Citrus tristeza virus (CTV) in Turkey has been known since the 1960s and the virus was detected in all citrus growing regions of the country. Even though serological and biological characteristics of CTV have been studied since the 1980s, molecular characteristics of CTV isolates have not been studied to date in Turkey. In this study, molecular characteristics of 15 CTV isolates collected from different citrus growing regions of Turkey were determined by amplification, cloning, and sequencing of their major coat protein (CP) genes. The sequence analysis showed that the CP genes were highly conserved among Turkish isolates. However, isolates from different regions showed more genetic variation than isolates from the same region. Turkish isolates were clustered into three phylogenetic groups showing no association with geographical origins, host, or symptoms induced in indicator plants. Phylogenetic analysis of Turkish isolates with isolates from different citrus growing regions of the world including well-characterized type isolates of previously established strain specific groups revealed that some Turkish isolates were closely related to severe quick decline or stem pitting isolates. The results demonstrated that although CTV isolates from Turkey are considered biologically mild, majority of them contain severe components potentially causing quick decline or stem pitting.The presence of Citrus tristeza virus (CTV) has been the most commonly observed viral disease of citrus causing significant economical losses in different countries for more than a century [ Closterovirus in Closteroviridae family. The virus has flexuous filamentous particles with 2000 nm length and 10-12 nm diameter with about 20 kb genome [Tristeza disease caused by century , 2. CTV The complete genome sequences of CTV isolates from different geographical origins and various biological properties were determined. Genome sequences showed that CTV has the largest plant virus genome ranging from 19226 to 19306 nt and organized into 12 open reading frames (ORF) potentially encoding 17 proteins. Comparison of these genomes also revealed that biologically and geographically different isolates contained a certain degree of sequence variation in different parts of their genomes . The p25As other RNA viruses, CTV isolates do not generally consist of a single unique RNA sequence, but they rather contain of a population of very closely related sequence variants. These variants may result from error made by the RNA-dependent RNA polymerase (RdRp) during replication, recombination in the population, and repeated inoculation in the filed by grafting or insect transmission. Genetic variations associated with insect transmission, different host plants, or host passages were observed in the CP gene of some CTV isolates –26. ThusSymptoms of tristeza disease were first observed in Turkey in the 1960s , 28, butMolecular characterization of the first identified Turkish isolate, Iğdır, by sequence analysis of the CP and the RdRp genes demonstrated that it contained a mixture of mild and severe strains showing homology to stem pitting causing and/or resistance breaking strains of CTV from other citrus growing regions of the world . AlthougCTV isolates previously collected from different citrus growing regions and identified by ELISA and RT-PCR  were useμl of sterile water and kept at -80°C until used. The CP genes were amplified by two-step reverse transcription polymerase chain reaction (RT-PCR) method. cDNA was synthesized from total RNAs isolated from CTV-infected plants using random hexamer primers and PrimeScript reverse transcriptase  as suggested by the manufacture. Then, the CP gene was amplified from the cDNA using ExTaq DNA Polymerase  with BC24 5′ ATGGACGACGAAACAAAG 3′ and BC25 5′ TCAACGTGTGTTAAATTTCC 3′ primers specific to the CP gene. PCR was conducted in 50 μl PCR mixture containing 1X PCR buffer , 2μl cDNA, 0.5 mM dNTP, 20 pmol BC24 and BC25 primers, and 2.5 units of ExTaq DNA polymerase . MJ Mini thermal cycler PTC1148  was programmed at 95°C for 3 min for initial denaturation and 40 cycles of 95°C for 30 s for denaturation, 55°C for 30 s for primer annealing, and 72°C for 1 min for primer extension followed by a final extension at 72°C for 5 min. The PCR products were separated 1% agarose gel with 100 bp DNA size makers by electrophoresis, stained with ethidium bromide, and visualized and analyzed by Doc-It system .Total RNA was isolated from bark tissue from young shoots of infected citrus plants. About 100 mg bark tissue was pealed and ground to powder in liquid nitrogen and total RNA was isolated from all samples using One Step RNA Solution  according to the manufacturer's instructions. Total RNA was dissolved in 50  Escherichia coli strain JM109 competent cells  were transformed with ligation mixture of pGEM-Teasy and the CP gene by heat shock procedure as described by the manufacturer. The recombinant colonies containing pGEM-Teasy plasmid vector with the CP gene were selected by blue/white screening on 2X YT medium. Then, bacterial colonies were screened with colony PCR using universal M13R and M13F primers to identify white colonies containing the cloned CP genes. At least two colonies carrying the pGEM-Teasy plasmid with the CP gene were identified for each isolate. Selected colonies were grown in liquid media containing 100 μg mL−1 ampicillin, then plasmids were isolated, and the presence of the CP gene was confirmed by EcoRI digestion.CTV CP genes amplified by RT-PCR method were cloned into the pGEM-Teasy plasmid vector  by the T-A cloning method using T4 DNA ligase.The cloned CTV CP genes were sequenced in both directions using universal M13 forward and reverse primers by automated cycle sequencing method. The sequences of the CP genes were assembled and analyzed using Vector NTI Suite program . Multiple alignments of nucleotide (nt) and amino acid (aa) sequences were performed in AlignX Module of Vector NTI Suite and their percent sequence identity was determined. The refined sequences of CTV CP genes were submitted to the GenBank databases (accession number KU365759 to KU365773). The aligned CP gene sequences were used for construction of phylogenetic trees in MEGA 6 program using neighbor joining algorithm. The constructed phylogenetic tree was tested by a bootstrap analysis with 100 replications to determine the statistical significance of the clustering of CTV isolates with each other or isolates from different citrus growing regions of the world.Evolutionary analyses of genetic distance and diversity were conducted in MEGA 6 . Average EcoRI restriction enzyme, majority of clones contained a 3000 bp plasmid and a 700 bp DNA corresponding to the cloned CP gene. However, some clones produced two smaller bands (500 and 200 bp) instead of 700 bp along with the plasmid DNA indicating that they contain an internal EcoRI site (data not shown). These results showed that the CP genes of all CTV isolates were cloned and indicated that sequence variations exist among the CP genes of some isolates.About a 700 bp band corresponding to the CP gene of CTV (672 bp) was amplified from total RNA isolated from Iğdır isolate which was used as positive control and 15 isolates collected from different regions using two-step RT-PCR method. However, no amplification was observed from healthy citrus tissue used as negative control . These rSequence analysis revealed that nt sequences identity in the CP genes of CTV isolates ranged from 91% to 100% . The higPhylogenetic analysis of the nt and aa sequences CTV isolates from different citrus growing regions of Turkey produced very similar phylogenetic trees. Phylogenetic trees showed that isolates were genetically related and divided into three distinct main groups . The braEstimation of the average evolutionary divergence of CTV isolates from different citrus growing regions of Turkey based on mean evolutionary distance and diversity is shown in The genetic diversity analysis revealed that while the mean evolutionary diversity for isolates from the same region (subpopulation) was 0.049±0.005, it was 0.007±0.001 for isolates from different regions (Interpopulation). The mean evolutionary diversity of entire population of CTV isolates was estimated to be 0.056±0.005 and evolutionary differentiation coefficient was 0.126±0.021  suggestiThe CP gene sequences of CTV isolates from different citrus growing regions of the world with different biological properties and type strains representing previously established strain specific groups were retrieved from gene GenBank databases. Comparison of the CP genes of Turkish CTV isolates with CP genes of international CTV isolates revealed 89-99% identity in their nt sequences indicating that Turkish isolates were similar to the international isolates.Phylogenetic analysis showed that all CTV isolates were divided into six different groups representing previously established strain specific groups . TurkishOn the other hand, five different isolates from two different regions of Turkey were closely related with previously identified mild isolates of CTV, group 6, from different regions of the world including South Korea, Greece, Italy, Spain, Portugal, Florida, Mexico, and China . While iThe remaining one isolate EM-15(DA-5) from a kumquat tree was clustered with previously identified QD isolates from Florida, Egypt, Mexico, Brazil, Tunisia, Albania, and Cyprus. This Turkish isolate was the most closely related to 141-58 isolate from Brazil and T36 from Florida, which is the type member of the QD strain group, was also closely related to this isolate. The results indicated that EM-15(DA-5) collected from a kumquat tree in eastern Mediterranean region is a potential QD isolate or it may contain a QD component .Genetic diversity of CTV isolates from different citrus growing regions of the world including Turkey was estimated as average evolutionary divergence based on mean evolutionary distance and diversity. The results of genetic diversity analyses of isolates from different countries are shown in The analysis also revealed that while the mean evolutionary diversity was 0.020±0.001 for isolates from the same strain group (subpopulation), it was 0.049±0.005 for isolates from different