya and Tanzania. The evolution of Str. ionanthus is perceived as complex due to morphological heterogeneity and unresolved phylogenetic relationships. Our study seeks to understand the molecular variation within Str. ionanthus using a phylogenomic approach. We sequence the chloroplast genomes of five subspecies of Str. ionanthus, compare their structural features and identify divergent regions. The five genomes are identical, with a conserved structure, a narrow size range (170 base pairs (bp)) and 115 unique genes . Genome alignment exhibits high synteny while the number of Simple Sequence Repeats (SSRs) are observed to be low (varying from 37 to 41), indicating high similarity. We identify ten divergent regions, including five variable regions  and five genes with a high number of polymorphic sites  which could be investigated further for phylogenetic utility in Str. ionanthus. Phylogenomic analyses here exhibit low polymorphism within Str. ionanthus and poor phylogenetic separation, which might be attributed to recent divergence. The complete chloroplast genome sequence data concerning the five subspecies provides genomic resources which can be expanded for future elucidation of Str. ionanthus phylogenetic relationships. Streptocarpus ionanthus (H. Wendl.) Christenhusz (Gesneriaceae) is a complex species, within Str. section Saintpaulia [Str. ionanthus extends from coastal Kenya to Tanga and Morogoro regions in Tanzania [Str. ionanthus is the only member of sect. Saintpaulia which has been recorded to occur in exposed habitats outside dense and closed canopy forests, environs which are prone to human activities. This has led to diminishing of population sizes and even the disappearance of most populations, leading to endangered status in taxa such as Str. ionanthus subspecies rupicola, velutinus, grandifolius and orbicularis according to the International Union for Conservation of Nature (IUCN) Red List of Threatened Species [ntpaulia , charactntpaulia  after a ntpaulia . The disTanzania , regionsTanzania . Str. io Species .Saintpaulia H. Wendl. has attracted research attention over the last two decades, witnessing inconsistent taxon classification for both molecular and morphological studies. Previous phylogenetic studies have applied few markers, both nuclear [Str. ionanthus group. Further, the 5S nuclear ribosomal DNA non-transcribed spacer (5S-NTS) data [Str. ionanthus. These observations challenge the narrow species concept used by Burtt [Str. ionanthus, this study made tremendous progress in Saintpaulia research by recognizing ten species under sect. Saintpaulia.The former genus  nuclear ,8,9 and  nuclear , aiming  nuclear , for insby Burtt ,11 to deby Burtt . Althoughttps://www.ncbi.nlm.nih.gov/genomes). The chloroplast sequence is characterized by uniparental inheritance and a substitution rate approximately half that of the nuclear genome [Recently, the amount of sequence data available has increased due to the advent of Next-Generation Sequencing (NGS) and relatively lower sequencing costs ,14. Presr genome . This lor genome . Furtherr genome ,18.Str. ionanthus suggests a case of a recently divergent group which needs to be investigated with methods other than gene-based approaches. Understanding the evolutionary relationship among such recently divergent lineages has been achieved using massive DNA data as opposed to a few genes [Str. ionanthus constituent taxa could elucidate its evolutionary relationship. Presently, only one chloroplast genome exists in sect. Saintpaulia and none in Str. ionanthus. Here, we sequence chloroplast genomes of five subspecies of Str. ionanthus aimed at (1) reporting the annotation and sequence variation, (2) screening for divergence hotspots, and (3) providing new genomic resources for future Str. ionanthus research.The poor resolutions and low bootstrap support values observed previously in ew genes ,20. ThusSaintpaulia taxa is presented in Str. ionanthus extended from 153,208 base pairs (bp) (Str. ionanthus subsp. grandifolius) to 153,377 bp (Str. ionanthus subsp. orbicularis)  to 84,115 bp (Str. ionanthus subsp. velutinus), while the SSC size exhibited a variation from 18,316 bp (Str. ionanthus subsp. grotei) to 18,332 bp in two subspecies (Str. ionanthus subsp. velutinus and Str. ionanthus subsp. grandifolius). The IR regions varied from 25,431 bp (Str. ionanthus subsp. velutinus and Str. ionanthus subsp. grandifolius) to 25,464 bp (Str. ionanthus subsp. orbicularis)  , exhibit3,207 bp . Similarcularis) . The fivcularis) .trnM-CAU, trnL-CAA, trnV-GAC, trnE-UUC, trnA-UGC, trnR-ACG and trnN-GUU), the four rRNAs, and seven PCGs . A total of 15 genes  