Teleopsis dalmanni. After an additional mating, females benefit from greatly increased fertility (proportion fertile eggs). Gains from multiple mating are not uniform across females; they are greatest when females have high fecundity or low fertility. Fertility gains also vary spatially, as we find an additional strong effect of the stream from which females were collected. Responses were unaffected by male mating history . Recent male mating may be of lesser importance because males in many species, including T. dalmanni, partition their ejaculate to maintain their fertility over many matings. This study highlights the importance of complementing laboratory studies with data on wild‐caught populations, where there is considerable heterogeneity between individuals. Future research should focus on environmental, demographic and genetic factors that are likely to significantly influence variation in individual female fecundity and fertility.Polyandry, female mating with multiple males, is widespread across many taxa and almost ubiquitous in insects. This conflicts with the traditional idea that females are constrained by their comparatively large investment in each offspring, and so should only need to mate once or a few times. Females may need to mate multiply to gain sufficient sperm supplies to maintain their fertility, especially in species in which male promiscuity results in division of their ejaculate among many females. Here, we take a novel approach, utilizing wild‐caught individuals to explore how natural variation among females and males influences fertility gains for females. We studied this in the Malaysian stalk‐eyed fly species  Both sexes in this species are highly promiscuous . Females and males were collected on day zero at dusk from lek sites on the edge of forest streams at several stream sites adjacent to tributaries of the Gombak River. Individuals were aspirated into plastic bags and within 1 hr of capture, males and females were transferred to individual 500‐ml containers lined with a moist cotton wool and tissue paper base. Flies were fed every 2 days with puréed banana.Female fecundity was recorded from counts of eggs deposited on the tissue paper base, which were collected and renewed every 2 days. Eggs were allowed to develop for a further 5 days in petri dishes containing a moist cotton pad. Fertility was estimated by scoring hatching success under a light microscope at 10 ×  magnification. Fertilized eggs that have hatched appear as empty chorion cases, while unfertilized eggs are full and show no signs of development. If fertilized eggs failed to hatch, but showed signs of development , they were recorded as fertile  or a nonsperm‐depleted male (N = 17). After the additional mating, females were rehoused and their subsequent reproductive output was recorded every 2 days from day 14 over the following 8 days, and morphometric measures taken as before.A second experiment was carried out using flies collected from five stream sites in the Ulu Gombak valley in July/August 2012. Individuals were collected as above. Females were housed individually in 500‐ml containers, and their reproductive output was recorded as in the first experiment. Males were placed in large 1,500‐ml containers either with a mix of males and nonfocal females allowing them to mate freely (sperm‐depleted), or only with other males (nonsperm‐depleted). Isolation from females allows males to replenish their sperm stores  as well as over the 8 days after the additional mating . To test whether an additional mating resulted in increased fecundity or fertility, the total individual reproductive output over the 8 days before and after mating was compared, as well as total individual reproductive output on the days immediately before (days 11–12) and after the additional mating . Lastly, we examined whether the direction of change in individual fertility was positive, or negative/unchanged, and tested the degree to which individual proportion fertility changed depended on female premating fecundity or fertility.lme4 package  was used in all models (except for those modeling change), as results can be unreliable when using both random effects and a quasi‐distribution . For both experiments, we repeated all analyses with female eyespan and thorax as covariates. This did not alter any of the results  after mating, followed by a peak (day 4) before settling to a more steady level  and postmating periods . Female fecundity did not change over the premating period .Fecundity was highly variable between females both in the premating , and was not different between the days immediately before (days 11–12) and immediately after (days 14–15) the extra mating  or postmating periods .Female fecundity did not differ when individual reproductive output was compared across the premating and postmating periods  and proportion fertility , and this extended into the postmating period (days 14–21) for female absolute fertility  but not proportion fertility . In contrast to fecundity, across the premating period there was a decline in absolute  and postmating periods .The pattern for individual female fertility in the premating period days 5–12), showed considerable variation among females, both in the absolute number of fertile eggs laid ; however, proportion fertility increased . The percentage of females with low fertility  dropped from 38% to 19%, whereas the proportion with high fertility  rose from 24% to 48% . However, when female fertility was accounted for, females with higher fecundity were more likely to have a positive change in fertility after the additional mating . In addition, females with low fertility were more likely to benefit from the additional mating . This greater effect of premating fertility persisted after accounting for differences in individual female fecundity .The direction of change in total individual fertility after the additional mating (increase or decrease/unchanged) did not depend on female fecundity , and this result remained when female fertility was accounted for . Female premating fertility had no effect on the change in proportion fertility . However, once fecundity was accounted for, female premating fertility did have an effect , as females that fertilized few of their eggs had a larger positive change in proportion fertility than females that were already fertilizing relatively more.A similar examination was made using the change in proportion fertility between the pre‐ and postmating periods Figure . Females2 = 5.8958, N = 84, p = .0152) and proportion fertility . After the additional mating, absolute fertility no longer differed between stream sites , but variation in proportion fertility persisted despite the extra mating .Finally, we examined differences in fertility across streams. In the premating period, there was variation between stream sites in absolute , or for the contrast of the days immediately before and after the additional mating, days 11–12 and 13–14 .The pattern for female fecundity was broadly similar to that of the previous experiment Figure . There w3.2.2SD, days 11–12). Proportion fertility was also similar to the previous experiment prior to mating . Comparing total fertility in the premating and postmating periods, absolute  and proportion fertility  increased after the additional mating. Likewise, between the days immediately prior (day 11–12) and immediately after (days 13–14) the additional mating, there was an increase in absolute , but not after female fertility was accounted for . Females with low premating fertility were more likely to have a positive change . However again, after accounting for fecundity, premating fertility did not predict the direction of change .The direction of change in individual fertility was more likely to be positive for more fecund females . Change in proportion fertility likewise did not depend on premating fertility . In addition, when the analysis was repeated and fecundity was accounted for, females with low fertility prior to mating also had a more positive change in proportion fertility .Change in proportion fertility between the premating and postmating periods did not depend on premating fecundity  and proportion fertility . Likewise comparing the days immediately before (day 12) and after the additional mating (day 14), male type had no effect on the increase in absolute  or proportion fertility . There was no effect of male type on either the direction of change in fertility  or the change in proportion fertility .Comparing the 8 days before and after the additional mating, male type was unrelated to the increase in absolute . Females from laboratory populations of T. dalmanni stalk‐eyed fly females collected from the wild. Females from the two collections, in 2011 and 2012, had mean female fertility of 46% or 32%, respectively, shortly after they were initially captured (days 5–6), and this declined to ~20% in both cases over the following week  prior to release for fertilization of an egg Kotrba, .The results here contrast with those of a previous study carried out on the same population (Harley et al., We can make several inferences from these studies of wild‐caught females. First, they confirm there is a cost of a failure to remate as the proportion of fertile eggs laid declines with time when females are unable to remate. Second, an additional mating has a greater beneficial effect when females already have low fertility. The most obvious proximate reason for this is that many wild females are sperm‐limited, either because they had not mated recently, not mated at a sufficiently high rate or because sperm allocation by males was considerably limited. These explanations could be directly assessed in the future by counting sperm in female sperm storage organs in wild‐caught females and after matings with wild‐caught males. This could be complemented by observing mating rates in the wild, and relating these measures to natural fertility levels. A third inference from the current experiments is that the fertility benefits to females vary between individuals, stream sites, across matings and fluctuate through time. In some contexts, individual females may be limited by the availability of mating opportunities, whereas in others, they may become increasingly limited by their own fecundity.The source of variation in fertility between individuals in the wild is currently undefined. It is likely that variable factors such as population density and sex ratio are important, particularly as they will affect female and male mating rates. Similarly, environmental conditions such as food availability can influence mating rates (Kotiaho, Simmons, & Tomkins, T. dalmanni (Kotrba, Other significant factors to consider are variation in male mating strategy and male quality as they may have a significant influence on the benefit that females obtain from remating. Males can adjust their ejaculate investment in response to female reproductive value (Engqvist & Sauer,  Kotrba, , and mal Kotrba, . Partiti Kotrba, . In addi Kotrba, . To cope Kotrba, . In thisT. dalmanni (Cotton, Földvári, Cotton, & Pomiankowski, Another cause of variation in male fertility and ejaculate allocation, other than recent mating history, is meiotic drive (Wilkinson, Johns, Kelleher, Muscedere, & Lorsong, T. dalmanni to further understand the environmental and population‐level variables that affect the benefits to additional matings. For example, we know that there is much variation in female fecundity and fertility between stream sites. What we have yet to elucidate is how streams differ—do they vary in food availability and quality, rainfall, humidity, temperature, population density, or sex ratio? Are these factors stable or fluctuating? Which have the most influence on female fecundity and fertility? We show that females with low fertility and high fecundity benefit the most from mating; improved knowledge of the conditions experienced by individuals throughout their lifetime will further our understanding of when and why it is beneficial for females to remate.We used wild‐caught flies to capture the natural variation between individuals, an approach that has been much neglected. It is important to dig deeper into the life history of In conclusion, this study has demonstrated that female sperm storage and depletion since the previous mating are key selection forces driving the benefits and evolution of mating rates in the wild. Females are generally sperm‐limited due to the minimal male sperm investment in individual copulations (Rogers et al., None declared.EH, AP, and KF conceived the original project and methodology; EH, AC, and JMH collected the data; LM analyzed the data; LM, AP, and KF led the writing of the manuscript. All authors contributed critically to the drafts and gave final approval for publication.https://doi.org/10.5061/dryad.pk5vd.Raw data have been archived in the Dryad Digital Repository:  Click here for additional data file.