strain groups (interpopulation) . The meaCitrus tristeza virus has been present in Turkey since 1963 without causing any major epidemics possibly due to the absence of the most efficient vector, brown citrus aphid (Toxoptera citricida), in the country [ country , 28. Bio country , 32, 37. country . TherefoSequence analysis showed that the CP genes of CTV isolates were generally conserved and showed about 10% variation in their nt and aa sequences. Two geographically separated isolates, EG28(EK-5) and EBS20(DK-4), collected from different citrus production regions were genetically the closest isolates with 100% identity in their nt and aa sequences. They were both isolated from Satsuma trees grafted on trifoliate orange rootstock and caused similar symptoms such as vein clearing and slight stem pitting on Mexican lime . In addiOverall genetic distance among CTV isolates was similar to genetic distance of isolates within each region except western Mediterranean region, which showed significantly lower genetic distance (0.006±0.003). The lower genetic distance estimated among isolates from this region is likely due to analysis of a fewer number of isolates from this region. Since we have identified only two isolates, no more isolates could be analyzed from this region. Genetic diversity analysis revealed that CTV isolates from the same region were genetically more diverse than the isolates from different regions suggesting that genetic diversity was not associated with geographical origin but may be related to biological characteristics such as host and/or symptom caused in indicator plants .Phylogenetic analysis showed that Turkish isolates were divided into three phylogenetic groups. Since two of these groups contained isolates from different citrus growing regions and different hosts and isolates inducing different symptoms on Mexican lime were in the same group, no association with geographical origin, host, or biological properties of Turkish isolates was observed. Contrary to previous findings showing that phylogenetic grouping of the CP gene is associated with biological properties of CTV isolates from different citrus growing regions , 39–41,  P. trifoliata. In addition, one isolate from kumquat was closely related with well-known QD isolate, T36, from Florida and other well-characterized QD isolates from different countries. These results suggested that isolates potentially causing SP and QD are present in Turkey. However, none of these isolates induced SP or QD on Madam Vinous orange grafted on sour orange rootstocks in biological indexing [Although the complete genome sequencing is the ideal and most accurate method for identification and strain typing , it is nindexing . Geneticindexing , 44.Thus, these results are more likely to suggest that Turkish isolate contains mixture of both mild and severe components as we previously demonstrated for one Turkish isolates, Iğdır . Since i


Preeclampsia is a multisystemic disorder leading to the development of a placental ischemic microenvironment with a resultant increase in oxidative stress. There is evidence that mitochondrial dysfunction and the innate immune system both play a role in the pathophysiology of this disease. Mitochondrial DAMPs such as mtDNA bind specific pattern recognition receptors such as Toll-like receptor 9 (TLR9) on the endosomal surface of immune cells, in particular neutrophils, subsequently activating them and triggering an innate response. We hypothesised that the exaggerated innate immune response seen in preeclampsia is provoked by dysfunctional mitochondria. Here we provide evidence that TLR9 activity is significantly increased at time of disease in women with preeclampsia. Furthermore, we show activation of neutrophil markers, Calprotectin, Myeloperoxidase (MPO), and IL-8 are significantly increased at time of disease compared to uncomplicated pregnancies. This research supports a potential role of TLR9 activation of an innate immune response evident in preeclampsia which may possibly be initially triggered by dysfunctional mitochondria. More recently, the cardiovascular system and its role in the development of preeclampsia is being explored3. Other pathological characteristics of preeclampsia include placental and systemic oxidative stress and dysfunction of the maternal vasculature5. In normal pregnancies there is evidence of a controlled systemic inflammatory response where cytokines promote the infiltration of the spiral arteries by invading trophoblast cells6. This controlled inflammatory response becomes dysregulated in preeclampsia resulting in abnormal activation of monocytes, neutrophils and the endothelium causing maternal inflammation7. There is overwhelming evidence that oxidative stress plays a key role in the pathophysiology of preeclampsia9. In turn oxidative stress, as a result of a placental ischemic microenvironment, releases reactive oxygen species into the maternal circulation, which can provoke a systemic inflammatory response10. The innate immune system acts as both a protector and effector during pregnancy. The innate system encompasses neutrophils, dendritic cells, natural killer cells and macrophages and these immune responders are activated to protect the mother from pathogens. Toll-like receptors (TLRs) are a family of type I transmembrane pattern recognition receptors (PRRs) that identify invading pathogens or endogenous damage signals and instigate an innate immune response. TLR9 can detect conserved sequences known as pathogen-associated molecular patterns (PAMPs) and also specifically respond to endogenous molecular structures known as damage-associated molecular patterns (DAMPs) via unmethylated CpG dinucleotide motifs as evident on mitochondrial DNA11. Activation of endosomal TLR9 involves an intracytoplasmic signalling cascade that leads to the up-regulation of pro-inflammatory transcription factors and subsequent liberation of pro-inflammatory cytokines12. There is evidence of increased TLR9 expression in both placental tissue and peripheral blood mononuclear cells (PBMC) from patients with preeclampsia compared with controls14.Preeclampsia is multifactorial disorder of pregnancy that is defined by the onset of hypertension and proteinuria after 20 weeks’ gestation. One of the most established characteristics of this disorder is the inability of the trophoblasts to invade the maternal uterine decidual arteries, resulting in poor placentation. Production of placental anti-angiogenic factors such as soluble fms-related tyrosine kinase 1 (sFlt-1) and soluble endoglin (sEng) have been extensively researched in pre-eclampsia15. Histopathological evidence has shown extensive infiltration of neutrophils in the systemic vasculature of women with preeclampsia compared to controls17. Neutrophils express a number of TLR’s on their surface including TLR918. Mitochondrial DNA (mtDNA) has been shown to activate neutrophils via a TLR9 signalling cascade, which elicits a neutrophil pro-inflammatory phenotype21. Neutrophil activation results in the secretion of a number of markers including reactive oxygen species (ROS), matrix metalloproteinase-8 (MMP-8), calprotectin, myeloperoxidase (MPO) and the pro-inflammatory cytokine IL-822.Neutrophils are short-lived effector cells of the innate immune system. These immune cells are activated in women during pregnancy and are further stimulated in preeclampsia23. Interestingly we also showed a significant increase in mtDNA at time of disease (TOD) in women with preeclampsia compared to controls24. The aim of this study was to investigate if the increase in mtDNA we previously reported in preeclampsia triggers activation of TLR9 signalling cascade. We also wanted to determine if a subsequent neutrophil pro-inflammatory phenotype was elicited in preeclampsia compared to controls by measuring a number of neutrophil activation markers longitudinally in pregnancy. We hypothesised that dysfunctional mitochondria provokes an exaggerated innate immune response in preeclampsia.Our research has previously provided evidence of a role for mitochondrial dysfunction in the pathophysiology of preeclampsia with an increase in ΔmtDNA evident between 15 and 20 weeks in women with preeclampsiaTo determine if TLR9 was activated by circulating mediators present in preeclampsia, HEK-TLR9 cells were treated with 3% plasma. The level of neutrophil activation markers, calprotectin, MPO, MMP8 and IL-8 were also examined. Firstly, there was no significant increase in TLR9 activity at 15 weeks’ gestation in cases compared to controls  Fig. . We subsThere was no significant increase in TLR9 activity at 20 weeks’ gestation in cases compared to controls  Fig. . While tThere was a significant increase in TLR9 activity at TOD in preeclampsia cases compared to controls  Fig. . SubsequWe investigated if the neutrophil-mediated innate immune response were altered longitudinally in pregnancy. Initially, there was no difference in TLR9 activity across gestation in controls. Fig. . SimilarIn cases complicated with preeclampsia, both TLR9 and calprotectin were significantly increased across gestation  and  respectively. Fig. . There wWe next explored if there was a difference in the neutrophil pro-inflammatory phenotype across all time-points in women who developed preeclampsia and delivered either before 37 weeks or after 37 weeks’ gestation Table . There w26. In this present study, we investigated if circulating mediators (including mtDNA)23, trigger TLR9 activity with the subsequent downstream activation of a neutrophil-mediated innate immune response in women with preeclampsia and healthy controls. We showed TLR9 activity is significantly increased in women with preeclampsia compared to healthy controls at time of disease. There was a corresponding increase in production of downstream neutrophil activation markers, calprotectin, myeloperoxidase, MMP8 and the pro-inflammatory cytokine IL-8 in women with preeclampsia compared to healthy controls at time of disease, indicating that complete activation of neutrophil pro-inflammatory phenotype only became evident late in pregnancy in preeclampsia.Pregnancy is associated with a maternal systemic inflammatory response; however, this response is exaggerated in preeclampsia. There