contained a single intron, whereas two genes (clpP and ycf3) contained two introns each. Compared to the congeneric Str. teitensis [Str. teitensis had 114 genes due to the absence of the gene ycf15.All five subspecies exhibited a duplication of 18 genes, including seven tRNAs  junction was characterized by the ycf1 gene while the IRB/LSC junction occurred between the genes rpl2 and trnH. The SSC/IRB junction extended into the ycf1 gene creating a ycf1 pseudogene with a conserved length (795–799 bp) in the IRA/SSC junction. To conclude, all junctions had similar genes with only slight variations in the distance between the junctions and adjacent genes.The structural alignment in Mauve revealed one synteny block (in red) with a conserved gene order, minimal structural disparity and no rearrangements among the six genomes . FurtherPi) across the analyzed coding and intergenic sequences of the five subspecies ranged from 0 (majority) to 0.00526 (psbC-psbZ) (Pi = 0.00050). The total alignment file was 153,533 bp, with 152,813 sites (99.53%) being monomorphic while only 184 sites were polymorphic of which subsp. rupicola had the majority of Insertion and Deletions (InDels). Twenty-six Protein-Coding genes (PCGs) were observed to contain polymorphic sites, with only five genes having more than five sites . The majority of the polymorphic sites (169) were singleton variable sites and there were only 15 parsimony informative sites, representing a relatively low variation among the subspecies. Despite the low variation, ten regions exhibited some polymorphism (hereafter termed as divergence hotspots), including five regions with Pi > 0.002  and five PCGs with more than five polymorphic sites.The values of nucleotide variability (bC-psbZ) , with a Saintpaulia cp genomes exhibited small variation in the number of SSRs with two subspecies (Str. ionanthus subsp. velutinus and Str. ionanthus subsp. grandifolius) having 40 SSRs, two subspecies (Str. ionanthus subsp. orbicularis and Str. ionanthus subsp. grotei) having 37 SSRs while Str. ionanthus subsp. rupicola and Str. teitensis have 41 and 28 SSRs, respectively  and Bayesian Inference (BI) tree approaches, as shown in Str. ionanthus subspecies chloroplast genomes. Generally, the angiosperm chloroplast genome is considered to be conserved [Str. ionanthus taxa used here reveal the typical angiosperm structure with identical genes, gene order and no structural reconfigurations. The genomes exhibit a narrow size range (170 bp) and do not deviate from the first reported chloroplast genome in sect. Saintpaulia [Hosta (<85 bp) [Pyrus hopeiensis (46 bp) [Str. ionanthus cp genomes can be termed as relatively variable. During this study, we sequence and compare the major features of five onserved . The Strntpaulia . However(<85 bp)  and Pyru (46 bp)  species Str. ionanthus subsp. orbicularis having the longest IR region and also being the largest of the five genomes in terms of complete genome size. The representative Str. ionanthus cp genomes in this study are characterized by similar genes in the Inverted Repeat/ Single Copy (IR/SC) boundaries, with slight variations in the length flanking or drifting away from the boundaries. Nonetheless, other reported Gesneriaceae genomes vary from Str. ionanthus in some junctions. The Large Single Copy/ Inverted Repeat A (LSC/IRA) occurs between rps19–rpl2 in sect. Saintpaulia and Harbelea [rpl22–rpl2 in Petrocodon [rps19 in Primulina [Dorcoceras [Lysionotus [Str. ionanthus genomes being similar to Petrocodon, Dorcoceras and Lysionotus, by having an overlap of ycf1 and ndhF genes, and different from Str. teitensis, Haberlea and Primulina which have ycf1. However, the other two junctions are similar within Gesneriaceae.Seen in the chloroplast genome, the Inverted Repeat (IR) region is reported to be stable  with borHarbelea , rpl22–rtrocodon  and insirimulina , Dorcocercoceras  and Lysisionotus  genomes.Str. ionanthus. Accompanying the absence of observable structural variations, the minor variations exhibited by the red/yellow lines in the single copy regions could be attributed to the presence of Insertions and Deletions (InDels) in those regions, especially the non-coding regions, as reported in another study [Notopterygium [Camellia [Prunus [Meconopsis [Arachis hypogaea (peanut) [Jacobaea vulgaris [Ulmus americana (elm) genotypes among others, reveal very high cp genome similarities. Thus, the high genome similarity among Str. ionanthus subspecies is expected. Interestingly, some studies such as Pyrus cultivars [Besides the similarity in the IR/SC junctions, the high genome synteny with minor variations reported in the Mauve alignment