Endocytic trafficking is a critical mechanism for cells to decode complex signaling pathways, including those activated by G-protein-coupled receptors (GPCRs). Heterogeneity in the endosomal network enables GPCR activity to be spatially restricted between early endosomes (EEs) and the recently discovered endosomal compartment, the very early endosome (VEE). However, the molecular machinery driving GPCR activity from the VEE is unknown. Using luteinizing hormone receptor (LHR) as a prototype GPCR for this compartment, along with additional VEE-localized GPCRs, we identify a role for the adaptor protein APPL1 in rapid recycling and endosomal cAMP signaling without impacting the EE-localized β2-adrenergic receptor. LHR recycling is driven by receptor-mediated Gαs/cAMP signaling from the VEE and PKA-dependent phosphorylation of APPL1 at serine 410. Receptor/Gαs endosomal signaling is localized to microdomains of heterogeneous VEE populations and regulated by APPL1 phosphorylation. Our study uncovers a highly integrated inter-endosomal communication system enabling cells to tightly regulate spatially encoded signaling.  •GPCRs that internalize to very early endosomes (VEEs) require APPL1 to recycle•Receptor recycling is driven by cAMP/PKA to phosphorylate serine 410 on APPL1•cAMP signaling from GPCRs, such as LHR, occurs from distinct VEE microdomains•APPL1 limits VEE cAMP signaling via opposing mechanisms required for GPCR sorting Sposini et al. report that G-protein signaling from specific GPCRs is spatially restricted in very early endosomes (VEEs), compartments distinct from classic early endosomes. Mechanistically, receptor signaling and sorting from VEEs occur via opposing phosphorylation states of APPL1 adaptor protein, providing novel decoding mechanisms for cells to tightly control signaling. Within any cellular signaling system, the spatial organization of signaling networks is a critical mechanism for cells to decode complex pathways to specific downstream responses. Thus, membrane trafficking and signaling are viewed as an integrated system that mediates diverse fundamental cellular programs . ControlFollowing endocytosis, cell-surface receptors are trafficked to early endosomes (EEs) that are classically considered to be the primary sorting compartment for all internalized cargo . We haveIn this study, we demonstrate a central role for APPL1 in directing receptor sorting and endosomal G-protein signaling from the VEE. Furthermore, we provide evidence of functional heterogeneity within VEEs and an inter-endosomal communication system that enables cells to tightly regulate and reprogram dynamic GPCR signaling within the endocytic network.Following ligand-dependent internalization, LHRs are sorted to VEEs, which are physically and biochemically distinct from EEs. Analysis of endogenous levels of the endosomal adaptor protein APPL1, demonstrated that this adaptor protein was localized to a subset of VEEs containing LHR . Therefode novo synthesis does not contribute to recycling and consistent with our confocal imaging of receptor recycling that tracks the fate of the internalized receptor  and a pH-sensitive GFP super-ecliptic pHluorin (SEP) tagged at the extracellular N terminus of LHR. This GFP variant is highly fluorescent when located at the cell surface , yet its fluorescence is rapidly quenched in the lumen of endocytic vesicles (acidic pH), thus enabling analysis of dynamic GPCR recycling as it reinserts into the plasma membrane at single-event resolution . TIR-FM receptor . Criticareceptor A and S3Breceptor . Co-locareceptor C.To examine whether APPL1-dependent recycling by LHR is conserved in cells that express LHR endogenously, we used primary human endometrial stromal cells (hESCs) . In hESCWe next examined the molecular mechanisms underpinning APPL1-dependent LHR recycling. GPCR activation and signaling are essential for subsequent intracellular trafficking of receptors . As LHR Given the absolute dependence of LHR recycling on APPL1, inhibition of PKA may impact recycling by altering trafficking of internalized LHR to APPL1 endosomes or by disrupting the endosomal localization of APPL1. The ability of APPL1 to localize to endosomes was unperturbed following treatment with KT5720 ; howeverPKA positively or negatively regulates recycling of other GPCRs via phosphorylation of receptors, or associated adaptor proteins . The humThe aforementioned data indicate that APPL1-dependent recycling of LHR is driven by PKA-dependent phosphorylation of APPL1 at S410. To ascertain whether LH stimulation induces phosphorylation of APPL1 in a PKA-dependent manner, and specifically on S410, cells expressing WT APPL1-GFP were treated with LH and immunoprecipitated using a GFP nanobody. Eluates were analyzed by western blot using a phospho-serine antibody and an APPL1 antibody. Immunoprecipitation of APPL1 was only detected in cells transfected with APPL1-GFP A. The phGiven that APPL1-dependent LHR recycling requires cAMP/PKA activation, we determined how APPL1 depletion reciprocally impacts LHR-mediated cAMP signaling. Agonist-induced cAMP production was measured in cells stably expressing FLAG-LHR and transfected with either non-targeting or APPL1 siRNA. There was no effect of APPL1 knockdown on the basal levels of cAMP; however, there was an unexpected increase in LH-induced cAMP levels following APPL1 knockdown A. This iAPPL1 is essential for LHR recycling from the VEE via a mechanism that involves cAMP/PKA activation and phosphorylation of APPL1. In turn, APPL1 negatively regulates LHR signaling. Therefore, we examined whether PKA activation and APPL1 phosphorylation alter cAMP signaling. LHR-expressing cells pretreated with the PKA inhibitor KT5720 showed a partial but significant reduction in the levels of LH-stimulated cAMP production when compared to DMSO-treated cells A, conditThe ability of APPL1 to negatively regulate cAMP levels of VEE-targeted GPCRs, yet positively regulate recycling via PKA, suggests that cAMP signaling occurs from endomembranes. To test this hypothesis, we assessed the proportion of LHR-mediated cAMP generated from intracellular compartments in cells treated with the inhibitor of the large GTPase, dynamin, a protein key in vesicle scission, including clathrin-coated vesicles, from the plasma membrane. Dyngo-4a is a potent dynamin inhibitor that blocks internalization of multiple GPCRs, including LHR and B2AR A McClus. LH-induAPPL1-positive LHR endosomes represent almost half of the total receptor-occupied VEE, when accounting that ∼70% of LHR endosomes are not EEA1 positive C and S3CEndocytic trafficking of GPCRs is recognized as a primary mechanism that cells use not only to define the pattern of cell-surface G-protein signaling but also to generate additional signaling platforms at the endomembrane . Our stuWe previously reported that certain GPCRs are sorted to the VEE, an endosomal compartment with physical and biochemical properties distinct from those of the classic EE or its intermediates. Despite the fact that APPL1 was identified as the only marker of the VEE, its role in LHR function at the VEE was unknown . APPL1 hThe inhibition of recycling by depletion of APPL1 is not due to redirecting LHR from VEEs to EEs. The small increase of LHR to EEA1-positive EEs following cellular depletion of APPL1 is likely a consequence of receptor accumulation and sequestration in endosomes due to the loss of the APPL1 compartment and inhibition of receptor recycling. APPL1 has been recently shown to occupy a small subpopulation of sorting endosomes, and even tubular structures from these endosomes, but these correspond to the morphologically larger EEA1-positive EEs, which are distinct from the smaller VEEs, and the role of APPL1 in the post-endocytic sorting was not assessed . We showDespite the divergent endosomal sorting of LHR and B2AR and their differential requirement for APPL1, the recycling of both GPCRs is regulated via their activation of Gαs-cAMP-PKA pathway. Interestingly, cAMP/PKA activation exerts opposite functions by phosphorylating distinct targets for these two GPCRs. Sequence-dependent recycling of B2AR is negatively regulated via PKA-dependent phosphorylation of the receptor C-tail . By contOur data also demonstrate a strong bidirectional relationship between GPCR signaling and endocytic trafficking. Not only does LHR-mediated cAMP signaling drive recycling via phosphorylation of APPL1, but APPL1 also exerts negative feedback on ligand-induced cAMP signaling from LHR. APPL1 is a known adaptor of signaling molecules, propagating signaling pathways such as AKT, GSK3-β, p38, and AMPK  rather tThere has been an increasing number of reports that GPCRs can continue, or reactivate, G-protein signaling from endosomes , althougIt is increasingly apparent that endocytic trafficking of GPCRs is critical to resolve, or decode, complex cellular signaling at a spatial level. The role of the endosomal network and machinery in regulating GPCRs is only just emerging. However, our discovery that endosomal heterogeneity and inter-endosomal communication are essential for coordinating GPCR signaling and sorting will enable the construction of cellular models that integrates GPCR signaling across multiple endosomal compartments, models that could provide insight into complex disease and therapeutic strategies for this highly significant superfamily of receptors.The antibodies used were: mouse anti-FLAG ; rabbit anti-APPL1 ; rabbit anti-EEA1 ; mouse anti-GAPDH (Millipore); rabbit anti-p42/44 ERK and phospho-p42/44 ERK ; rabbit anti-phosphoserine (Millipore); goat anti-rabbit and anti-mouse AlexaFluor 488, 555, 568, and 647 (Thermo Fisher); and goat anti-rabbit and anti-mouse horseradish peroxidase (HRP) (Thermo Fisher Scientific). The inhibitors/activators used were: Dyngo-4a (Abcam) at 30 μM (45 min pre-treatment), KT5720 (Abcam) at 10 μM (15 min pre-treatment), and 8-Br-cAMP (Sigma) at 0.5 mM (15 min pre-treatment). LH and follicle-stimulating hormone (FSH)  were used at 10 nM, and isoproterenol  was used at 10 μM. All concentrations of ligands used (LH and FSH at 10 nM and isoproterenol at 10 μM) induce maximal cAMP responses from dose-response curves published previously .FLAG-hLHR, FLAG-hB2AR, FLAG-hFSHR, and GFP-WT APPL1 have been previously described . Nb37-GFFLAG-hB1AR was purchased from Addgene. mCherry-WT APPL1 was generated via subcloning mCherry into GFP-WT APPL1 using SalI and XhoI sites. mCherry-S410A APPL1 and -S410D APPL1 were generated by site-directed mutagenesis  using oligos corresponding to GCAGAGGCACGAGGCCCTGCGGCCAGCAGC and GCAGAGGCACGAGGACCTGCGGCCAGCAGC, respectively. siRNA-mediated knockdown of APPL1 was achieved by transfection of duplex RNA oligos (Life Technologies) corresponding to GACAAGGTCTTTACTAGGTGTATTT. Control cells were transfected with non-sense duplex RNA oligos (AATTCTCCGAACGTGTCACG).2. Primary hESC cultures were established from endometrial biopsies, taken randomly in the cycle, as previously described  and 17α-medroxyprogesterone acetate (MPA) (10−6 M) for 72 hr. For both HEK293 cells and hESCs, transient and stable transfections of DNA were performed with Lipofectamine 2000 (Life Technologies). Transfection of siRNA was performed using RNAiMAX (Life Technologies). For transient expression, cells were assayed 48 and 96 hr post-DNA and -siRNA transfection, respectively.HEK293 cells (ATCC) were maintained in DMEM containing 10% FBS and penicillin/streptomycin (100 U/mL) at 37°C in 5% COescribed . ProlifeFlow cytometry was used to quantitate the internalization and recycling of receptors by measuring the levels of cell-surface FLAG-tagged receptors as described previously . BrieflyReceptor imaging in live or fixed cells was monitored by “feeding” cells with Alexa-Fluor 488- or 555-conjugated FLAG antibodies  in phenol-red-free DMEM prior to agonist