has been a number of studies which have provided evidence of increased neutrophil activation in women with preeclampsia13. We have shown a significant increase in TLR9 activity using a reporter cell assay incubated with plasma taken at time of disease in preeclampsia cases compared to healthy controls. Furthermore, previous work in our lab using the same study group samples examined mtDNA  and reported that mtDNA was also significantly increased at time of disease in preeclampsia cases in the same study cohort23. This indicates that the increase in mitochondrial DAMP (mtDNA) at time of disease may activate a TLR9 mediated innate immune response in preeclampsia cases only. Exciting new work by He et al. has identified an additional role for TLR9 in preeclampsia where they established that TLR9 supressed angiogenesis in part by increasing sFlt-1 expression in a murine model of preeclampsia27. Furthermore, research carried out in spontaneous hypertensive rats, illustrated circulating mtDNA may lead to the activation of the innate immune system through TLR921. Research by Goulopoulou et al., has also shown preliminary data indicating that TLR9 activation provokes preeclampsia–like symptoms in pregnant rats20 emphasising the pathogenic role of TLR9 in preeclampsia.The innate immune response plays a well described role in the pathophysiology of preeclampsia. Mitochondrial DAMPs such as mtDNA bind specific pattern recognition receptors such as TLR9 on the endosomal surface of the immune cells, in particular, neutrophils and activate an innate response. TLR9 receptors are also localised to trophoblasts and the villous stromal vascular endothelium and their expression is increased in preeclampsia26. Akçum et al., reported increased circulating calprotectin levels at term in women with preeclampsia, and interestingly found an additional increase in women with severe preeclampsia.Calprotectin is a calcium binding protein and is located in the cytosol of neutrophils and is released upon neutrophil activation. In our study, calprotectin was significantly increased at time of disease compared to controls. This is in agreement with recent studies which have reported increase in calprotectin at term in preeclampsia29, whereas Gandley et al., reported a 3-fold increase in circulating MPO levels in women with preeclampsia compared to matched healthy controls at 32–38 weeks’ gestation30. The variation in MPO levels in previous preeclampsia studies may be due to small sample size. In our study we compared 60 cases and 120 matched controls, which to our knowledge is one of the biggest studies carried out in relation to MPO and preeclampsia.MPO is a lysosomal enzyme mainly produced and released by activated neutrophils. In this study, we showed a significant increase in MPO in cases as early as 20 weeks’ gestation when compared with controls and this significant increase was equally evident at time of disease in preeclampsia. Previous studies measuring MPO in preeclampsia have reported conflicting results; some studies have reported no difference in MPO in preeclampsia in samples taken at 24 weeks or later in the third trimester32. Recent research shows strong evidence that MMP8 play an important role in mediating endothelial cell angiogenesis32. Furthermore, endothelial dysfunction is a pathogenic characteristic of preeclampsia, dysregulated MMP8 expression may play a crucial role in the disruption of angiogenesis in preeclampsia leading to endothelial dysfunction. In our study, there was an increase in circulating levels of MMP8 expression at TOD in women with preeclampsia cases compared to healthy controls.MMP8 is part of the MMP family which consists of 23 zinc and calcium dependent proteases that effect different mechanisms of the extracellular matrix. In recent years, MMPs have become a target of interest in preeclampsia due to its role in vascular function and remodelling33. A recent study reported a significant increase in IL-8 in early gestation (5–15 weeks’) in women who subsequently went on to develop preeclampsia. However, a limitation of their study was the small sample size (n = 9)34. Other studies investigating IL-8 throughout pregnancy reported similar results to ours with no significant increase evident in the second trimester36 but a significant increase in IL-8 is evident in the third trimester in women with preeclampsia compared to healthy controls37.Cytokines and chemokines have been extensively studied as markers of inflammation involved in the pathophysiology of preeclampsia. In this study, we showed a significant increase in circulating IL-8 levels at time of disease in cases compared to controls. IL-8 has previously shown to be increased in women with preeclampsia in the third trimesterPreterm preeclampsia occurs before 37 weeks’ gestation and is frequently regarded as a slightly different phenotype to preeclampsia occurring at term. Therefore, we investigated whether neutrophil activation triggers an altered innate immune response in preterm preeclampsia compared to term preeclampsia. In this study, there was no statistically significant difference in the concentration of neutrophil activation makers in preterm versus term in our cohort.40. This current study shows that the TLR9 activation of the innate immune system may play a role in the pathophysiology of preeclampsia in late gestation.When investigating the activation of a TLR9 mediated innate immune response across pregnancy, there was no significant difference in expression of any of the neutrophil markers measured in healthy controls. However, in preeclampsia, significant increases were seen in neutrophil activation markers, TLR9 and calprotectin, illustrating that the innate immune response may be initially triggered earlier in preeclampsia but complete activation is not significantly evident until later in pregnancy. This is in agreement with recent studies of maternal inflammation  in preeclampsia, whereby low level inflammation is evident early in pregnancies but is amplified in the third trimester of pregnancyHere we provide evidence that circulating plasma mediators may activate a TRL9-mediated innate immune response in preeclampsia. We show that the activity of TLR9, a receptor for mtDNA, is significantly increased at time of disease in preeclampsia. Subsequently we have shown increased production of neutrophil activation markers particularly late in pregnancy in preeclampsia. Finally, we have shown that possible activation of TLR-9 by dysfunctional mitochondria may provoke an exaggerated neutrophil-mediated innate immune response in preeclampsia.42. A nested case-control study within SCOPE Ireland was conducted which included all preeclampsia cases in SCOPE Ireland and matched controls with a case-to-control ratio of 1:2. Preeclampsia cases was defined as a systolic blood pressure ≥140 mm Hg and/or diastolic blood pressure ≥90 mm Hg on at least two occasions 4 hrs apart after 20 weeks’ gestation and with proteinuria (24 hour urinary protein ≥300 mg or urine dipstick protein ≥+2). Randomly selected controls were taken from healthy pregnant women who had uncomplicated pregnancies which were defined as pregnancies not affected by preeclampsia, preterm birth or growth restriction and delivered at >37 weeks. All blood pressure readings were <140 and/or <90 mmHg prior to the onset of labour. These were matched with the cases for maternal age, body mass index (BMI) and gestational age. Both 15 and 20 week samples were taken from the SCOPE study from women who subsequently went onto develop preeclampsia (n = 60) and controls (n = 120). Samples were also taken from a subset of women (n = 25) at the time of disease (TOD) with preeclampsia and matched controls. The 60 women with preeclampsia were composed of 39 women who developed term preeclampsia and 21 preterm preeclampsia cases. The SCOPE study was conducted according to the guidelines laid down in the Declaration of Helsinki, and all the procedures were approved by the Clinical Research Ethics Committee of the Cork Teaching (EMC5(10)05/02/08), and all women provided written informed consent.Subjects were recruited from the Screening for Pregnancy Endpoints (SCOPE) study Ireland which is an international multicentre prospective cohort study of nulliparous singleton pregnancies aimed to develop a screening test to predict adverse pregnancy outcomes including preeclampsia, SGA infants and spontaneous pre-term birthPlasma samples were collected in BD Heparin Vacutainer tubes, placed on ice, and centrifuged at 2,400 g for 10 minutes at 4 °C according to a standardised protocol. Plasma samples were stored at −80 °C until analysis.TLR9 ligand activity was monitored with HEK-blue TLR9 Reporter Cell assay (InvivoGen). All experiments were performed using a cell density of 50,000 cells in a 96 well plate. Cells were initially seeded and left overnight prior to treatment for 24 hrs with 3% plasma taken at 15 and 20 weeks’ gestation  and TOD . The supernatant was incubated with Quanti-Blue detection medium (InvivoGen) and the activity was read on a Varioskan Flash plate reader (Thermo Scientific) at 630 nm.Plasma calprotectin (S100A8/S100A9) concentrations were measured by enzyme-linked immunosorbent assay (ELISA) using human S100A8/S100A9 Quantikine kit. Heparin plasma samples from all time-points were initially diluted a 100-fold in assay buffer and then directly added to a pre-coated plate. The ELISA was performed as per manufacturers’ instructions.Myeloperoxidase (MPO), Matrix metalloproteinases-8 (MMP8) and interleukin-8 (IL-8) concentrations were measured respectively at all time-points by individual ELISA DuoSet kits (R&D SYSTEMS. USA & Canada). ELISA was carried out as per manufacturer’s instructions.Analysis was performed using GraphPad Prism. Data were presented using median (±Interquartile range [IQR]) and comparisons of data between cases and controls were performed using a non-parametric Mann Whitney U test or Wilcoxon signed rank test as appropriate when data was not normally distributed. Data that was normally distributed were represented as mean (±SEM) and comparisons of data between cases and controls were performed using an unpaired t-test. P values < 0.05 were considered as statistically significant.