portray a conserved cp genome in er study . Mixed oer study  or familer study  have repterygium , CamelliCamellia , Prunus  [Prunus , Meconopconopsis , just to(peanut) , seventevulgaris , two Ulmultivars  report aStr. ionanthus, with the subspecies rupicola having the highest quantity.Simple Sequence Repeats (SSRs) are important sources of information for genetic diversity and polymorphism testing  due to mStr. ionanthus cp genomes is comparatively lower (Pi = 0.0006) than in some other reported taxa  [psbC-psbZ, psaA-ycf3, atpF-atpH, psbM, rps3, rps16, rpoC2, rpoB, ycf1 and ndhA). Although these divergence regions were discovered in Str. ionanthus, the majority of them occur in Str. ionanthus subsp. rupicola which limits their ability to separate the Usambara taxa. However, this result should be interpreted with caution and more sampling could reveal interesting details about the variation of these genome regions. The extremely high polymorphism of Str. ionanthus subsp. rupicola may be partly due to long-term isolation of the subspecies from the others.The overall nucleotide variability in = 0.009) ,42, an e= 0.009) . Here, IStr. ionanthus classification. The coding and non-coding sequences have varied substitution rates [Str. ionanthus exhibit higher nucleotide diversity than the coding regions, with the most variable region being psbC-psbZ. Studies in higher plants have reported a high variability of matK, rps16 and rbcL [Pyrus specie chloroplast genomes [petN-psbM, psbM-trnD, rps4-trnT-trnL, and psaI-ycf4) having an average variation of Pi = 0.00054. However, in our study, most of these regions exhibit very low or no polymorphism. The divergence hotspots detected here could be tested further for utility in the phylogenetic analyses using all subspecies and more samples. Our results are valuable for future studies on estimating the variation within Str. ionanthus.The observed low variability means that a majority of the genome regions are of limited capacity for phylogenetic studies, thus previously applied chloroplast regions could not resolve on rates . Non- coon rates ,43. Simion rates , the intand rbcL  and otheand rbcL ,47, thus genomes  identifySaintpaulia phylogenetics, previous phylogenetic studies [Str. ionanthus, although these studies applied few markers. Here, we report the first genome-scale phylogenetic analysis in sect. Saintpaulia by comparing the phylogenetic relationship among the six sequenced taxa and within Gesneriaceae. However, we admit the fact that our study might not make entirely conclusive remarks on Str. ionanthus phylogeny due to the limited number of genomes. Nevertheless, our observations are consistent with most earlier studies and sets the blueprint for future phylogenomic analyses in understanding Str. ionanthus.The relative stability of molecular data makes them useful in estimating phylogenetic relationships among species . Despite studies ,7 were uStr. ionanthus using both nuclear [Str. ionanthus. Str. ionanthus subsp. rupicola is presented as distinct from the other four subspecies in all datasets used here, although this is not a new finding as similar outcomes have been reported in previous studies. This can be geographically explained in that Str. ionanthus subsp. rupicola occurs in Kenya while the other four subspecies are distributed in the Usambara mountains (Tanzania).Rapid evolution leads to poorly-resolved phylogenies  and prod nuclear  and chlo nuclear ,51 and aStr. ionanthus (illustrated in We collected leaf samples of five subspecies of Str. teitensis (GenBank Accession: MF596485) using the program Bowtie ver. 2.2.6 [Filtration was performed on the raw Illumina reads using an NGS QC tool kit  to elimir. 2.2.6 , followir. 2.2.6  set at kr. 2.2.6  with parr. 2.2.6 .https://chlorobox.mpimp-golm.mpg.de/index.html), combined with manual corrections to confirm the start and stop codons. The program tRNAscan-SE ver. 1.21 [https://rocaplab.ocean.washington.edu/tools/cpbase/). The annotated genomes were submitted to the National Center for Biotechnology Information (NCBI) GenBank database  junctions, structural re-organization and the loss or pseudogenization of genes have been used in previous studies to inform an evolutionary history of species . Compari genomes . The IR/Str. ionanthus genomes were identified using nucleotide diversity values (Pi) of the aligned sequence, executed in DNA Sequence Polymorphism (DnaSP) ver. 6.0 [Str. ionanthus were evaluated by analyzing the number of Insertions and Deletions (InDels) using DnaSP and, eventually, confirmed manually from the aligned sequences.Intraspecific variations within the five ver. 6.0 . The setSaintpaulia