treatment. Fixed cells were washed three times in PBS/0.04% EDTA to remove FLAG antibody bound to the remaining surface receptors. Cells were imaged using a TCS-SP5 confocal microscope (Leica) with a 63× 1.4 numerical aperture (NA) objective. Leica LAS AF image acquisition software was utilized. All subsequent raw-image files were analyzed using ImageJ or LAS AF Lite (Leica) to measure endosome diameter size or level of co-localization. Pearson’s colocalization coefficient was calculated for at least 3 regions of interest (ROIs) per cell using the ImageJ plugin JACoP.Immunoprecipitation of GFP-APPL1 constructs was conducted using GFP-Trap agarose beads (ChromoTek) as per the manufacturer’s protocol. HEK293 cells transfected with GFP-APPL1 constructs were washed with ice-cold PBS three times, collected, and homogenized with lysis buffer  for 30 min. Lysates were centrifuged, and the supernatant was incubated for 2 hr with GFP-Trap agarose beads. Beads were washed three times and resuspended in elution buffer . Samples were separated on a 12% SDS-PAGE gel.For measurement of ERK activation by western blot, cells were treated and lysed as described previously . MeasureCells were imaged using a Elyra PS.1 AxioObserver Z1 motorized inverted microscope with a scientific complementary metal-oxide-semiconductor (sCMOS) or electron-multiplying charge-coupled device (EMCCD) camera and an alpha Plan-Apochromat 100×/1.46 Oil DIC M27 Elyra objective (Zeiss), with solid-state lasers of 488 nm, 561 nm, and/or 642 nm as light sources. For live-cell imaging, cells were imaged live for 1 min at a frame rate of 10 frames per second (fps) at 37°C in phenol-red-free Opti-MEM supplemented with HEPES (Life Technologies). ZEN Lite image acquisition software was utilized to collect time-lapse movies and analyzed as tiff stacks using the ImageJ plugin Time Series Analyzer. The number of recycling events counted was normalized by cell area. For fixed-cell imaging, cells were prepared as for confocal imaging.Cells were imaged using an Elyra PS.1 AxioObserver Z1 motorized inverted microscope with a EMCCD camera and Plan-Apochromat 63×/1.4 Oil DIC  M27 Elyra objective (Zeiss) with solid-state lasers of 488 nm and 561 nm as light sources. ZEN lite software was used for both acquisition of z stacks (5 phases and 3 rotations grating) and reconstruction. Quality of raw and reconstructed data was determined using the ImageJ plugin SIM check . Cells wThe study was approved by the National Health Service National Research Ethics-Hammersmith and Queen Charlotte’s and Chelsea Research Ethics Committee (1997/5065). Pre-menopausal women were recruited from the Infertility Clinic at Hammersmith Hospital, Imperial College London NHS Trust. Written informed consent was obtained from all participants in accordance with the guidelines in the Declaration of Helsinki 2000. Samples were obtained using a Wallach Endocell sampler , starting from the uterine fundus and moving downward to the internal cervical ostium. In total, 8 endometrial biopsies were processed for primary cultures in this study. The average age (±SD) of the participants was 32.5 ± 4.4 years.Data are given as means ± SE. Statistical significance was determined using GraphPad Prism v5 . An unpaired Student’s t test, one-way ANOVA followed by Dunnett post-test, or two-way ANOVA followed by Bonferroni post-test was used when comparing two groups, more than two groups, or at least two groups under multiple conditions, respectively. Differences were considered significant at p < 0.05.S.S., F.G.J.-A., M.A.A., A.O., C.W., and A.C.H. performed experiments. S.L. and J.J.B. provided human endometrial biopsies. S.S., F.G.J.-A., M.A.A., E.R., S.L., J.J.B., and A.C.H. designed research, analyzed data, and wrote the paper.


Giardia duodenalis  is a flagellated protozoan that parasitizes the small intestine and is a common causal agent of zoonotic infections in humans and animals. To assess the genetic diversity and zoonotic transmission potential of G. duodenalis in stray dogs, 159 fecal specimens were collected from dogs in Chengdu, Yaan, and Leshan in Sichuan province, China. Of the 159 fecal samples from stray dogs, 18 (11.3%) were G. duodenalis-positive based on nested PCR amplification of the beta giardin (bg) gene, and the occurrence varied from 1.8% to 35% in different cities. Dog-specific assemblages C (n = 9) and D (n = 9) were identified. The glutamate dehydrogenase (gdh) and triosephosphate isomerase (tpi) genes of all bg-positive isolates were characterized. A total of 16 and 8 isolates were positive for the gdh and tpi genes, respectively. Two novel sequences of the bg locus were detected among genetic assemblage D isolates, and one novel gdh sequence and four novel tpi sequences were identified among genetic assemblage C isolates. Mixed infections of assemblages C and D were also detected. Assemblages A and B, which have high zoonotic potential, were not detected. Our results show that G. duodenalis is prevalent and a cause of diarrhea in dogs in Sichuan province, China. Giardia duodenalis, also known as G. intestinalis or G. lamblia, is an important zoonotic intestinal parasite that infects humans and a variety of domestic and wild animals [G. duodenalis has high potential for zoonotic transmission via water or feces; diarrhea is a major clinical sign of infection. In humans, G. duodenalis can infect immunocompromised hosts [G. duodenalis has been reported in livestock [ animals . G. duoded hosts , such asivestock , 16, 26,ivestock , 20, andivestock , 15, 27;G. duodenalis [There are eight known genotypes (A–H) of odenalis . Assemblodenalis , cattle odenalis , sheep [odenalis , dogs [1odenalis , and catodenalis . Specifiodenalis . Hence, odenalis , 11, 27.odenalis . Assemblodenalis , 20, andodenalis . Assemblodenalis .G. duodenalis infections in dogs in China [G. duodenalis in stray dogs in China. In developing countries, stray dogs are common and have a wide distribution. In this study, nested PCR was used to amplify the beta giardin (bg), glutamate dehydrogenase (gdh), and triosephosphate isomerase (tpi) loci to investigate the prevalence and genotypes of G. duodenalis in stray dogs in Sichuan province, China.Many recent epidemiological studies have reported in China , 19, 27,in China , and Brain China . In Heilin China , 27, zooFrom November 2016 to January 2017, 159 fecal specimens were collected from stray dogs in shelters in Chengdu, Yaan, and Leshan cities in Sichuan province, China. Forty specimens whose hosts had diarrhea were obtained from Chengdu. Sixty-three and 56 specimens whose hosts did not exhibit diarrhea were obtained from Yaan and Leshan, respectively. All dogs were stray and abandoned and were housed in shelters for at least 2 months. Fecal samples were collected, numbered in plastic containers, and transported to our laboratory in ice packs on the day of collection. These fecal specimens were stored in a 4 °C refrigerator.Fecal specimens (50–100 mg) were removed from each plastic container. DNA was extracted directly from fecal samples using an E.Z.N.A. Stool DNA Kit , according to the protocol recommended by the manufacturer. DNA samples were stored at −20 °C until use for PCR.G. duodenalis was detected by nested PCR amplification of an approximately 530-bp fragment of the bg locus. The bg-positive products were further characterized by amplification of gdh and tpi. The primers for PCR amplification and annealing temperatures for the three genes were obtained from Zhang et al. [g et al. . Each rebg, tpi, and gdh of G. duodenalis available in the GenBank database using BLAST and Clustal X.All positive secondary PCR products were sent to Sangon Biotech Company  for sequencing. Genotypes with mutations, including single nucleotide substitutions, deletions, or insertions, were confirmed by DNA sequencing of at least two PCR products. Assemblages and subtypes were identified by the alignment of the nucleotide sequences with known reference sequences for G. duodenalis genotypes and previously published reference sequences in GenBank, a phylogenetic analysis was performed. A neighbor-joining tree was constructed using Mega 6 based on evolutionary distances calculated with the Kimura 2-parameter model. The reliability of trees was assessed using a bootstrap analysis with 1,000 replicates.To assess the genetic relationships among 2 test was used to compare the infection rates of G. duodenalis at three stray dog shelters in different cities, and differences were considered significant when p < 0.05.The χbg locus. Among positive specimens, 14 were obtained from dogs with diarrhea and 4 from dogs without diarrhea. The occurrence rates in dogs with and without diarrhea were 35.0% and 3.4%, respectively. The infection rates of dogs with diarrhea were highly significantly different from dogs without diarrhea. . Multilocus sequence typing at bg, gdh, and tpi revealed the presence of G. duodenalis assemblages C (13) and D (9). The infection rate in Chengdu was highest , followed by Yaan  and Leshan  based on amplification of the bg locus. The genotypes of all positive specimens are listed in In this study, 18 (11.3%) positive specimens were obtained from 159 fecal specimens by nested PCR amplification of the bg, gdh, and tpi is summarized in A total of 18 positive specimens were identified by nested PCR, and a phylogenetic analysis based on KX164009 with two SNPs (substitution: A/C and C/A), and YA16 was similar to KF958111 with one SNP (substitution: C/A).All isolates obtained from dogs were assemblages C and D, including nine that were assemblage C and nine that were assemblage D . Among tgdh locus, YA16 was similar to EF507635 with two SNPs (substitution: A/G and T/C).Among 16 isolates from dogs, 12 were identified as assemblage C and 4 as assemblage D . The YA1tpi locus . CD18 was similar to KX014801 with two SNPs (substitution: G/A and G/A). CD21 and YA49 were similar to KX014804 with a SNP (substitution: A/G). CD28 was similar to KF993723 with a SNP (substitution: A/C).Eight isolates were identified as assemblage C at the pi locus . Among eG. duodenalis in dogs has been reported in Heilongjiang [G. duodenalis in dogs in Japan [In China, the prevalence of ongjiang , Shanghaongjiang , Guangdoongjiang , and Henongjiang . Four geongjiang . For strongjiang . Assemblongjiang . In totain Japan , Brazil in Japan , Poland in Japan , the Netin Japan , and Engin Japan  are showG. duodenalis in dogs, assemblages C and D, host-specific genotypes, are considered dominant [G. duodenalis has been reported in previous multilocus analyses [tpi, gdh, and bg loci) is widely used for the detection of G. duodenalis co-infection in humans and animals [G. duodenalis-infected feces of the owner.In most studies of dominant , 19, 27.dominant , have zo animals , 27. In  animals  suggestsG. duodenalis in this study. The bg, gdh, and tpi loci varied with respect to PCR amplification rates, consistent with most previous multilocus typing studies of G. duodenalis [G. duodenalis, indicating a minimal role in zoonotic transmission in Sichuan province, China.Multilocus sequence typing was used for the genetic characterization of odenalis , 19, 25.G. duodenalis are present in stray dogs in Sichuan province, China. Zoonotic genotypes (assemblages A and B) were not found, suggesting that these genotypes are not prevalent in stray dogs in Sichuan province, China. Moreover, new subtypes were identified. Nevertheless, G. duodenalis is a prevalent protozoan parasite, and although zoonotic assemblages were not found in stray dogs in this study, potential transmission should not be overlooked. Certain measures should be taken to reduce the possibility of intraspecific transmission.The results obtained in this study demonstrate that genetic assemblages C and D of The authors declare that they have no conflict of interest.