Sequencing of 16S rDNA V3–V4 region is widely applied for food community profiling. However, two different universal forward primers (named here MUYZER-primer1 and KLINDWORTH-primer2) targeting an identical conservative sequence upstream of the V3 region of 16S rRNA gene, and only distinguished by a single mismatch are both used. This study was carried out to compare whether the accuracy of food microbiota analysis would depend on the choice of one of these two primers.Leuconostoc, Oenococcus and Fructobacillus species but not Weissella species. Food products containing either Leuconostoc and/or Weissella were selected for a detection test. As expected from our in silico analysis, our study showed that this mismatch induced a strong biased amplification specifically associated to the OTUs belonging to the genus Leuconostoc but not to the genus Weissella. In presence of Muyzer-primer1, none of the sequences expected for Leuconostoc genus was detected whereas those sequences were correctly amplified with Klindworth-primer2. Since Leuconostoc is an important genus in food, agro-environments and in digestive tract of animals, we recommend that Muyzer-primer1 should thus be abandoned for the bacterial characterization of their associated microbiota.Alignment of both primers with common food-borne bacteria 16S sequences revealed that the mismatch between both primers might specifically affect the amplification of  Bacterial 16S ribosomal DNA (rDNA) is widely used to characterize bacterial communities associated with a wide range of ecosystems. For this purpose, primers targeting conservative regions of the 16S rRNA gene are commonly designed and used to sequence hypervariable regions, which are informative for taxonomic assignment. Thus, the most critical step for accurate 16S rDNA amplicon analysis is the choice of primers. Using suboptimal primer pairs can induce serious amplification biases such as under-representation or selection against single species or even whole groups , packaged under modified atmosphere, were selected for this study. Two mock communities (MC) containing notably one strain um DSM557 and one um DSM557 and one AGCAGT-3′) (MUYZER-Primer1) according to Muyzer et al., [WGCAGT-3′) (KLINDWORTH-Primer2) according to Klindworth et al., [Two distinct amplifications of the ~ 450-bp V3–V4 hypervariable regions of the bacterial 16S rRNA gene were performed in parallel: one with the primer V3F  on 50–200 ng of purified amplicons from the first PCR using 2.5 U of a DNA-free Taq DNA Polymerase and 1xTaq DNA polymerase buffer as detailed in Poirier et al. . AmplicoThe quality of the sequencing was first evaluated using FastQC , 10. TheLeuconostoc, Oenococcus and Fructobacillus genera. As shown in Table Weissella genus. This A nucleotide being highly conserved in bacterial 16S rDNA, in particular within species belonging to Firmicutes. However, all other sequences of the three remaining genera of Leuconostoceae have a T nucleotide. By contrast, Klindworth-Primer2 sequence, which has a degenerated base W (A/T) at this position, should not induce any bias during the amplification of sequences belonging to all Leuconostocaceae.We first carried out a comparative alignment of both primers sequences to 16S rRNA encoding gene sequences of about 450 strains commonly found in food microbiota (data not shown). This alignment revealed that the mismatch differentiating both primers could have a specific impact on the amplification of Leuconostoc was detected within the samples amplified with Muyzer-Primer1. By contrast, proportions of OTUs assigned to Leuconostoc varied from 3% in CS2 up to 44% in GB3 within samples amplified with Klindworth-Primer2. This result clearly highlights that the use of Muyzer-Primer1 to monitor bacterial communities induces a serious bias for samples that contain Leuconostoc species. Moreover, results also showed that sequences of Weissella were amplified by both primers. However, the use of Muyzer-Primer1 leads to an overestimation of the relative abundance of this genus probably due to the non-amplification of Leuconostoc. This result was also confirmed by the 16S rRNA gene (region V3–V4) sequencing of mock communities. While no sequence of Leuconostoc was detected in both mock communities with Muyzer-primer1, Klindworth-primer2 allowed the amplification of 1.9% and 1% of sequences assigned to Leuconostoc gelidum subsp. gelidum in MC1 and MC2, respectively.The relative bacterial composition within Leuconostocaceae of our chosen food samples and mock communities is detailed in Fig. Exploring the diversity of microorganisms in foodstuffs has become a broad focus of research in food microbiology. However, the recent and rapid adoption of new generation sequencing techniques caused a certain backlog in proper evaluation of the primers used for diversity surveys. Notably, the percentage coverage and application scope of the primers used in previous studies are largely unknown.Leuconostoc species was missed.Our study shows that even commonly used single primers only differentiated by a single mismatch could exhibit significant differences in the sequencing results. In some samples, 44% of the total microbial community carried by Leuconostoc in particular. For instance, Zhang et al. did not detect Leuconostoc in Yucha samples (a fermented food made with cooked rice and fresh fish) [Dicentrarchus labrax without detecting OTUs for this genus [Leuconostoc are common on seafood products [Leuconostoc with this primer whereas they could observe its presence in the same cheeses by targeting V4–V5 or V6–V8 regions [Regarding the bibliography, we pointed out that this unappropriated Muyzer-Primer1 was still used in recent studies to describe food products, a situation that is somewhat problematic since these products may contain sh fish)  while itsh fish) , 21. Simis genus  whereas products . Ercolin regions , 25.Leuconostoc, Oenococcus and Fructobacillus species play a relevant role either in food spoilage or in food and beverage fermentation in which they are of interest as a starter culture [Oenococcus oeni is responsible for malolactic fermentation in wine, Leuconostoc mesenteroides is responsible for acetoin and butanediol production in cheese through citrolactic fermentation, whereas Leuconostoc gelidum pyruvate metabolism would trigger spoilage of fresh meat. Their good estimation within food microbiota is therefore crucial for understanding how food communities function during meat, cheese or wine fermentation but also for elucidating whether there is any possibility to develop new strategies to interfere with the growth and to postpone spoilage of packaged and refrigerated foods products. Furthermore, they are also widespread in the environment, and have been isolated from plant matter [Despite its high coverage rate of 98% highlighted by Wang et al. , Muyzer- culture . For inst matter  and humat matter . ConsequLeuconostoc species suggesting the general failure of Muyzer-Primer1 to amplify this genus. Furthermore, our study focused on a comparative analysis between Leuconostoc and Weissella but not on Oenococcus and Fructobacillus. However, it is very likely that these genera might not be amplified with Muyzer-Primer1.Although the number of samples tested in this study were few, our result was confirmed by the analysis of microbial community from various food products , which were amplified with Muyzer-Primer1. Although these samples were not sequenced with Klindworth-Primer2 as a control, they revealed to be deprived of


Talaromyces marneffei has a large expansion of these proteases identified through genomic analyses. Here we characterize the expansion of these genes  and their role in T. marneffei using computational and molecular approaches. Many of the genes in this monophyletic family show copy number variation and positive selection despite the preservation of functional regions and possible redundancy. We show that the expression profile of these genes differs and some are expressed during intracellular growth in the host. Several of these proteins have distinctive localization as well as both additive and epistatic effects on the formation of yeast cells during macrophage infections. The data suggest that this is a recently evolved aspartyl protease gene family which affects intracellular growth and contributes to the pathogenicity of T. marneffei.Aspartyl proteases are a widely represented class of proteolytic enzymes found in eukaryotes and retroviruses. They have been associated with pathogenicity in a range of disease-causing microorganisms. The dimorphic human-pathogenic fungus  Plasmodium, and Candida [Proteases are a diverse and ubiquitously distributed group of enzymes that are important for a variety of biological processes including morphogenesis, cellular function, immunity, and nutrition. The aspartyl protease (aspartic peptidase) class of proteases similarly serve many roles but in particular have been implicated in virulence and pathogenicity of microorganisms such as HIV,  Candida –3. In seBatrachochytrium dendrobatidis, responsible for amphibian infection and death worldwide, one aspartyl protease family is expanded to 77 genes (4.5 fold) relative to a closely related, less pathogenic, species [Metarhizium, M. anisopliae, which has a broad host range, has significantly more aspartyl protease-encoding genes than its locust-specific relative, M. acridum [The acquisition or expansion of genes is a potential source of variation that can lead to the generation of new virulence factors. For example in the chytrid fungus 77 genes .5 fold rCandida genus. These genes, known as secreted aspartyl proteases (SAPs), have been identified and/or characterized in C. glabrata, C. tropicalis, C. dubliniensis and C. albicans [C. albicans, and several of the 10 members of this expansion have been associated with distinct cellular traits, including hyphal formation, adhesion, dimorphic switching and host cell apoptosis [bona fide roles in immune responses, biofilm metabolism and mucosal infection [C. parapsilosis, production of three SAP proteins varies with respect to the tissue infected as well as between clinical isolates; thus multiple factors influence a single process such as infection [The most thoroughly studied families of aspartyl proteases belong to species in the albicans . SAP genpoptosis –8. Despipoptosis , a numbenfection . In C. pnfection ,14.Aspergillus fumigatus has two aspartyl protease-encoding genes and one of these, pep1, is expressed during infection in several animal models [Candida species and A. fumigatus, this may be due to functional redundancy between members of the expanded families or other protease classes. An important distinction between Candida and many other morphologically complex fungi is that the latter also have other secreted protease classes besides the SAPs.l models ,16. Mutal models . TherefoCandida species and A. fumigatus, the function of aspartyl proteases in most other pathogens, including T. marneffei has not been examined. T. marneffei (formerly Penicillium marneffei) is a thermally dimorphic pathogen of humans that is endemic to Southeast Asia. The incidence of talaromycosis (formerly penicilliosis) peaked with the AIDS epidemic in this region [T. marneffei is capable of growing as both multinucleate septate hyphae and uninucleate yeast cells dividing by fission [in vitro occurs via an arthroconidial hyphal phase consisting of uninucleate, branched and septate cells. In vivo the transition from conidia to yeast cells is direct. The yeast form predominates during infection, where T. marneffei grows and proliferates within host phagocytic cells thereby avoiding exposure to, and clearance by, the immune system.Despite these studies in s region . T. marn fission . The traT. marneffei, orthologous to the A. fumigatus pep1 gene. The expansion, named paralogue of pep (pop), was found to be monophyletic and is associated with