genomes using MISA (Microsatellite Identification tool) on the web version [Simple Sequence Repeats (SSRs) were identified from the six sect.  version . The selStr. ionanthus, the phylogenetic relationship was explored at the family level using the other nine Gesneriaceae chloroplast genomes and two outgroups already deposited in the National Center for Biotechnology Information (NCBI) (http://tree.bio.ed.ac.uk/software/figtree/).Since the focus of this study was on understanding n (NCBI) . We applStr. ionanthus to understand the species evolution and inform development of horticultural cultivars. We performed comparative analysis to estimate the level of variation in gene arrangement, mutation spots, repeat sequences and phylogenetic relationships among five Str. ionanthus taxa and other Gesneriaceae. The majority of the phylogenetic markers developed as barcodes for angiosperm classification have proven useful in resolving phylogenetic relationships in higher taxonomic levels but are rarely informative at lower levels. Seen in Str. ionanthus, the nine subspecies exhibited poor resolutions and mixed signals in previous phylogenies which used few molecular markers. No clear phylogenetic distinction has been reported among the subspecies, except subspecies rupicola which exhibits a clear monophyly within the complex. This implies a case of recent divergence in Str. ionanthus, especially in the Usambara mountains taxa. To the best of our knowledge, this study presents the first genome-scale analysis in the group and the findings exhibit a close phylogenetic relationship and low sequence variation among the five subspecies investigated. However, our study identified some divergent hotspots which could be explored for polymorphism with more sampling and applied to shed more light on the evolution of Str. ionanthus. Our work can be a blueprint for progressive molecular research in Str. ionanthus, especially phylogenomic analysis which should incorporate the entire species’ taxon representation and increased sampling for each taxon. To conclude, this study provided a first glimpse into the evolution of Str. ionanthus complex using a phylogenomic approach and opened the species to more research opportunities.It is undoubtedly crucial to expound on the genetic relationships within


Pronodularia japanensis  from Tochigi Prefecture, Japan. The complete F-type mitochondrial genome  contains 13 protein-coding genes, 2 rRNA genes, and 22 tRNA genes. Molecular phylogenetic analyses using complete F-type mitochondrial genomes of 56 Unionida species revealed the phylogenetic position of P. japanensis in Unionidae. This study should be basic data to investigate the genetic diversity in this species.We have sequenced the female-type (F-type) complete mitochondrial genome of  Pronodularia japanensis  inhabits in Japan, but the population of this species has been decreasing. Therefore, it is necessary to conserve this species.The freshwater mussels of the family Unionidae are benthic bivalves that inhabit rivers and lakes widely. Unionidae consists of four subfamilies: Gonideinae, Ambleminae, Anodontinae, and Unioninae  was collected in Tochigi Prefecture, Japan . Genomic DNA Extraction from the mantle, library preparation, next-generation sequencing, trimming of nucleotide sequences, de novo sequence assembly and annotation of mitochondrial genome were performed as previously described (Fukata and Iigo Unio japanensis (AB055625) identified the complete mitochondrial genome of P. japanensis from Tochigi, Japan . The results of BLAST search at NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi) showed that the mitochondrial genome is the F-type. The complete mitochondrial genome contains 13 protein-coding genes, 2 rRNA genes, and 22 tRNAs. The sequence has been submitted to DDBJ/EMBL/Genbank with an accession number of LC505454.Gibbosula crassa, NC_037942; Margaritifera marocana, NC_034911; Cumberlandia monodonta, NC_034846) as an outgroup. The sequences were aligned by ClustalW and molecular phylogenetic tree was constructed by the maximum likelihood method using MEGA X .The molecular phylogenetic tree  revealedFormer molecular phylogenetic analyses of the Unionidae based on partial mitochondrial genomes showed inconsistent relationships among studies (Bolotov et al. P. japanensis, and defined its phylogenetic position among Unionidae. This research would facilitate further investigations of the evolution of Unionidae. This research would also provide valuable information for investigating the genetic diversity of local populations of P. japanensis.In conclusion, our study described the complete mitogenome of