However, data on patients  are controversial, so the debate on the role of hyperuricemia as a causative factor for CVD is still ongoing. Increasing evidence indicates UA as more meaningful to assess CVD in women, even though this aspect needs deeper investigation. It will be important to identify thresholds responsible for UA “biological shift” from protective to harmful effects in different pathological conditions, and according to possible gender-related differences. In any case, UA is a low-tech and inexpensive biomarker, generally performed at patient’s hospitalization and, therefore, easily accessible information for clinicians. For these reasons, UA might represent a useful additive tool as much as a CV risk marker. Thus, in view of available evidence, progressive UA elevation with levels higher than 6 mg/dL could be considered an “alarm” for increased CV risk.Uric acid (UA) is a potent endogenous antioxidant. However, high concentrations of this molecule have been associated with cardiovascular disease (CVD) and renal dysfunction, involving mechanisms that include oxidative stress, inflammatory processes, and endothelial injury. Experimental and  Uric acid (UA) is a known endogenous scavenger, which provides a major part of the antioxidant capacity against oxidative and radical injury . HoweverFor its anti- and pro-oxidant effects, hyperuricemia has been associated with CVD, and its functional role is still controversial. Interestingly, recent data indicate a stronger association between UA and CVD in women than in men. The aim of the present paper is to summarize dietary sources and metabolism of UA, and to discuss available evidence on the role of UA in CVD.Serum UA concentration reflects the interaction of purine intake with diet, endogenous purine metabolism, altered UA excretion  and intestinal degradation ,14.Food intake may significantly affect the development of hyperuricemia. A high intake of red meat and seafood, sugar sweetened drinks, and alcohol increase the risk of hyperuricemia . Most ofAlthough many fruits and vegetables can contain high purine levels, their natural macro- and micro- nutrients in natural ratios and proportions may likely act to optimize the biological advantages of UA natural intake with diet ,19. In fInstead, the diffusion of (1) Western diet, with high fats and fructose content; (2) street-food (SF), rich in saturated fats and poor in fibers, vitamins, and antioxidants; and (3) sedentary habit, which characterizes our actual lifestyle, may favor hyperuricemia. In particular, two studies from Italian researchers showed evidence that SF consumption is associated directly with body mass index, larger waist circumference, higher levels of cholesterol, and UA concentrations, and inversely correlated with high-density lipoproteins cholesterol ,25. MoreIn a recent mice experimental model, the Western diet induced cardiomyocte hypertrophy, myocardial oxidative stress, interstitial fibrosis, and impaired diastolic relaxation, and enhanced activation of the S6 kinase-1 growth pathway and the profibrotic transforming growth factor-β1/Smad2/3 signaling pathway and macrophage proinflammatory polarization . Conversn = 39) evidenced that subjects on MeD showed a statistically significant increase of UA compared either to baseline and to German diet group, which is quite surprising since the German diet is characterized by high amounts of meat and pork, sausages and butter, moderate amounts of whole-meal bread, potatoes, fruit and vegetables, dairy products, fish and eggs, moderate to high amounts of beer, sweet foods, and sugar [The scientific community has become increasingly interested in the Mediterranean diet (MeD) and lifestyle, which includes high fruit and vegetable intake, low meat consumption, use of extra virgin oil and red wine, and daily physical activity. The main reason of this interest has to be attributed to MeD’s favorable cardiometabolic, neuronal, and beneficial health effects when compared with other dietary patterns ,31. The nd sugar .UA has been primarily identified as a powerful antioxidant; therefore, UA elevation in CVD may represent a compensatory mechanism in response to a pro-oxidative and pro-inflammatory status . NonetheA possible difference in gender-related UA effects is intriguing, because although UA concentration is physiologically lower in women than in men, it is emerging to be more related with CVD in women than men ,49,50. Hn = 843, UA cut off corresponding to 5.7 mg/dL; n = 2177, UA cut off 7 mg/dL for males), it does not seem to be an independent risk factors for hard events in both sexes [Moreover, in view of the known association between hyperuricemia and insulin resistance and diabetes, the gender-specific difference in the association of UA with CVD risk may be compatible with the stronger relationship between UA and diabetes in women, given that diabetes confers a greater risk for CVD in women than in men . Accordith sexes . Conversth sexes ,58. Thisth sexes .in vitro data suggest that UA induces monocyte apoptosis through the activation of both death receptor and mitochondrial-mediated pathways, and stimulates mononuclear cells to produce TNFα [Presently, whether UA represents an independent risk factor for cardiovascular events with a direct and causal role or if it is just a marker for an adverse risk profile is still conjectural ,61. UA iuce TNFα . Moreoveuce TNFα .In addition, a new mechanism for urate-induced CVD has been proposed . In partMany other mechanisms may account for the atherogenic role of UA, although a clear causal relationship between UA and CVD has never been confirmed. UA may stimulate vascular smooth cell proliferation, platelet hyper-reactivity and cyclooxigenase-2 activity, and immunity response, whereas it reduces NO availability . In partInterestingly, it has been hypothesized that circulating UA exerts protective effects on endothelial cells from oxidative stress, whereas its entry into the cell favors oxidative stress and reduces NO, evidencing the “shuttle” capacity of UA . In thisHowever, other data confirmed UA protective roles, in view of its antioxidant capacity. In fact, UA intravenous infusion (1000 mg) improves endothelial dysfunction in type 1 diabetes and smokers . InteresIn any case, the identification of UA as a CVD culprit may be useful because UA pharmacological reduction is possible, but clearly effective only in identifying hyperuricemia as a cause rather than a consequence of clinical factors related to CVD and adverse prognosis. There are recent preliminary indications of cardiovascular and renal effects of UA lowering ,74,75. IUA is a low-tech and inexpensive biomarker which is generally performed at a patient’s hospitalization, therefore being easily-accessible information for clinicians. Moreover, UA provides repeatable results, without presenting significant diurnal variations, although meals may affect its concentration, with significant postprandial effects. For this reason, fasting before a withdrawal is always preferable ,78,79. TAvailable data on the role of hyperuricemia as CV risk factor are not definitive . HoweverIncreasing evidence indicates UA as a more meaningful marker to assess CV risk in women. This fact is particularly relevant due to the lack of appropriate biomarkers to assess female CV risk, and the easy availability of this biomarker in clinical practice.In any case, progressive elevations of UA and levels higher than 6 mg/dL should be considered an “alarm” and should induce the clinician to activate a global CV risk reduction program  to improve outcomes, especially in female subjects.


Infection with the human T-cell lymphotropic virus type 1 (HTLV-1), although asymptomatic in most cases, can lead to potentially grave consequences, such as adult T-cell leukemia-lymphoma and HTLV-1-associated myelopathy / tropical spastic paraparesis. Its prevalence varies widely across different populations and geographic regions. A population-based study in the city of Salvador, located in the Northeast region of Brazil, showed an overall prevalence of HTLV-1 seropositivity of 1.7%. Blood borne virus infections are recognized as important hazards for patients and staff in maintenance hemodialysis (MHD) units but most studies focus on hepatitis B, hepatitis C and human immunodeficiency viruses. There are scarce data about HTLV-1 infection in the MHD population. We aimed to determine the prevalence and risk factors for HTLV-1 infection among MHD patients in the city of Salvador-Bahia, Brazil.We conducted a multi-center, cross-sectional study nested in a prospective cohort of MHD patients enrolled from four outpatient clinics. HTLV-1 screening was performed with ELISA and positive cases were confirmed by Western Blot. Factors associated with HTLV-1 seropositivity were identified by multivariable logistic regression.605 patients were included in the study. The overall prevalence of HTLV-1 infection was 2.48% (15/605), which was similar to that of hepatitis B [1.98% (12/605)] and C [3.14% (19/605)] viruses in our sample. HTLV-1 seropositivity was positively associated with age , unmarried status , and history of blood transfusion .The overall prevalence of HTLV-1 infection in a sample of MHD patients was similar to that of other viral infections, such as hepatitis B and C. Our data revealed that MHD patients who are older, unmarried or who have received blood transfusions are at higher risk for HTLV-1 infection. The human T-cell lymphotropic virus type 1 (HTLV-1), the first human retrovirus identified –5. HAM/THTLV-1 infection has become a worldwide epidemic, but studies focusing on population prevalence are scarce and stem mainly from endemic areas . The disIn Brazil, most of the prevalence data have been obtained from serological screening of asymptomatic blood donors. These data show a great variation in prevalence within the country, being highest in the northeast region –23. In nBlood borne virus infections are recognized as important hazards for patients and staff in maintenance hemodialysis (MHD) units but most studies focus on hepatitis B (HBV), hepatitis C (HCV) and human immunodeficiency virus (HIV). Few studies were specifically designed to estimate the prevalence of HTLV-1 infection in MHD patients –30. ThesGiven the high prevalence of HTLV-1 infection in the general population in Salvador-Bahia and the scarcity of data about this infection in the MHD population, we conducted a cross-sectional study nested in a prospective, multicenter cohort of MHD patients, to identify the prevalence and factors associated with HTLV-1 seropositivity in this population.We screened MHD patients enrolled in the Prospective Study of the Prognosis of Chronic Hemodialysis Patients (PROHEMO) cohort. The current phase of the PROHEMO study cohort began in 2010; it includes MHD patients from four  outpatient dialysis units in the city of Salvador, the capital of Bahia, located in the northeast region of Brazil , 33. We Cross-sectional study nested in a prospective cohort study of MHD patients. Information on socio-demographic, laboratorial and clinical variables ware obtained directly from the PROHEMO database, which contains data prospectively collected from patients and medical records. Information on HTLV-1 status was obtained by screening stored blood samples. Between August and October 2011, at the time of blood collection for monthly dialysis labs, additional 10 ml samples of blood were drawn and stored at −20 °C for future analyses. These samples were screened for antibodies against HTLV 1/2, using an enzyme immunoassay (EIA) - Gold ELISA HTLV-1/2 (REM Indústria e Comércio LTDA) and the positive cases were confirmed by a Western Blot (WB) immunoassay .The economic class of the patients  was determined using the classification system of the Brazilian Institute of Market Research (Abipeme) that considers consumer goods, such as refrigerators, televisions and telephones. Patients in classes D and E were categorized as poor or very poor .A variable “history of blood transfusion” was available in the dataset. Participants of the cohort were asked the following question “Did you receive blood transfusion over the past three months?” There were no data on transfusions past this period or on the number of units transfused.All participants signed an informed consent form agreeing with the use of their clinical information and collection and testing of blood samples for HTVL serology for research purposes. The medical director of each participating dialysis unit signed a consent letter approving the study protocol. The Institutional Review Board of the Medical School of Bahia of the Federal University of Bahia approved the study protocol.www.openepi.com), we estimated that 548 patients would be needed to detect a prevalence of HTLV-1 seropositivity in MHD patients in Salvador of 2%, with 95% confidence limits extending 1% on either side.In December 2011, the population of MHD patients of the city of Salvador was estimated to be around 2000 patients . Using the software Openepi  or Fischer’s exact test. Variables that showed an association with HTLV-1 seropositivity characterized by a p value ≤ 0.25 in univariate analyses were selected for a multivariable logistic regression model to identify independent predictors of HTLV-1 seropositivity. We used an automated multivariable logistic regression method called forward stepwise. Briefly, in this method the computer tests the variables one by one for entry into the model, based on the significance level of the score statistic. The variable with the smallest p value is entered into the model (entry criterion: p < 0.05). After each entry, variables that are already in the model are tested for possible removal. The variable with the largest p value is removed , and the model is re-estimated. Variables in the model are then evaluated again for removal. When no more variables satisfy the removal criterion, covariates that are not in the model are evaluated for entry. Model building stops when no more variables meet entry or removal criteria or when the