repetitive elements and subtelomeric regions of the T. marneffei genome. This family shows evidence of significant positive selection as well as copy number variation amongst T. marneffei isolates. Despite these rapid evolutionary changes, the functional domains are conserved in the majority of the proteins. Based on their expression profiles, five pop genes were selected for functional analysis. Single and double deletions of these genes revealed cumulative and interdependent impacts on yeast cell formation during macrophage infection, while localization studies revealed unique cellular targeting during in vitro and ex vivo growth. The data suggest that the expansion of these genes may have been an important step in the evolution of pathogenicity in T. marneffei.In this study, we describe a major expansion of aspartyl protease-encoding genes in T. marneffei (unpublished data). Reciprocal BLAST comparisons suggested that T. marneffei pepA (PMAA_090410) was least diverged from the A. fumigatus pep1 gene (Supplementary Table 1). To understand the nature of the expansion, genes found in this family and their orthologues in other species were used to generate a relatedness tree . Long branch lengths in the expansion relative to branch lengths of the Talaromycete orthologues point to a rapid generation of variation within the group.A comparative genomics study of 17 Eurotiomycetes revealed an increase in the number of genes encoding aspartyl proteases in ess tree . The 23 pop expansion. Using the genome sequencing data from a different study, which investigated 20 clinical and environmental isolates of T. marneffei (unpublished data), we mapped the depth of reads from these strains for each pop gene. This metric can be used as a proxy for the copy number of these genes in each strain  substitution rates across this family. Codon-based alignments for each of these genes were generated, as were those of their orthologues in three closely related Talaromyces species . A negative correlation was observed between surface exposure and variation indicating that positive selection occurring in these genes is predominantly associated with the protein surface ).In order to determine whether this positive selection was confined to particular amino acid residues or domains, the location of variable residues within the expansion was mapped on to the known 3D structure of the gue Pep1  ). The pepA product shows a moderate reduction in conservation in both sequences while popP and popH products show additional residues in the secretion signal as well as differences in the activation peptide compared to the other pop proteins. The popE activation peptide is disrupted which may impact function. Expression of popK has not been detected under any condition tested to date so the lack of conservation in this gene is likely to be unimportant. The core catalytic residues of aspartyl proteases are made up of two DTG or DSG motifs, which are highly conserved across taxa [pop genes while the second is conserved in 19, with 15 showing conservation of both motifs. Five other proteins show conservative substitutions from G to A in the third residue of the first catalytic core, which is unlikely to affect function. As with the activation and secretion signals of popK the catalytic sites of this gene are disrupted. Cysteine residues, which form the structurally important C-terminal disulphide bridge, are absolutely conserved in pop proteins.Despite the changes in protein sequence due to positive selection the residues and domains that define aspartyl proteases are conserved in the oss taxa   located less than 100 kb from genome scaffold ends, while this was true for only 5.3% of all genes .In order to understand the mechanism for the expansion of these genes in e genome . In manye genome . Therefopop genes are important for pathogenicity of T. marneffei, we interrogated a previously generated RNAseq dataset from saprophytic  and pathogenic (37°C in vitro and ex vivo human and murine macrophage infections) growth forms (unpublished data). Expression patterns for the 16 pop genes showed a strong bias towards the pathogenic growth form with the remainder showing minimal expression in the conditions tested . At 24-h post infection, wild-type T. marneffei within macrophages produced numerous yeast cells that divide by fission. Similar to the wild-type, the ∆popH strain showed no phenotypic effect while the ∆pepA strain had a minor but insignificant effect during growth in J774 murine macrophages  showed no significant difference from the ∆popO single mutant (60.7 ± 2.4% compared to 66.8 ± 8.8%). In contrast, the ∆popS/∆popP and ∆popS/∆popO mutants showed additive to near-additive effects  while the ∆popS/∆pepA mutant exhibited synergistic effects (39.6 ± 6.2%) with respect to the single ∆popS mutant (60.7 ± 3.5%). The ∆popO/∆popP double mutant shows no significant difference from ∆popO (65.2 ± 2.0% compared to 66.8 ± 8.8%) in yeast cell formation despite the fact that both single mutants show significant independent effects. These data clearly demonstrate a complex relationship with both overlapping and unique functions for these pop genes.All strains were examined for phenotypic variation rophages . In contormation . As popHpop gene products, carboxyl-terminal mCherry fusion constructs driven by the native promoter were generated and used to transform the respective deletion strains. Phenotypic characterization of these strains suggested that the fusion alleles complemented the mutant phenotype  compared to wild-type   . The datT. marneffei during macrophage infection, western blot analysis was used to identify the presence of these proteins in fractions of T. marneffei infected macrophages . J774 macrophages were infected with conidia from each of the pepA::mCherry, popP::mCherry, popH::mCherry, popS::mCherry and popO::mCherry strains and protein was extracted from purified yeast cells isolated from within macrophages and the remaining macrophage lysate lacking T. marneffei yeast. A strain carrying a histone H1 mCherry fusion (H1::mCherry) allele was used as a control. Western blot analysis revealed the presence of the H1::mCherry protein in the T. marneffei yeast extract but not in the macrophage fraction . In contrast, the Pop::mCherry fusion proteins were detected in both T. marneffei yeast and macrophage factions. This was most clearly evident for the popP::mCherry strain , consistent with the expression profiling data that showed the popP gene has the highest expression of all the pop family members. A higher level of PopP::mCherry fusion protein is observed in the T. marneffei yeast cell faction. Coupled with the co-infection results, these data strongly suggest that at least a number of the proteases encoded by the pop gene family are secreted and function outside of T. marneffei cells.To examine the secretion of these proteases by The evolution of traits that permit exploitation of new environments can proceed by the repurposing of genes with or without loss of the starting function, or the evolution or acquisition of new genes. While the former is constrained if there is selection acting to maintain the ancestral function of a gene, this is not so for the latter. The evolution or acquisition of new genes can occur by horizontal gene transfer or by gene amplification followed by neofunctionalisation. Gene amplification as a mechanism for evolving new traits provides redundancy for maintaining the original function, relaxes selection on individual genes and increases the level of the gene product, which may in itself have selective advantages.T. marneffei that may have been critical in the evolution of pathogenicity. T. marneffei is the only identified animal pathogenic species in the Talaromyces genus and the only species in the Eurotiomycetes order that exhibits dimorphic switching; an essential trait for intracellular growth. This study showed that the genes in this expansion form one monophyletic clade, with only single orthologues of the proteases present in other species. In most cases, these genes are associated with subtelomeric regions and surrounded by repetitive sequences, including transposable elements, suggesting mechanisms for the genesis of the expansion. Such mechanisms may have included transposon-driven duplication or unequal crossing-over between repetitive sequences. The role of transposons in the evolution of this gene family is further supported by the presence of a sub-clade of pop genes that lack introns, and which are likely to have arisen by retrotransposition. Furthermore, these genes vary in copy number amongst T. marneffei isolates, indicating an ongoing dynamic process in the evolution of this family. Given these observations, we hypothesized that this family may have evolved early in the acquisition of pathogenicity in T. marneffei.Here we have described the identification of a novel expansion of aspartyl protease-encoding genes in Plasmodium falciparum, the VSG genes of Trypanosoma bruscei and the TLO family in Candida species [T. marneffei, variation in pop protein sequence showed clear positive selection and was primarily observed in residues exposed to the host. Given it is predicted that pop gene products are secreted, this is consistent with the hypothesis that this may provide a source of antigenic variation for T. marneffei to allow escape from detection or help modulate immune interactions during infection.The activity of genes within expanded gene families is associated with virulence in a number of protozoan and fungal species. These include the VAR genes of  species –25. In sT. marneffei pop genes, it is not clear if the expression of each gene shows significant cell to cell variation, as would be expected if they were functioning similarly to the VAR and VSG genes, where switching expression from one member of the gene family to another permits immune evasion and affects infection outcome. Expression profiling suggests that this may not be a significant factor in the action of these genes as each gene showed distinct cell type and growth condition dependent patterns. Fluorescent tagging of members of the Pop family confirmed the expression profiles and revealed novel localization for PopH and PopP. Collectively, these data suggest that at least some pop genes in this family are more likely to require the enzymatic function of the gene product to assist the growth of T. marneffei under various environmental conditions. This does not preclude some or all of these genes also serving a more structural antigenic variation role.For the Candida genus, the number of secreted aspartyl protease (SAP) genes differ, with larger numbers hypothesized to confer greater pathogenicity [Candida albicans, SAPs are associated with distinct stages of pathogenesis. Mutations in SAP 1–3 show virulence defects in murine vaginitis models while SAP 4–6 are implicated in peritonitis [T. marneffei that are expressed during ex vivo macrophage growth  led to reductions in yeast cell formation under these conditions, and combinatorial deletions of these genes showed more severe or synthetic phenotypes. This suggests that these genes play a role in virulence and the strong conservation of the functional domains in these gene products is consistent with the idea that their enzymatic activity is important. Moreover, the strong positive selection signature that was restricted to the pop expansion, the partially redundant function for some of these genes and the ongoing variation in copy number amongst T. marneffei strains suggests that the biological roles(s) of these genes may be a recent development in the evolution of pathogenicity of T. marneffei and that these genes may act as a source of new effectors to allow T. marneffei to respond to new environments. Some genes such as popP and popH showed minor or no phenotypes when deleted despite strong expression and may therefore be functionally redundant with one or more other pop genes.Aspartyl protease expansions are found in a small number of other pathogenic fungi, where they are proposed to influence pathogenicity and infection. In species of the genicity . In Canditonitis ,28. Delepop gene products have conserved pre-pro peptide sequences suggesting that they are secreted proteins. Localization of PopH and PopP in vitro was to the septa of arthroconidial filaments, which go on to produce yeast cells, and dividing yeast cells, suggestive of a role in the division of yeast cells by fission, a process that is unique to T. marneffei amongst the Eurotiomycetes. However, single and double mutants of these two genes showed no defects in this process, indicating that their role is non-essential or redundant. Localization of PopP, PopS, PopH and PopO ex vivo was perinuclear and vesicular, typically representing secretory machinery such as the endoplasmic reticulum and Golgi. While none of the gene products were detected extracellularly by fluorescence microscopy, we showed that some of these gene products could be detected in macrophage lysates after infection with T. marneffei, even after T. marneffei had been removed from the infected cells, and that complementation of the mutant phenotypes could be achieved by coinfection of macrophages with two mutant strains. These data strongly support the prediction that the pop gene products are secreted and that the lack of detection extracellularly by fluorescence microscopy indicates a sensitivity issue due to either diffusion or degradation of these proteins. This is consistent with the PepA data, which showed localization immediately adjacent to the cell wall, thus likely outside the cell membrane. In C. albicans SAP9 is localized to the cell wall and mediates interactions with phagocytic cells of the immune system [pep1, the orthologue of pepA in A. fumigatus is classified as an allergen and as such interacts with the host immune system [pepA gene may also mediate intracellular interactions between T. marneffei and host immune cells such as macrophages.The e system . Similare system . The peppop proteins, which need to be proteolytically cleaved for function  was generated with primers QQ87 and QQ88 and cloned into pBluescript II SK+ to generate pMP7880. To make the deletion construct pMP7886, PCR primers QQ89 and QQ90 were used to generate an inverse PCR product (which removes from -25 to +1409 of pepA) for GatewayTM reaction. Wild-type PCR product of popH (PMAA_058850) was generated with primers QQ81 and QQ82 and cloned into pBluescript II SK+ to generate pMP7876. To make the deletion construct pMP7881, PCR primers QQ83 and QQ84 were used to generate an inverse PCR product (which removes from -110 to +1328 of popH) for GatewayTM reaction. Wild-type PCR products of popO (PMAA_062510) was generated with primers QQ98 and QQ99 and cloned into pBluescript II SK+ to generate pHW7877. To make the deletion construct pHW7882, PCR primers QQ100 and RR1 were used to generate an inverse PCR product (which removes from -59 to +1212 of popO) for GatewayTM reaction. The deletion constructs of the following gene were created using pHW7772 containing the pDONR-bar cassette. Wild-type PCR products of popS (PMAA_089390) was generated with primers QQ75 and QQ76 and cloned into pBluescript II SK++ to generate pHW7879. To make the deletion construct pHW7884, PCR primers QQ83 and QQ84 were used to generate an inverse PCR product (which removes from -121 to +1225 of popS) for GatewayTM reaction. The deletion constructs of the following gene were created using pHW7773 containing the pDONR-riboB cassette. Wild-type PCR products of popP (PMAA_065480) was generated with primers QQ93 and QQ94 and cloned into pBluescript II SK+ to generate pMP7878. To make the deletion construct, pMP7883, PCR primers QQ95 and QQ96 were used to generate an inverse PCR product (which removes from -144 to +1979 of popP) for GatewayTM reaction. Complementation constructs were generated by ligating the wild-type PCR products of each gene into EcoCRI digested pHB7615 (niaD targeting vector) to generate pMP7891 (pepA), pMP7887 (popH), pHW7888 (popO), pHW7890 (popS) and pMP7889 (popP).escribed . Souther methods . SequencM method . The delSpeI digested pHW7911 (mCherry::niaD targeting vector). These sequences excluded the stop codon and native terminator sequence of each gene. For the pepA::mCherry fusion construct, PCR product generated using QQ87 and XX20 was cut with XbaI and ligated into SpeI digested pHW7911 to generate pMP8062 For the popH::mCherry fusion construct, PCR product generated using QQ81 and XX19 was cut with SpeI and ligated into SpeI digested pHW7911 to generate pMP8058 For the popO::mCherry fusion construct, PCR product generated using QQ98 and XX17 was cut with XbaI and ligated into SpeI digested pHW7911 to generate pMP8059 For the popS::mCherry fusion construct, PCR product generated using QQ75 and XX18 was cut with XbaI and ligated into SpeI digested pHW7911 to generate pMP8061 For the popP::mCherry fusion construct, PCR product generated using QQ93 and XX18 was cut with SpeI and ligated into SpeI digested pHW7911 to generate pMP8060.The mCherry tagged constructs were generated by inserting promoter and open reading frame sequences of each gene into A. nidulans minimal medium (ANM) supplemented with 10 mM ammonium sulfate ((NH4)2SO4) as a sole nitrogen source. At 37°C strains were grown on Brain Heart Infusion (BHI) medium (3.7% brain heart infusion).At 25°C strains were grown on pepA (G1004), ∆popH (G994) and ∆popO (G997) strains were generated by transforming strain G816  [pepA), pHW7881 (popH), pHW7882 (popO) and selecting for uracil prototrophy. The ∆popS (G1002) strain was generated by transforming strain G809  with linearised deletion construct pHW7884 (popS) and selecting for glufosinate resistance. The ∆popP (G999) strain were generated by transforming strain G829  [popP) and selecting for riboflavin prototrophy. The ∆pepA, ∆popH, ∆popO, ∆popS and ∆popP mutations were complemented by transforming the respective strains with pMP7891 (pepA), pMP7887 (popH), pHW7888 (popO), pHW7890 (popS) and pMP7889 (popP) to generate ∆pepA pepA+ (G1005), ∆popH popH+ (G995), ∆popO popO+ (G998), ∆popS popS+ (G1003) and ∆popP popP+ (G1000) strains. To generate ∆popP ∆popH (G1007), ∆popP ∆pepA (G1011), ∆popS ∆popP (G1013), ∆popS ∆popO (G1009), ∆popS ∆pepA (G1012), ∆popO ∆pepA (G1010) and ∆popO ∆popP (G1008) double mutants, ∆pepA, ∆popH, ∆popO, ∆popS and ∆popP single mutant strains were grown on 1 mg/ml 5-FOA supplemented with 10 mM ((NH4)2SO4) and 5 mM uracil to select for the loss of the pyrG marker. These were then transformed with the respective linearised deletion constructs corresponding to the combination required for the strain. The mCherry tagged strains were generated by transforming the ∆pepA+, ∆popH, ∆popO, ∆popS+ and ∆popO strains with circular pepA::mCherry (pMP8062), popH::mCherry (pMP8058), popO::mCherry (pHW8059), popP::mCherry (pHW8060) and popS::mCherry (pMP8061) constructs into the respective strains, to generate ∆pepA pepA::mCherry (G1006), ∆popH popH::mCherry (G996), ∆popO popO::mCherry (G1061), ∆popS popS::mCherry (G1060) and ∆popO popO::mCherry (G1001) strains.Transformation was performed previously described using PEG-mediated protoplast fusion . Full de, pyrG1)  with linB::pyrG)  with lin5 cells/m into a 6 well microtitre tray containing sterile coverslips and 2 mL of DMEM (J774) or RPMI (THP-1) medium and incubated at 37°C for 24 h. Macrophages were activated with 0.1 μg/mL lipopolysaccharide (LPS)(J774) or differentiated by the addition of 32 μM phorbol 12-myristate 13-acetate (PMA)(THP-1) for 24 h at 37°C. Cells were washed with PBS and 2 mL of complete DMEM or RPMI medium containing 1 × 106 conidia was added. A control lacking conidia was also performed. Macrophages were incubated for 2 h at 37°C to allow conidia to be engulfed, washed once in PBS to remove non-phagocytosed conidia and incubated a further 24 or 48 h at 37°C. Macrophages were fixed in 4% paraformaldehyde and stained with 1 mg/mL fluorescent brightener 28  to observe fungal cell walls. Mounted coverslips were examined using differential interference contrast (DIC) and epifluorescence optics for cell wall staining and viewed on a Reichart Jung Polyvar II microscope. Images were captured using a SPOT CCD camera (Diagnostic Instruments Inc) and processed in Adobe Photoshop. The numbers of ungerminated conidia, germlings or yeast cells were recorded in a population of approximately 100 macrophages in three independent experiments.J774 and THP-1 macrophage infection assay was performed as per the previously described protocol . Briefly5 cells/mL in a flask containing Dulbecco’s modified Eagle medium (DMEM) and grown for 2 days. Macrophages were activated with 0.1 μg/mL lipopolysaccharide (LPS), incubated for 24 h and infected with 3 × 108 conidia of T. marneffei. After 2 h the unphagocytosed conidia were washed off, fresh DMEM added and incubated for an additional 24 h. The media was removed and RIPA buffer  containing cOmpleteTM Protease Inhibitor (Roche) and 1mM PMSF, was added to lyse the macrophages. The cell lysate was centrifuged at 2000xg and the pellet predominantly consisting of T. marneffei yeast cells was separated from the J774 macrophage protein supernatant. T. marneffei cells were homogenized in a FastPrep®-24. Approximately 15 μg of each protein extract, as determined by Bradford assay, was used for SDS-PAGE. Western blot transfer was performed using Immobilon-P polyvinylidene difluoride (PVDF) filters (Merck Millipore Ltd). The mCherry-tagged protein was detected using an anti-mCherry monoclonal rat primary antibody (ThermoFisher Scientific) and an anti-rat IgG horseradish peroxidase (HRP)-linked secondary antibody . HRP activity was detected using ClarityTM Western enhanced chemiluminescence (ECL) substrate (Bio-Rad) on a Bio-Rad ChemiDocTM MP imaging system.The macrophage infection assay was performed as previously described (46). Briefly, J774 murine macrophages were seeded at 1 × 10Immunofluorescence localization of the mCherry-tagged strains was performed with an anti-mCherry rat monoclonal primary (Life Technologies) and a goat anti-rat ALEXA 488 secondary antibody (Molecular Probes) using standard protocols [