current model is the same as a previous model. A p value ≤ 0.05 in final analyses was considered statistically significant. The analyses were performed using SPSS for Windows version 21.0 and the 95% confidence interval of the HTLV-1 prevalence was calculated using the bootstrap (50 replications) of the STATA, version 12.0.Continuous data were summarized using mean ± standard deviation (SD) or median and interquartile range (IQR); categorical variables were summarized using relative frequencies. Comparisons of continuous variables between seropositive and seronegative groups were performed using the Student’s Sixteen patients were HTLV-1 seropositive by the ELISA technique but one was considered a false positive due to a negative Western Blot. Therefore, 15 out of 605 MHD patients studied had confirmed HTLV-1 infection, for an overall prevalence of 2.48% (95% CI 1.22–3.74%). There were no cases of HTLV-2 seropositivity in our sample.In two out of these fifteen cases (13.3%), the ESRD was attributed to urological complications of HTLV-1 infection, specifically neurogenic bladder leading to obstructive kidney failure. The first case was a 70 year-old female, wheelchair-bound due to HAM/TSP secondary to HTLV-1. She had a well-documented case of neurogenic bladder and was followed by a team of Neurologists and Urologists at an ambulatory clinic specialized in HTLV-1 at the University Hospital of the Federal University of Bahia. She did not have diabetes or hypertension and other causes of ESRD were excluded on clinical grounds. The second case was a 47 year-old man who initially presented to the emergency department of a public general hospital with uremia. He was found to have a distended bladder and bilateral hydronephrosis on ultrasound, with a normal-sized prostate gland. A Foley catheter was placed, but renal failure did not improve and he remained dialysis-dependent. He had no prior history of hypertension or diabetes.Table Figure Table Our initial hypothesis was that the prevalence of HTLV-1 in the MHD population would be greater than that observed in the general population. This hypothesis was based on older literature data from other countries –29, 35 aIn their study, Dourado et al. studied individuals from 1 to 89 years. The prevalence of HTLV-1 infection increased with age, going from 0.3 to 1.1 to 1.1 and 8.4% among those 0–15, 16–30, 31–50 and > 50 years old, respectively. In the present study of MHD patients, the prevalence of HTLV-1 infection also increased with age, going from 1.6% in those 18–30 to 2.9% among those > 50 years old; for every increase in one year of age, we found a 4% increase in the odds of having a positive HTLV-1 serology. A potential explanation for this association is that increasing age provides a greater length of exposure to events that might result in acquiring the virus, such as sexual activity, intravenous drug use or blood transfusion. Since there is no cure for HTLV-1, once acquired, the infection is carried forward over time (cohort effect).The association between HTLV-1 infection and blood transfusion is well established and has been confirmed in several studies using blood donors in different countries –41, inclIn Brazil, however, there is still a considerable need for blood transfusions among patients who are recent on MHD, since many of them start dialysis emergently, without the benefit of pre-dialysis medical care. This was illustrated in a study conducted at the largest public hospital in Salvador , which iIn their population-based study, Dourado et. al showed that the prevalence of HTLV-1 was greater among females, those with lower income, worse living conditions and less education . In the In Brazil, the main etiologies of ESRD are hypertension (35%), diabetes mellitus (30%), chronic glomerulonephritis (12%) and polycystic kidney disease (4%) . Herein,Our study has several limitations. An inherent limitation of cross sectional studies is that associations do not imply causality. We utilized the database of an ongoing multicenter cohort study of MHD in our area and all of our analyses were limited to the variables that were present in their database. We did not have information on sexual practices, living conditions, intravenous drug use or neurologic symptoms. Moreover, our data on blood transfusion was categorical (yes/no) and restricted to the previous three months; we had no information on lifetime exposure to blood products. Finally, our study provides no information about HIV co-infection as none of the four MHD units studied admit HIV positive patients.The prevalence of HTLV-1 infection in MHD patients from four hemodialysis units in northeast Brazil was not higher than that encountered in a population-based study of the same geographic area. HTLV-1 seropositivity in MHD patients was similar to that of other viral infections, such as HBV and HCV. Older age, unmarried status and blood transfusions were independently associated with HTLV-1 infection.


Chemokine (C-C motif) ligand 1 (CCL1) is produced by activated monocytes/ macrophages and T-lymphocytes, and acts as a potent attractant for Th2 cells and a subset of T-regulatory (Treg) cells. Previous reports have indicated that CCL1 is overexpressed in adult T-cell leukemia cells, mediating an autocrine anti-apoptotic loop. Because CCL1 is also known as a potent chemoattractant that plays a major role in inflammatory processes, we investigated the role of CCL1 in the pathogenesis of human T-cell leukemia virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP).CCL1 gene promoter, (4) plasma levels of CCL1 were significantly higher in patients with HAM/TSP than in HTLV-1-seronegative patients with multiple sclerosis and HTLV-1-infected asymptomatic healthy carriers, and (5) minocycline inhibited the production of CCL1 in HTLV-1-infected T-cell lines.The results showed that: (1) CCL1 was preferentially expressed in HAM/TSP-derived HTLV-1-infected T-cell lines, (2) CCL1 expression was induced along with Tax expression in the Tax-inducible T-cell line JPX9, (3) transient Tax expression in an HTLV-1-negative T-cell line activated the The present results suggest that elevated CCL1 levels may be associated with the pathogenesis of HAM/TSP. Although further studies are required to determine the in vivo significance, minocycline may be considered as a potential candidate for the long-term treatment of HAM/TSP via its anti-inflammatory effects, which includes the inhibition of CCL1 expression. All samples were examined in triplicate. TaqMan® real-time RT-PCR assays were performed to quantify the differences in the expression of tax and HBZ mRNA as previously reported [Tax, HBZ, and CCL1 mRNA expression levels were normalized to the expression of human hypoxanthine phosphoribosyltransferase 1 (HPRT1) . Human CCL1 gene-specific assays (Applied Biosystems Hs00171072 m1) were used for CCL1 quantification. All assays were performed in triplicate.To examine the HTLV-1 proviral load (PVL), quantitative PCR was performed using 100 ng of genomic DNA , −401 to +60, −281 to +60, and −221 to +60, PCR was carried out using the appropriate primer sets with restriction sites . The expression vector pCG-Tax and the control vector pCG-BL were kindly provided by Dr. J. Fujisawa .The following pGL3-based plasmids were constructed for luciferase reporter gene assays. To amplify the 5 cells per well, and were transfected with 1 μg of the reporter plasmid and 30 ng of the Renilla luciferase control plasmid phRL-TK, with or without 1 μg of the Tax expression plasmid pCG-Tax, using Lipofectamine LTX with PLUS reagent . Firefly and Renilla dual luciferase assays were performed 48 h post-transfection as described previously [Renilla activity.To compare the relative promoter activity, the CD4+ T-cell leukemia cell line Jurkat was used for dual luciferase assays. Jurkat cells were seeded on 12-well plates at 2.0 × 10eviously . Each exp < 0.05 were considered statistically significant.To test for significant differences among the four different groups of subjects, i.e., HAM/TSP, HTLV-1-seronegative MS, HCs, or NCs, the Kruskal-Wallis test was employed. For multiple comparisons, we used Sheffe’s F to analyze statistical differences. Correlations between variables were examined using Spearman’s rank correlation analyses. The results are presented as the mean ± SD where applicable. Values of CCL1 mRNA was preferentially expressed in HTLV-1-infected human T-cell lines derived from patients with HAM/TSP (4 out of 4 tested), compared with HTLV-1-transformed T-cell lines (1 out of 3) and ATL cell lines (1 out of 4). Real-time PCR analysis revealed that the expression levels of the viral RNAs tax and HBZ in these HAM/TSP-derived cell lines were relatively high, in comparison with the levels in ATL cell lines  to the culture medium . As shown in Fig. 2 induced the expression of Tax at the RNA and protein levels  of these T-cell lines were not associated with the status of HTLV-1 infection Fig. . HoweverCCL1 gene is responsible for the expression stimulated by Tax, the Firefly luciferase gene was used to monitor the activity of the CCL1 gene promoter - and the cAMP response element-binding protein (CREB)-binding sites were essential and sufficient for CCL1 transactivation by HTLV-1 Tax.To examine whether the 5′-flanking sequence of the human ter Fig. . For thitax mRNA expression in PBMCs . Also, there was no statistical correlation between plasma CCL1 levels and the disease severity  of HAM/TSP patients.To investigate whether CCL1 expression is associated with the in vivo pathogenesis of HAM/TSP, we performed ELISAs to measure the plasma levels of CCL1 in 31 patients with HAM/TSP, 13 HTLV-1-seronegative patients with MS, 19 HCs, and 10 NCs Fig. . We obseIt has been reported that minocycline, a widely used tetracycline antibiotic, has neuroprotective properties against excitotoxicity in several experimental models of neurological diseases by inhibiting the activation and proliferation of microglia , 26. TheAlthough neurological symptoms of HAM/TSP are certainly progressive and can lead to a deterioration in the quality of life, no ideal therapeutic strategy has been shown to significantly modify the long-term disability associated with HAM/TSP. It is therefore particularly important to develop an effective therapy to improve the long-term prognosis of HAM/TSP. Previous reports have indicated that CCL1 is one of the specific chemoattractants that induce cytokine secretion and direct the trafficking of immune cells that are more likely to cross the blood-brain barrier, enter the CNS, and attract other cells including pro-inflammatory virus-specific CD8+ cells, resulting in bystander damage of CNS tissue . MeanwhiCCL1 gene promoter by acting concurrently on AP-1- and on CREB-binding sites. More importantly, we also found that CCL1 plasma levels were significantly higher in patients with HAM/TSP than in HTLV-1-seronegative patients with MS and HCs, and CCR8 was expressed at higher levels in Tax-positive PBMCs than those of Tax-negative PBMCs derived from HAM/TSP patients. It is noteworthy that there was no correlation between serum CCL1 levels and HTLV-1 PVL or tax mRNA expression in PBMCs, and there was no statistical significance between plasma CCL1 levels and the disease severity  of HAM/TSP patients, suggesting that the plasma CCL1 levels of HAM/TSP patients were not reflected by changes in levels of HTLV-1 PVL or tax mRNA expression in the systemic circulation. This is probably because other factors may also be affecting the increased plasma levels of CCL1 and clinical manifestations of HAM/TSP patients, or systemic circulation does not reflect the tissue microenvironment at the spinal cord. Nonetheless, our data suggest that the CCL1-CCR8 axis may play a role in the pathogenesis of HAM/TSP. Indeed, Enose-Akahata et al. (2012) have reported that minocycline directly inhibited activated mononuclear phagocytes (MPs) and that the downregulation of MP function can modulate CD8+ T cells  function in HAM/TSP patients [Our data showed that CCL1 was preferentially expressed in HTLV-1-infected T-cell lines derived from patients with HAM/TSP, whereas its sole receptor CCR8 was broadly expressed in both HTLV-1-infected and non-infected T-cell lines. Interestingly, high levels of CCL1 were detected in the culture supernatants of HTLV-1-infected T-cell lines derived from patients with HAM/TSP (3 out of 4 tested), whereas it was not detectable in any of the other cell lines tested except for the HTLV-1-transformed C5MJ cell line. Experiments using JPX-9 cells, which is a subline of the Jurkat HTLV-1-negative human T-cell leukemia cell line that expresses biologically active Tax protein under the control of the metallothionein promoter, showed that CCL1 was expressed exclusively in Tax-positive cells, suggesting that CCL1 expression was driven exclusively by HTLV-1 Tax at the single-cell level likely because of the Tax transactivation function. Furthermore, when HTLV-1-uninfected cell lines  were incubated with 10 U/ml of IL-2, the levels of CCL1 expression were unaffected. Thus, a Tax-dependent mechanism seems more likely. Indeed, our reporter gene assays showed that Tax transactivates the patients . BecauseThere is growing evidence that certain antibiotics exert their clinically beneficial effects not only by killing or inhibiting the growth of bacterial pathogens, but also by their anti-inflammatory or immunomodulatory activities . For insIn conclusion, we demonstrated that CCL1 was preferentially expressed in HTLV-1-infected T-cell lines derived from patients with HAM/TSP. Higher CCL1 plasma levels were found in patients with HAM/TSP in comparison with HCs and HTLV-1-uninfected patients with MS. Moreover, minocycline inhibited the production of CCL1 in a dose-dependent manner in HTLV-1-infected T-cell lines. Taken together, our results show that CCL1 may be a key molecule in the pathogenesis of HAM/TSP as well as a potential target for immunotherapy. Therefore, further analyses must be performed in CSF samples to confirm the significance of minocycline.


Low-density lipoprotein receptor-related protein-1 (LRP1) plays multifunctional roles in lipid homeostasis, signaling transduction, and endocytosis. It has been recognized as an endocytic receptor for many ligands and is involved in the signaling pathways of many growth factors or cytokines. Dysregulation of LRP1-dependent signaling events contributes to the development of pathophysiologic processes such as Alzheimer’s disease, atherosclerosis, inflammation, and coagulation. Interestingly, recent studies have linked LRP1 with endothelial function and angiogenesis, which has been underappreciated for a long time. During zebrafish embryonic development, LRP1 is required for the formation of vascular network, especially for the venous development. LRP1 depletion in the mouse embryo proper leads to angiogenic defects and disruption of endothelial integrity. Moreover, in a mouse oxygen-induced retinopathy model, specific depletion of LRP1 in endothelial cells results in abnormal development of neovessels. These loss-of-function studies suggest that LRP1 plays a pivotal role in angiogenesis. The review addresses the recent advances in the roles of LRP1-dependent signaling during angiogenesis. A . A 10). 2-macroglobulin (α2M), apoE, and matrix metalloproteinase 9 (MMP9)] may activate cytoplasmic pathways in an LRP1-dependent manner  polymerases (PARPs) play a pivotal role in DNA repair, apoptosis, chromatin modulation, and cell cycle regulation. They catalyze the transfer of ADP-ribose units from NAD+ to acceptor proteins or on PARP itself . PARP inPARP-1 promotes angiogenesis through its promoting role in cell cycle progression. During hypoxia-induced angiogenesis, two events happen with PARP-1, one is an increase in PARP-1 enzymatic activity and the other is its translocation out of the nucleus. It is still a puzzle whether the change in PARP-1 activity is linked with its nuclear export. It is also unclear whether PARP-1 dissociation from LRP1-ICD is required for the nuclear export of LRP1-ICD. The last 33 amino acids within LRP1-ICD are responsible for PARP-1 interaction, where a phosphorylation site for PKA and a dileucine motif are located. However, the effect of PKA and dileucine motif on their interaction and nuclear export remains to be determined.i to activate phosphatidylinositide 3-kinase and endothelial nitric oxide synthase (eNOS) in order to regulate the integrity of adherens junction and dynamic assembly of focal adhesion complexes. On the other hand, S1P2 and S1P3 can couple to multiple G proteins such as Gi, Gq/11, and G12/13. Although S1P has been detected inside of cells, the exact role of intracellular S1P remains elusive.Sphingosine-1-phosphate is generated from sphingosine, the backbone of sphingolipids on cell membrane. It is an important signaling molecule in many cellular processes including cell growth, anti-apoptosis, and cell migration –54. The i inhibition, which is suggested by the observation that pertussis toxin restores the inhibitory effect of S1P on PDGF-induced cell migration. Moreover, in HUVECs, LRP1 promotes the inhibitory effect of S1P on PDGF-BB-induced Gi activation. It indicates that LRP1 plays a key role in the cross talk between S1P and PDGF-BB signaling and, likely, the final outcome of endothelial migration and angiogenesis. However, it remains a puzzle how LRP1 promotes the inhibitory effect of S1P signaling, specifically, the decrease in Gi-GTP level. One reasonable explanation is that LRP1 might interact with mediators of S1P/Gi pathway, which remains to be identified. It is also intriguing to determine whether LRP1-dependent endocytosis is required for this crosstalk of S1P and PDGF-BB signaling pathways.Mice with LRP1 depletion in embryo proper display dramatic vascular defects, including interrupted endothelial integrity, a thin and disorganized smooth muscle cell layer, and an embryo with extensive hemorrhage . Mechanis activation, resulting in the upregulation of cAMP level, and subsequently the activation of PKA and eNOS , and inhibitor PAI-1 play a requisite role in pericellular proteolysisis. Upon activation, uPA initiates a series of signaling events to modulate cell adhesion, migration, and survival. Once PAI-1 forms a complex with uPA and uPAR, LRP1 binds to this complex and leads to a decrease of uPAR steady-state level –63. Recein vivo studies are needed for fully understanding the significant roles of LRP1 in VEGF–VEGFR2–uPAR signaling at pathophysiologic settings. In addition, the role of LRP1 has only been tested with its internalization inhibitor RAP, which also blocks ligand binding of other LDLR family members (Low-density lipoprotein receptor-related protein-1 also forms a multireceptor complex with VEGFR2, β1-integrin, and uPAR on the cell membrane of ECs and targets this multiprotein complex for endocytosis (Mechanism #1 in Figure  members , 69. Morin vivo pathophysiologic experiments is necessary for the dissection of accurate roles for each signaling pathway in different angiogenic models or pathological conditions of vascular growth. These studies will surely provide useful insights about how to design potent and specific LRP1-based therapeutic strategies for angiogenesis-related diseases.The role of LRP1 in angiogenesis has just emerged in recent years. Given that LRP1 facilitates the endocytosis of many ligand–receptor complexes and is involved in growth factor or other cytokine-dependent signaling in different pathophysiologic conditions, it is not surprising that various signaling pathways are involved in LRP1-regulated EC growth, migration, and angiogenesis. In multiple angiogenic models, different output of LRP1 loss of function is likely a balanced effect of intricate signaling cascades mediated by LRP1 in ECs as well as in response to different microenvironment settings. Besides the aforementioned signaling pathways that are regulated by LRP1, other pathways are likely involved as well. For example, mouse embryonic fibroblasts or neuronal cells with a knockin mutation of LRP1’s NPxY motif that is responsible for β1-integrin interaction display impaired migratory capability . Given tAll authors listed have made substantial, direct, and intellectual contribution to the work and approved it for publication.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.