Immunotherapies are rapidly being integrated into standard of care (SOC) therapy in conjunction with surgery, chemotherapy, and radiotherapy for many cancers and a large number of clinical studies continue to explore immunotherapy alone and as part of combination therapies in patients with cancer. It is evident that clinical effectiveness of immunotherapy is limited to a subset of patients and improving immunotherapy related outcomes remains a major scientific and clinical effort. Understanding the immune cell subset phenotype and activation/functional status (cellular immunome) prior to and post therapy is therefore critical to develop biomarkers that (1) will predict if a patient will respond to immunotherapy and (2) are a result of immunotherapy. In this study, we investigated local (tumor) and peripheral (blood) cellular immunome of patients with melanoma, breast cancer, and brain cancer using a rapid and reliable standardized, multiparameter flow cytometry assay. We used this approach to monitor changes in the peripheral cellular immunome in women with breast cancer undergoing SOC therapy. Our analysis is unique because it is conducted using matched fresh tumor tissue and blood from patients in real-time, within 2–3 h of sample acquisition, and provides insight into the innate and adaptive immune cell profile in blood and tumor. Specific to blood, this approach involves no manipulation and evaluates all immune subsets such as T cells, B cells, natural killer (NK) cells, monocytes, dendritic cells (DCs), neutrophils, eosinophils, and basophils using 0.5 ml of blood. Analysis of the corresponding tumor provides much needed insight into the phenotype and activation status of immune cells, especially T and B cells, in the tumor microenvironment vs. the periphery. This analysis will be used to assess baseline and therapy-mediated changes in local and peripheral cellular immunome in patients with glioblastoma, breast cancer, and melanoma in planned immunotherapy clinical studies. Over the past 7 years, immunotherapy has dramatically changed the treatment landscape for many solid tumor and hematologic malignancies –5. CanceIntratumoral injection of an oncolytic virus to promote local recruitment of immune cells to the tumor is being developed as a strategy to overcome resistance to immune blockade , 16. We Toward the goal of utilizing the cellular immunome to predict response to immune-based therapies, we analyzed matched blood and tumor tissue from melanoma, breast cancer and brain cancer patients. Previous studies have shown that peripheral immune subtypes can be predictive of intratumoral immune responses and serve as a prognostic factor in cancer patients . Analysin = 15) were diagnosed with localized disease and received SOC neoadjuvant chemotherapy followed by surgery. Melanoma patients (n = 9) included in this study had stage I-IV disease. Primary tumors or metastatic lymph nodes were resected during SOC therapy. Brain tumor patients (n = 3) were newly-diagnosed with grade III glioma or grade IV glioblastoma (GBM) and received SOC surgery. De-identified matched blood and tumor tissue from consented patients was obtained by the BRPC and BTBR and used for examination of the cellular immunome.Eligible patients, 18 years or older, who underwent SOC surgical resection of melanoma, breast cancer, and brain cancer were consented to research tumor tissue and blood collection though the Duke BioRepository and Precision Pathology Center (BRPC) or the Brain Tumor BioRepository (BTBR) at Duke University School of Medicine. All patients were selected based on ability to get both tumor tissue and blood at the same time. Breast cancer patients (Blood was obtained by venipuncture and collected in Vacutainer collection tubes containing EDTA anticoagulant (BD Biosciences). Blood was rotated on shaker until flow cytometry analysis (10 min-4 h post collection).Surgically resected tumors were collected and stored in MACS tissue storage solution at 4°C (Miltenyi). Storage time was 2–16 h post-tumor collection. Tumor cells and tumor infiltrating immune cells were analyzed after tumors were processed using the Tumor Dissociation Kit and Gentle MACS mechanical dissociator (Miltenyi) following the manufacturer's recommendations. Single cell suspension was immediately analyzed by flow cytometry.6 cells were added to each tube and cell staining was performed as described above for peripheral blood. Tumor cells were filtered three times using a 70 μM cell strainer to remove dead cell debris. All processed samples were then analyzed on a 13-color CytoFlex flow cytometer (Beckman Coulter). Data were analyzed using Kaluza Software (Beckman Coulter).All analysis was performed using DuraClone IM (Immune Monitoring) panels: Basic , B cell , T cell , Granulocyte , T cell Receptor , Regulatory T cell/Treg , and Dendritic cell/DC . For peripheral blood analysis, 100 μl of blood was added to the appropriate tubes and cells were processed according to the manufacturer's instructions. In brief, blood was incubated with the antibodies for 15 min in the dark, followed by red blood cell lysis using VersaLyse (Beckman Coulter) for 15 min in the dark. Cells were then washed twice in PBS prior to data acquisition. Treg cell analysis was performed according to the manufacturer's protocol. Briefly, 50 μl of peripheral blood was incubated for 15 min with DuraClone IM Treg Tube 1, followed by a wash in 3 ml of PBS and centrifugation at 500 × g for 5 min. Cells were then resuspended in 50 μl of 100% fetal calf serum followed by 5 μl of fixative reagent. After a 15 min incubation, cells were resuspended in 400 μl of permeabilizing reagent and immediately transferred to DuraClone Treg Tube2 for a 60 min incubation. Cells were then washed in PBS prior to data acquisition. For tumor cells and tumor-infiltrating immune cells, 1 × 10(1A), breast cancer (1B), and brain cancer (1C) patients. We examined lymphocytes including B cells (CD19+), CD3+/CD4+ T cells, CD3+/CD8+ T cells, and NK T cells (CD3+CD56+). Total monocytes (CD14+CD16+)  , were th cells CD+, CD3+/CEMRA cells). CD27 and CD28 (receptors involved in T cell activation) are used to study cellular activation history and are present on naïve and central memory T cells but absent on effector memory and effector T cells . Al. AlEMRA Cell surface T cell receptor expression was analyzed in melanoma, breast cancer, and brain cancer patient blood using the DuraClone IM TCR panel . LymphocRegulatory T cells (Tregs) and the ratio of regulatory T cells to conventional T cells in blood are used to measure therapy outcomes in patients treated with immunotherapy. To examine Tregs, lymphocytes were identified based on their CD3, CD4, and CD8 expression . CD3+CD4Because we analyzed whole blood, we were able to examine populations that are excluded when analysis is conducted on peripheral blood mononuclear cells (PBMCs), namely granulocytes lowCD38low B cells, transitional B cells (IgM+CD38+), and plasmablasts (IgM–CD38+). Increased numbers of memory B cells and plasmablasts in blood and tumor would indicate B cell responses in patients with cancer  are potent antigen presenting cells  and haveNext we examined the local (tumor) cellular immunome in patients with melanoma, breast cancer and brain cancer. Surgically resected fresh tumor tissue (matched to blood from same patient) was processed into a single cell suspension within 16 h of tumor harvest followed immediately by flow cytometry. We analyzed T cell subsets to examine the phenotype and differentiation status of these T cells. In Regulatory T cells are an important CD4+ T cell subtype that can impact anti-tumor immune responses . In the In the two melanoma patients with high percentage of B cell infiltrates in the tumor, we further phenotyped the infiltrating B cell subpopulations. Data presented in Breast cancer analysis was conducted with tumor tissue from 15 women presenting with localized disease who underwent surgery as part of SOC therapy. We processed all samples fresh in order to avoid cell death and cell surface marker alteration as a result of the freeze-thawing cycle . In FiguNext we analyzed CD4+ and CD8+ T cells for expression of CD57 and PD1. Data presented in We were able to conduct a comprehensive analysis of the T cell subset in breast tumors obtained from patients who underwent SOC therapy, however, our analysis was limited with regards to B cell analysis. Although, a fraction of the breast tumors were infiltrated by significant numbers of B cells, we lacked sufficient tumor sample to complete a detailed B cell analysis. In future studies, we plan to focus specifically on the B cell subsets that are found in breast tumors.Brain tumor immune analysis was conducted in three patients whose tumors were resected as part of SOC therapy. In Analysis of T cell subsets in brain tumor revealed that infiltrating CD4+ and CD8+ T cells expressed CD57 . PD1 wasWe analyzed melanoma, breast, and brain tumors for the presence of granulocytes and dendritic cells and observed that these cells were not present in the tumors.The analysis that we have presented was developed to examine baseline and therapy-mediated changes in the peripheral (blood) cellular immunome in the context of therapy along with companion analysis in tumors before and after therapy when feasible. Therefore, to validate our peripheral cellular immunome analysis, we longitudinally examined blood from women with breast cancer undergoing SOC therapy. Analysis of blood was performed as described above using the DuraClone IM basic panel The ability to precisely and reproducibly understand immune cell phenotype and activation status (cellular immunome) in tumor and blood is critical to assess if a patient has responded to therapy and to develop biomarkers of response to therapy. The analysis presented in this manuscript was done using paired tumor and blood samples from patients with cancer with the following objectives: (1) examine blood and tumor cellular immunome in patients with cancer; (2) determine feasibility of this approach across different tumor types, and (3) assess if the experimental protocol is reproducible and precise to allow longitudinal monitoring of the peripheral cellular immunome to measure changes in response to therapies. Although cost of real time analysis can be a concern during immune monitoring, a side by side comparison between real times analysis of samples and analysis of frozen PBMCs revealed comparable overall costs.In this study we present analysis conducted in patients with melanoma, breast cancer, and brain cancer. Our data demonstrate that we can indeed reliably and accurately monitor patient cellular immunome in tumor and blood in a matter of 2–5 h (blood) and 2–16 h (tumor) after sample acquisition and get a complete profile that encompasses 77 immune subsets. The value of developing an assay that has a quick turn-around and can be conducted in real-time is important in biomarker development; specifically if we want to use blood and tumor cellular immunome to drive decisions related to therapy. Conducting analysis of blood in real-time is important for many reasons. The use of fresh blood eliminates the need to purify and freeze peripheral blood mononuclear cells (PBMCs), which introduces variability during isolation, freezing, storage, and thawing procedures. The use of whole blood permits analysis of all cell populations in blood . For example, our analysis of granulocytes in n = 100). Specifically, we are monitoring the peripheral cellular immunome at baseline, post-chemotherapy, and pre-surgery and 2 months post-surgery to comprehensively address how the peripheral cellular immunome is impacted during SOC therapy. Also part of this planned study is the investigation of the local/tumor cellular immunome, in patients undergoing SOC surgery. An example of longitudinal analysis of the peripheral cellular immunome in women with breast cancer undergoing SOC therapy is presented in Our data in in vivo immune competence than PBMC function , breast cancer , and melanoma .These studies were initiated to develop a robust method for cellular immunome monitoring in the context of our current immunotherapy clinical trials. In a recent clinical study with a novel recombinant oncolytic poliovirus, PVSRIPO, we reported unprecedented responses in the treatment of recurrent glioblastoma, with 21% of patients experiencing durable, long-term (>3 years) survival . Our teaThis study was approved by the Duke Institutional Review Board as an exempt protocol to access blood and human tissue through the Duke BioRepository and Precision Pathology Center (BRPC) or the Brain Tumor BioRepository (BTBR).EKH designed study, administered the project, performed and analyzed experiments, and wrote manuscript. VF and KL performed and analyzed experiments and reviewed manuscript. GB provided clinical samples and edited manuscript. ESH provided clinical samples and reviewed manuscript. SN designed study, administered the project, analyzed experiments, and wrote manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Thus, while jumping performance appears to be unaffected when leg extensor muscle strength and tendon stiffness are maintained, the reduced muscular power output during lower limb multi-joint tasks seen with aging may be due to age-related changes in motor task execution strategy rather than due to muscle weakness.Reductions in muscular power output and performance during multi-joint motor tasks with aging have often been associated with muscle weakness. This study aimed to examine if matching