Mutations in an enzyme involved in protein degradation affect a signaling pathway that stimulates the development of the digestive tract. Related research article Galeone A, Han SY, Huang C, Hosomi A, Suzuki T, Jafar-Nejad H. 2017. Tissue-specific regulation of BMP signaling by Drosophila N-glycanase 1. eLife6:e27612. doi: 10.7554/eLife.27612NGLY1 in patients with an inherited disorder  signaling is disrupted in cells that will give rise to portions of the digestive tract.Now, in eLife, Hamad Jafar-Nejad and colleagues at Baylor College of Medicine and the RIKEN Global Research Cluster – including Antonio Galeone as first author – report new insights into the role of this enzyme in the development of the fruit fly, nogaster . Fruit fPngl. This leads to defects in BMP signaling in two layers of tissue that are required for the digestive system to form properly (The BMP pathway is of particular interest because it regulates many aspects of development in both fruit flies and mammals . Cells sproperly .NGLY1 gene. Whether patients have similar alterations in BMP signaling within specific cells or tissues remains to be determined, but this study will help to inform future investigations.This study is the first to identify a specific signaling pathway that is disrupted in the absence of the Pngl enzyme and may provide insight into the underlying causes of some of the symptoms typically seen in patients with mutations in the Pngl mutants are due to defects in BMP signaling, suggesting that the Pngl enzyme also affects other proteins and pathways. Direct examination of the N-glycans attached to Dpp and other proteins in wild type and Pngl-deficient tissues will begin to address some of these questions.The findings of Galeone et al. raise a number of questions regarding how this enzyme works. An enzymatically inactive version of Pngl did not rescue specific digestive system defects, suggesting that Pngl activity is important for BMP signaling. However, it is not known whether the enzyme acts directly on Dpp to affect its ability to form homodimers. It is also possible that the loss of Pngl could affect BMP signaling indirectly, by influencing how other proteins within these cells are modified or degraded. BMP signaling within the affected cells may be uniquely sensitive to disruptions in the normal systems that regulate proteins at this stage of development. Additionally, not all phenotypes within the Pngl results in tissue- and stage-specific alterations in the BMP signaling pathway and provides a new lens through which we can begin to dissect how it influences other systems.Finally, studies on the role of NGLY1 in other organisms suggest that this enzyme may have roles beyond the removal of N-glycans on proteins destined for degradation . There i


Candida. Actually, issues with Candida infections and fungal resistance compromise the use of Poly(methyl-methacrylate) (PMMA) as dental material. The challenge remains to turn PMMA into an antifugal material, which can ideally affect both sessile (attached) and planktonic (free-floating) Candida cells. We aimed to tackle this challenge by designing PMMA-UA composites with different UA concentrations (3–12%). We studied their physico-chemical properties, the antifungal effect on Candida and the cytotoxicity toward human cells. We found that UA changes the PMMA surface into a more hydrophilic one. Mainly, as-preparation composites with ≥6% UA reduced sessile Candida for >90%. After six days, the composites were still efficiently reducing the sessile Candida cells (for ~70% for composites with ≥6% UA). Similar results were recorded for planktonic Candida. Moreover, the inhibition zone increased along with the UA concentration. The antifungal effect of UA was also examined at the surface of an UA-loaded agar and the minimal inhibitory concentration (MIC90) was below the lowest-studied 0.0125% UA. Furthermore, the embedded filamentation test after 24 h and 48 h showed complete inhibition of the Candida growth at 0.4% UA.Undecylenic acid (UA), known as antifungal agent, still cannot be used to efficiently modify commercial dental materials in such a way that this affects  Candida is among the four fungal species that are mostly causing these mortalities [Candida species, one of the most found in clinical cases is Candida albicans (C.a.) [C.a. [Fungal infections are recognized as one of the major health issues with about 1.2 billion people suffering from it (as reported in 2012) . Candidas (C.a.)  and thus.) [C.a. ,14,15.C.a. pathogenicity have been known for a long time, the current state-of-the-art clinical treatments, i.e., mainly the treatment with echinocandins and fluconazole, still cannot combat candidiasis  [C.a. [C.a. biofilms composed of multiple cell types  enclosed in an extracellular matrix [C.a., the mostly studied ones, which are also recognised as the most critical ones, are the following two: (i) the adherence of yeast-form cells to a surface and (ii) the ability to switch between yeast and hyphal growth forms (dimorphism) [C.a. cells, and inhibit both the adherence of the yeast-form cells to a surface and the yeast-to-hyphae transition [Although issues with the ns ~50%) . This is%) [C.a. . With thr matrix . In factorphism) ,17. Theransition ,13,17,18C.a. were not known until one recent detailed study in that direction, which found that UA reduces the hypha-to-yeast ratio (even at 1 millimolar), causes the deformation of the surface of C.a. cells (at one millimolar) and inhibits numerous other factors, such as hyphal formation, adhesion, mitochondrial activity, cell proliferation, transcriptional regulation of the cell membrane virulent factors, and biofilm formation [C.a., it is of interest to use UA as a filler in polymers, which are often used in implants for medical applications, especially in dentistry. In that respect, the main challenge is to achieve that UA, which is incorporated into such a polymer, gets released in the amount sufficient to show an ultimately simultaneous anti-Candida effect on both sessile and planktonic cells.One of the fungicidal substances known for decades for its potential to combat fungus is undecylenic acid (UA) . The medormation . Knowingw/w) were studied and compared to the PMMA only (sample 0% UA). To study the influence of UA on the physico-chemical properties of the composites’ surface, the surface of the composites was characterized by Fourier transform infrared spectroscopy (FTIR), by measuring the water contact angle and by high resolution scanning electron microscope (HR SEM). In order to evaluate the potential use of PMMA-UA composites as a fungistatic and fungicidal materials, we examined the morphology of sessile C.a. cells on the composites’ surfaces by HR SEM and measured at two timepoints : the inhibition zone, the percentage of metabolically active both sessile and planktonic C.a. cells, and their toxicity towards human cells. Moreover, we investigated the effect of UA on the morphology and growth of sessile C.a. cells in embedded conditions, as well as after the incubation of C.a. (at two different concentrations) with agar loaded with different concentrations of UA to find the minimal inhibitory concentration (MIC).In the present study, we developed composite materials by incorporating UA into a commercial cold-polymerized acrylic resin Poly(methyl-methacrylate) (PMMA) widely used in biomedical applications, especially in dentistry . The pro−1 region for its antisymmetric vibrations, and in the 1350–1450 cm−1 region for its symmetric vibrations [−1 and 908 cm−1), =C–H (at 3078 cm−1) and O–H (at 1411 cm−1). Both characteristic C=C peaks of UA were evident in PMMA-UA composites, confirming the presence of UA in the composites. Nevertheless, the =C–H bend of UA was visible in the composites with 9% and 12% of UA only as a week peak shifted towards lower wavelengths (3074 cm−1), while it was not seen in composites with 3% and 6% of UA. This indicates the interaction of the unsaturated UA tail with PMMA, probably via hydrogen bonding, because C=C peaks were clearly visible. However, the O–H peak of UA at 1411 cm−1 was not observed in the composites, suggesting the interaction of this part of the carboxylic group with PMMA. Moreover, a broad peak with the appearance of multiple peaks in the 3300–2500 cm−1 region (which corresponds to both O–H and various C–H vibrations) was observed in both PMMA and UA. However, the CH3 peak is more characteristic of PMMA (see peaks at 2985 cm−1 and 2964 cm−1), while CH2 is more characteristic of UA, due to a long C11 chain (see peaks at 2927 cm−1 and 2854 cm−1) [Firstly, PMMA-UA composites were studied by FTIR by comparing the spectra of native components (UA and PMMA) with composites containing different UA concentrations . As expebrations . However54 cm−1) . All fouThe wetting properties of the composites were studied by measuring their water contact angle. It has to be noted that all studies were done on the native (non-polished) composites’ surface and hence, there are no parallel lines and cracks characteristic for polished PMMA . The reIn fact, besides the chemical groups at the materials’ surface, other factors, such as the surface topology, influence the hydrophilic/hydrophobic appearance of that surface. For instance, the surface with convex structures  would appear as hydrophobic , while the smooth non-structured surface behaves as hydrophilic. Therefore, we examined the surface of the studied composites by HR SEM. In fact, it has been often reported that a polished PMMA surface is either rough or contains voids due to the lines and cracks from the polishing process ,27,28. OC.a. Particular attention was given to the composites with zero percent and three percent of UA, because of the above observed relatively small differences in both the water contact angle  firstly grow in the “valley” by leaving “protrusions” unoccupied  were firstly found in the “valley”  not duemolar UA , suggestst cells .C.a. cells could be seen on the surface of the composites with 6%, 9% and 12% of UA  prevent the attachment of C.a. cells and (b) have fungicidal effect, because the rare sessile C.a. cells were often dead.Moreover, rare 2% of UA a–c. ThisC.a. cells. After 48 h of incubation at 37 °C, the inhibition zone comprising the disk and the clear region (where C.a. growth was inhibited) was measured for the different composites after their preparation (T0) and six days after their preparation (T6). The results , where a disk of the studied material is placed on the surface of an agar plate seeded with microorganisms, here  results  showed aC.a. cells, which were attached to the surface of the studied materials, we performed an XTT test, which measures the number of metabolically active C.a. cells. The XTT test was performed after the composites’ preparation  and six days after that (marked as T6). The results given in C.a. cells on the surface of the studied PMMA-UA composites (as compared to the surface of native PMMA). We found that the percentage of metabolically active C.a. cells was less than five percent on composites with 9% and 12% of UA as compared to native PMMA for both studied timepoints, i.e., T0 and T6. In other words, the number of metabolically active C.a. cells was reduced by more than 95% in composites with 9% and 12% of UA as compared to native PMMA for both timepoints. On the surface of the composites with six percent of UA, 3.7% and 29.6% of metabolically active C.a. cells were measured for T0 and T6, respectively; while on composites with three percent of UA, the corresponding values were 59.4% and 68.6%, respectively . Overall, the obtained XTT results showed a considerable reduction of the number of metabolically active C.a. cells on the surface of PMMA-UA composites in both T0 and T6. This reduction was more than 95% on the composites: with ≥6% UA in T0 and with ≥9% UA in T6. Therefore, the developed PMMA-UA composites showed an antifungal surface activity towards C.a. sessile cells at an early stage of colonisation (after 24 h), which has not been previously observed in composites of acrylic-denture liners with UA, which was explained by probably low concentrations of the released UA [C.a. cells measured on the composites’ surfaces are in agreement with the above given HR SEM observation of the PMMA-UA surfaces, where very few C.a. cells were observed, often deformed and with loss of their cell integrity  and this is reduced in the presence of cell media and C.a. cells. In order to study this, we performed an XTT test on the supernatant, which was above the surface of the composites during 24 h incubation with C.a. cells. The obtained results (C.a. cells. This could be a consequence of the opposing effects of: (a) an expected lower drag-to-cell ratio (drug is here UA) in medium than on the surface of the corresponding composites, and (b) the previously reported lower drag resistance for the planktonic as compared to the sessile C.a. cells at the comparable cell concentration [C.a. cells, by reducing the number of metabolically active both sessile and planktonic C.a. cells. Therefore, we plotted in C.a. cells, which include both the sessile and the planktonic C.a. cells, as a function of the UA concentration. We found that the percentage of the total metabolically active C.a. cells was less than five percent for composites with 9% and 12% of UA for both timepoints . On the other hand, for composites with 3% and 6% of UA, the percentage of the total metabolically active C.a. cells was 68.6% and 5.2%, respectively, after T0 . This also confirms the above given fungicidal activity of the here developed PMMA-UA composites.Beyond the anti- results b showed ntration ,33. SurpC.a. planktonic cells was also studied on agar containing different concentrations of UA . Firstly, after the incubation (for 24 h at 37 °C) of C.a. cells at a concentration of 106C.a. cells/mL, we measured the absorbance of the supernatant containing the planktonic C.a. cells at 620 nm, A620. The results expressed as average percentages of A620 readings compared to control (0% UA) are given versus UA concentration in agar in w/w) in agar was sufficient to reduce more than 90% of the C.a. planktonic cell growth, i.e., 0.0125% was already the concentration above the one necessary to reduce C.a. growth of 90% with respect to the control  for 90% growth reduction, i.e., MIC90). Similar results were obtained with the XTT test done on the same suspensions  by dividing the total mass of UA in the agar substrate by the number of incubated C.a. cells. The so-obtained results , we performed the same experiment with ten times higher concentration of C.a. cells (107C.a. cells/mL). The results . However, even the lowest UA concentration in agar of 0.0125%  was higher than MIC for 50% growth reduction (MIC50). Thus, only a limit to what MIC90 could be was observed. However, the value for a limit below which is MIC90 found here  is much lower than the 256 μgUA/mL, which was previously reported for planktonic C.a. cells incubated at 107C.a. cells/mL [C.a. growth in-between samples with the similar drag-to-cell ratios  was typically higher at higher C.a. cells’ concentration (107C.a. cells/mL). This is in agreement with the previous reports that the resistance of C.a. cells to drags increases with a higher cells’ concentration [C.a. cells.The effect of UA on the growth of pensions . Since w results b showed  results c,d showecells/mL . We haventration ,33. ThesCandida effect of UA on both sessile and planktonic C.a. cells, the only study technique that gave us information on the C.a. cell type was HR SEM  would be toxic to human cells. Therefore, in order to assess the effect of PMMA-UA composites as a final material, we performed a preliminary cytotoxicity study by using the 3--5-(3-carboxymethoxypenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) test according to the previously reported method [0 and T6) with respect to composites preparation. The results , which was even more pronounced for the composite with nine percent UA . Moreover, even at T0, meaning zero days after composite preparation and after 24 h immersion of composite-discs in the medium, there was release of UA from composites with greater than six percent, which was sufficient to affect cells. Therefore, composites with 9% and 12% of UA cannot be used as antifungal materials in or on patients due to their toxic effects on human cells . However, based on the here-obtained values for cells viability in the preliminary toxicity test, PMMA-UA composites with UA concentrations of up to six percent look still promising for such applications.The above given study demonstrates that the developed PMMA-UA composites and UA itself are efficient anti-d method . The tes results  showed aC.a. strain, inoculum concentration, temperature and time of incubation, planktonic or sessile cells, etc.). McLain et al. reported a sevenfold reduction of the C.a. germ-tube formation without an effect on the growth rate in the presence of 10 μM UA (as compared to control) (C.a. strain ATCC 28367 at concentration 106C.a. cells/mL incubated at 39 °C for two hours) [C.a. cells were yeast form [C.a. cells to change their form was suppressed, the adherence of C.a. cells and their growth were not inhibited. However, one other study reported the reduction of a C.a. biofilm formation, but a higher number of hyphae on Coe Soft as compared to the PMMA surface (C.a. strain ATCC 90028 at concentration 107C.a. cells/mL incubated at 37 °C for 24 h) [C.a. biofilms (48 and 72 h) by reducing both the cell counts and their metabolic activity, but not sufficient to influence C.a. at an early stage of the colonization, i.e., 24 h (C.a. strain ATCC 90028 at concentration 107C.a. cells/mL incubated at 37 °C for 24–72 h) [C.a. planktonic cells growth at concentration of released UA of 256 μg/mL; but at the same concentration of released UA, the fungistatic effect on C.a. growth was shown only until eight hours [So far, there have been reported only a very few studies of one commercial acrylic denture liner , which according to the authors contain UA  . In the ast form . Althoug24–72 h) . The samht hours . None ofCandida materials by incorporation of UA into commercial PMMA, which allowed UA to be released in a sufficient amount to show simultaneously the inhibition of the adherence at early stage of the colonization (24 h), morphogenesis (yeast-to-hyphae transition) and growth of both sessile and planktonic C.a. cells. Therefore, we here present several pieces of evidence that novel PMMA-UA composites have strong fungistatic and fungicidal anti-Candida effect. We have also shown that beyond fungicidal effect, UA at larger concentrations (as in composites with ≥9% of UA) can be cytotoxic for human cells, and thus, care has to be taken when UA is used in or on patients.Here, we have developed new anti-C.a., thanks to the development of novel fungicidal and/or fungistatic materials in such a way that a sufficient amount of the active antifungal component can reach both sessile and planktonic fungal cells.By taking into consideration global health issues with fungal infections ,2,3, thiw/w) of UA in the final composite materials. The so-obtained composites were prepared in the different form according to the tests’ requirements either as free-standing solid disks  or as solid disks on the bottom of the wells in the 24-well plates (for biofilm formation and XTT assay). In all experiments, the samples with 0% of UA were used as controls. For all experiments, the as-prepared composites were sterilized under the UV-C lamp for 15 minutes prior to any study.Commercial Triplex Cold  components, solid  and liquid  were mixed according to the manufacturer’s instructions (the mixing ratio corresponding to 13 g of solid on 10 mL of the liquid component). Undecylenic acid (UA)  was physically incorporated into the polymer to obtain 3%, 6%, 9% and 12% . Transmittance from 4600 cmThe water contact angle on the surface of the composites was measured by the sessile drop method  using double distilled water . All measurements were performed minimum in triplicates. C.a. ATCC90028 strain . C.a. stock was kept at −80 °C and after recovery, kept on Sabouraud 4% Glucose Agar  and stored at 4 °C during the experiments. For the Kirby–Bauer test, the determination of the minimum inhibitory concentration and the filamentation assay: the strain was sub-cultured on SGA for 24 h at 37 °C and an inoculum was prepared from freshly grown colonies on SGA at a concentration of 106C.a. cells/mL in 0.9% sterile NaCl . For HR SEM analysis, the determination of the minimum inhibitory concentration and XTT assay: an inoculum was adjusted to a concentration of 106C.a. cells/mL in RPMI 1640 medium .In this study, we used C.a. (at concentration 106C.a. cells/mL) placed on the surface of the composite disks were incubated at 37 °C for for 24 h. After the incubation, the C.a. suspension was removed, and the composite discs were washed with sterile PBS and dried naturally. After drying, the composite discs were coated with 3 nm thick Iridium coating  and as such studied by high resolution Scanning Electron Microscope, HR SEM  operating at an acceleration voltage of 3 kV.Sample preparation: 100 μL of C.a. cells (at concentration 106C.a. cells/mL) were spread onto Yeast Extract Peptone Dextrose agar  plates (Petri dishes filed in with YPD-agar). The studied PMMA-UA composite disks were placed on the YPD-agar plates seeded by C.a. and incubated at 37 °C for 48 h. After incubation, the inhibition zones were obtained by measuring the diameter, which include the disk (dimeter 20 mm) and the clear zone with inhibited C.a. growth (minimum of 5 measurements per disk and a minimum of 3 disks per UA concentration were studied). Kirby–Bauer test was performed at two timepoints: after the composites’ preparation  and six days after the preparation of the composites (T6).H-tetrazolium-5-carboxanilide) reduction assay, the XTT  saturated solution at 0.5 g/L was prepared in sterile PBS , sterilized by filtration using a 0.22 μm pore-size filter, aliquoted into working volumes, and stored at −20 °C when not used. The stock of the XTT solution was thawed before every assay, and the menadione  solution previously prepared in the acetone as a 10 mM stock solution was added to the XTT to have a final menadione concentration of 1 μM. The so-obtained solution will be referred to as XTT/menadione.The preparation of XTT/menadione solution: For the XTT -2C.a. incubation with composites for XTT assay: 400 μL of a C.a. inoculum (at a concentration 106C.a. cells/mL in RPMI 1640 medium) was added to the wells (in 24-well plates), which contained the studied composite disks at the wells’ bottom (disks with 0% of UA were used as controls). The so-prepared well plates were covered with a sterile seal plate film, covered with a lid, sealed with parafilm and incubated for 24 h at 37 °C. After incubation, two different XTT assays were performed:(a)C.a. sessile cells: After 24 h of C.a. incubation, the supernatant (liquid above the biofilm) was removed and wells were washed with sterile PBS to remove the remained non-adherent planktonic cells in the wells. After draining these plates in an inverted position, 200 μL the XTT/menadione was added to every well. The plates were covered with a lid, sealed with parafilm, wrapped with aluminum foil and incubated in the dark for 3 h at 37 °C.XTT assay on (b)C.a. planktonic cells: After 24 h of C.a. incubation, 200 μL of supernatant (liquid above the sessile cells) was taken from each well and transferred in new 24-well plates. Subsequently, 200 μL the XTT/menadione was added to each of these wells. The plates were covered with a lid, sealed with parafilm, wrapped with aluminum foil and incubated in the dark for 3 h at 37 °C. XTT assay on C.a. cells (%) was calculated with the following equation:Abs, Abs and Abs(medium) denote the absorbance of liquid containing XTT: with C.a. for PMMA-UA composites and PMMA, and without C.a. for RPMI medium, respectively.XTT measurement: The absorbance of the so-obtained solutions (100 μL placed in the 96-well plate) was measured with a microtiter plate reader  at 490 nm. As a control, RPMI medium was tested under the same conditions. From the obtained absorbance values, the percentage of metabolically active C.a. sessile and C.a. planktonic cells) were performed at two timepoints: after the composites’ preparation  and six days after the preparation of the composites (T6).Both XTT assays  was added and incubated at 37 °C for 24 h. Upon incubation, MIC was determined by two tests: (a) by measuring the absorbance of 50 μL of the so-obtained C.a. suspensions at 620 nm, using a microplate reader , and (b) by measuring the absorbance at 490 nm (with the same microplate reader) of 50 μL of the so-obtained C.a. suspensions where 50 μL of XTT/menadione was added after their incubation in the dark for 3 h at 37 °C.One millilitre of dispersion (as described in the previous C.a. (concentration 106C.a. cells/mL) were added, mixed and poured into sterile Petri dishes (diameter 30 mm). After natural cooling down, Petri dishes were incubated at 37 °C and studied by optical microscope  after 24 h and 48 h. Photomicrographs were taken through the agar matrix.In the 5 mL of the two UA dispersions (as described in the previous H-tetrazolium (MTS) toxicity test, we used a modified protocol from ref. [0 and T6, the composites were immersed for 24 h 0 days and 6 days after their preparation, respectively . Human A594 cells were cultured in RPMI-1640 medium (Sigma-Aldrich) supplemented with 10% foetal bovine serum and 2% 5000 U mL−1 Penicillin, 5 mg mL−1 Streptomycin and 10 mg mL−1 Neomycin (Sigma-Aldrich). Four thousand A549 cells per well were cultured in 96-well plates at 37 °C for 24 h, and afterwards exposed to 100 μL media containing 50% fresh growth media and 50% (v/v) the “disc conditioned media” for an additional 24 h. Cells treated only with medium, with 50% of growth medium and 50% of PBS without disc immersion, as well as with 50% of growth medium and 50% of PBS with PMMA disc immersion served as controls. After 24 h incubation, the supernatant of each well was removed. 100 μL of MTS solution  was added to the cells. After 2 h incubation in the dark, the absorbance of the formazan product was measured with a microplate reader  at a wavelength of 490 nm. All experiments were performed in four repetitions. Results are given as means (with standard deviations) of the values obtained in these four repetitions.For the 3--5-(3-carboxymethoxypenyl)-2-(4-sulfophenyl)-2rom ref. . In brie