younger and middle-aged adults for triceps surae (TS) muscle strength and tendon stiffness eliminates age-related differences in muscular power production during drop jump. The maximal ankle plantar flexion moment and gastrocnemius medialis tendon stiffness of 29 middle-aged (40–67 years) and 26 younger (18–30 years) healthy physically active male adults were assessed during isometric voluntary ankle plantar flexion contractions using simultaneous dynamometry and ultrasonography. The elongation of the tendon during the loading phase was assessed by digitizing the myotendinous junction of the gastrocnemius medialis muscle. Eight younger (23 ± 3 years) and eight middle-aged (54 ± 7 years) adults from the larger subject pool were matched for TS muscle strength and tendon stiffness  and then performed series of drop jumps from different box heights  onto a force plate (sampling frequency 1000 Hz). The matched young and middle-aged adults showed similar drop jump heights for all conditions . However, middle-aged adults showed longer ground contact times (on average 36%), lower vertical ground reaction forces (36%) and hence lower average mechanical power  independent of box height. Further, leg stiffness was lower (48%) in middle-aged compared to younger adults for all jumping conditions and we found significant correlations between average mechanical power and leg stiffness (0.70 ≤  Lower limb muscular power production is crucial for an effective and safe locomotion in sport and during activities of daily living. For example, rapid muscle force generation is required during various daily lower limb multi-joint tasks and has been identified as a predictor for mobility  and fallPrevious research suggests an age-related decline in muscular power production of the leg extensor muscles during single  and kinetics  would be eliminated when young and middle-aged adults are matched for triceps surae (TS) isometric muscle strength and tendon stiffness, in order to test the hypothesis that age-related differences in muscular power production during lower limb multi-joint tasks cannot solely be explained by alterations in leg-extensor muscle strength and tendon stiffness seen with aging. Therefore, the second aim of this study was to determine to what extent leg stiffness  is associated with the average mechanical power output during maximal vertical jumping in young and middle-aged adults with the hypothesis that leg stiffness may be a major factor of the age-related decline in muscular power production during lower limb multi-joint tasks. As it has been shown that the mechanical properties of the TS MTU in particular are crucial for lower limb multi-joint tasks  and 26 younger (18–30 years) physically active healthy male adults gave their written informed consent to participate in this study. Participants were included when two participants, one from the young and one from the middle-aged groups had similar TS MTU mechanical properties. Finally, eight middle-aged (range: 41–67 years) and eight younger (19–28 years) adults were matched for TS muscle strength and tendon stiffness. None of the participants had experience in regularly performing DJs. Exclusion criteria were any previous Achilles tendon ruptures, Achilles tendon injuries  or pain within the last 12 months, or musculoskeletal impairments in the lower limbs , which could have influenced the outcomes of this investigation. The study was approved by the university’s human ethics committee and was conducted according to the Declaration of Helsinki.TMOne, Esaote, Genua, Italy). All sustained contractions were guided by visual feedback displayed on a computer screen showing the produced joint moment. In the current study, force values of ±5% of the target force were accepted (when the force was held for 3 s). If this was not achieved, the specific trial was repeated. During all maximal contractions, strong verbal encouragement was given to the participants in order to attain their actual MVC  onto a force plate  in a randomized order. In order to avoid any effects through muscle fatigue, about 90 s of rest was provided between each trial. Since ground contact time influences leg stiffness during maximal vertical jumping ,b, partimaxF is the maximal vertical ground reaction force and ΔCoM is the maximum vertical displacement of the body’s center of mass during ground contact . The level of statistical significance was set at α = 0.05. All results provided in the text, tables and figures are presented as mean and standard deviation (SD).For all participants, the trial with the greatest jumping height for each jumping condition (box height) was taken into account for further analysis. A two-way repeated measures analysis of variance  was performed in order to detect potential effects of age or box height on DJ height or kinetics with respect to average mechanical power, leg stiffness, maximal vertical ground reaction force and ground contact time. Detected significant main effects or interactions were further analyzed using Duncan’s Figures 2), which meant that there were no significant differences in maximal isometric ankle joint plantar flexion moments and GM tendon stiffness between young  and middle-aged adults (3.2 ± 0.5 Nm/kg and 572 ± 100 N/mm). Paired samples t-tests revealed a significant group difference in age , but no significant differences in body height (178.3 ± 6.5 cm vs. 179.9 ± 4.7 cm) and mass (79.3 ± 9.6 kg vs. 75.3 ± 4.9 kg).The eight young and eight middle-aged adults were matched for TS isometric muscle strength and tendon stiffness  = 3.04, p < 0.05]. Duncan’s post hoc analysis revealed no differences in jumping height between TS muscle strength and stiffness-matched young and middle-aged adults independent of jumping condition . Jumping height was significantly lower for the 13 cm compared to all other box heights in both age groups. However, a lower DJ performance for the 23 cm compared to the 39 cm condition was only found in younger adults.Concerning the DJ performance, the implemented two-way ANOVA revealed a statistically significant age group × box height interaction for jumping height  and maximal vertical ground reaction force during support phase , with the middle-aged adults showing longer ground contact times and lower forces for all box heights . The two-way ANOVA revealed a statistically significant age × box height interaction for average mechanical power . Duncan’s post hoc analysis revealed significant differences in average mechanical power between age groups independent of box height (1.39 ≤ d ≤ 3.16) and higher power values with increasing box height . However, the middle-aged adults showed no significant difference in average mechanical power for the 23 and 33 cm conditions. Further, there was a statistically significant age group × box height interaction for leg stiffness . Duncan’s post hoc analysis revealed lower leg stiffness values in the middle-aged compared to younger adults for all jumping conditions (1.18 ≤ d ≤ 2.57) and lower leg stiffness values with raising box height only in the younger adults . However, no significant difference in leg stiffness was found between the 33 and 39 cm conditions.Considering the DJ kinetics, there were significant age effects on ground contact time [p < 0.001) effect of jumping condition on maximal vertical ground reaction force during support phase [F = 17.23], with higher vertical ground reaction forces with increments in box height. However, no significant difference in maximal vertical ground reaction force could be detected for the 33 and 39 cm conditions. Significant positive correlations were found between average mechanical power and leg stiffness over all analyzed participants .Regarding a potential box height effect on DJ kinetics, there was a significant , meaning that approximately 50 to 70% of the variability in average mechanical power during support phase can be related to the variance in leg stiffness within the pooled group of middle-aged and younger adults. These results support previous findings showing a significant relationship between muscular power output and the stiffness of the lower limb joints during maximal vertical jumping , indicating that leg-extensor isometric muscle strength and tendon stiffness play an important role for the performance in explosive motor tasks . The current finding, that a similar performance during maximal vertical jumping can be achieved by different motor task execution strategies i.e., levels of leg stiffness is in accordance to previous results ,b. In thper se. Despite the fact that seven of our middle-aged adults were between 41 to 59 years, we observed functionally relevant (≥36%) differences for the main outcome parameters between the age groups, indicating that including a higher number of participants over the age of 59 years would have even strengthened the findings of the current study. Finally, a limitation of the current study is the relatively low number of participants in each group  which reduces the potential for detecting statistical differences between age groups. Therefore we cannot exclude that a higher number of subjects might have led to significant differences in jumping height between young and middle-aged adults. However, this drawback has no effect on our observation that matching young and middle-aged adults for TS muscle strength and tendon stiffness does not eliminate age-related differences in mechanical power output during jumping.A limitation of the current study may be that we did not use optical motion capture analysis but used a more convenient approach in order to assess mechanical power during maximal DJs. However, average mechanical power was calculated by the ratio of total work and ground contact time for all age groups and, therefore, we believe that this drawback potentially affects our results more in absolute terms than the validity of the corresponding comparative data. Regarding our ankle-knee joint configuration for GM tendon stiffness assessment, the contribution of the GM muscle to the net joint moment may be reduced compared to a fully extended knee joint angle since the GM operates on the ascending limb of the force-length relationship. However, in a previous study  we foundIn conclusion, independent of task demand, matching younger and middle-aged adults for TS isometric muscle strength and tendon stiffness eliminates age-related differences in jumping performance but not in mechanical power production during maximal DJs. Leg stiffness during the support phase was found to be significantly associated with muscular power production during maximal DJs, with lower leg stiffness values in middle- compared to younger-aged adults. Thus, while jumping performance appears to be unaffected when leg extensor muscle strength and tendon stiffness are maintained, the reduced muscular power output during lower limb multi-joint tasks seen with aging may be due to age-related changes in motor task execution strategy rather than due to muscle weakness.The data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.This study was carried out in accordance with the recommendations of the German Sport university’s human ethical commitee. The protocol was approved by the German Sport university’s human ethical commitee. All subjects gave written informed consent in accordance with the Declaration of Helsinki.KK, SH, and MK conceived the work. SH, TA, and MK acquired the data. MK drafted the manuscript. All authors contributed to analysis and interpretation of the work, prepared the figures, contributed to final approval of the version to be published, and agreed to be accountable for the work.KK has equity in Protendon GmbH & Co. KG, whose device and software was used for the data collection, processing, and analysis in this study. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling Editor declared a shared affiliation, though no other collaboration, with one of the authors CM.