Abstract3-staining and fluorescence in situ hybridization (FISH) with telomeric (TTAGG)n and 18S rDNA probes. Standard chromosomal complements are reported for the first time for Deraeocoris rutilus  (2n=30+2m+XY) and Deraeocoris ruber (2n=30+2m+XY) from the family Miridae. Using FISH, the location of a 18S rDNA cluster was detected in these species and in five more species: Megaloceroea recticornis  (2n=30+XY) from the Miridae; Oxycarenus lavaterae  (2n=14+2m+XY) from the Lygaeidae s.l.; Pyrrhocoris apterus  (2n=22+X) from the Pyrrhocoridae; Eurydema oleracea  (2n=12+XY) and Graphosoma lineatum  (2n=12+XY) from the Pentatomidae. The species were found to differ with respect to location of a 18S rRNA gene cluster which resides on autosomes in Oxycarenus lavaterae and Pyrrhocoris apterus, whereas it locates on sex chromosomes in other five species. The 18S rDNA location provides the ﬁrst physical landmark of the genomes of the species studied. The insect consensus telomeric pentanucleotide (TTAGG)n was demonstrated to be absent in all the species studied in this respect, Deraeocoris rutilus, Megaloceroea recticornis, Cimex lectularius Linnaeus, 1758 (Cimicidae), Eurydema oleracea, and Graphosoma lineatum, supporting the hypothesis that this motif was lost in early evolution of the Heteroptera and secondarily replaced with another motif (yet unknown) or the alternative telomerase-independent mechanisms of telomere maintenance. Dot-blot hybridization PageBreakanalysis of the genomic DNA from Cimex lectularius, Nabis sp. and Oxycarenus lavaterae with (TTAGG)n and six other telomeric probes likewise provided a negative result.Eight species belonging to five true bug families were analyzed using DAPI/CMA The 18S rDNA location provided the ﬁrst physical landmark of the genomes of the species studied. The species Deraeocoris ruber and Deraeocoris rutilus were studied here for the first time likewise in terms of their standard chromosomal complements.To fill this gap and learn more about bug genomes, we applied FISH technique with telomeric (TTAGG)Megaloceroea recticornis, Deraeocoris rutilus, Cimex lectularius, Eurydema oleracea, and Graphosoma lineatum,we used a (TTAGG)n telomeric probe to justify a hypothesis that this telomeric motif is absent in the true bugs  ) and Pyrrhocoris apterus n and (TTGGGG)n, nematode (TTAGGC)n, insect (TTAGG)n, shrimp (TAACC)n, vertebrate (TTAGGG)n, and plant (TTTAGGG)n.PageBreakAdditionally, we carried out a dot-blothybridization of the genomic DNA from Deraeocoris ruber, Deraeocoris rutilus, Megaloceroea recticornis, Nabis sp., Cimex lectularius, Oxycarenus lavaterae, Pyrrhocoris apterus, Eurydema oleracea, and Graphosoma lineatum were collected in the vicinities of Plovdiv and Sofia, Bulgaria in 2009-2011  and AT- specific 4-6-diamidino-2-phenylindole (DAPI) was used according to 2 at room temperature and then an incubation in 2xSSC at 60°C for 1 h. Furthermore, the preparation (without Giemsa) were stained first with CMA3 (0.4 μg/ml) for 25 min and then with DAPI (0.4 μg/ml) for 5 min. After staining, the preparations were rinsed in the McIlvaine buffer, pH 7 and mounted in an antifade medium .To reveal the base composition of C-heterochromatin, staining by GC-specific chromomycin APyrrhocoris apterus  was isolated using a Chelex-100 extracted method. FISH using a 18S rRNA gene probe was carried out on the chromosomes of Deraeocoris ruber, Deraeocoris rutilus, Megaloceroea recticornis, Oxycarenus lavaterae, Pyrrhocoris apterus, Eurydema oleracea, and Graphosoma lineatum. FISH using a telomeric (TTAGG)n probe was carried out on the chromosomes of Deraeocoris rutilus, Megaloceroea recticornis, Cimex lectularius, Eurydema oleracea,and Graphosoma lineatum. The target 18S rDNAgene was PCR amplified n was PCR amplified and labeled using primers TTAGG_F and TTAGG_R  dextran sulfate, 1% (w/v) Tween-20 and 10 µg salmon-sperm DNA was added on preparations. Slides were mounted using glass coverslips and rubberPageBreak cement. The slides were denaturated for 5 min at 75°C. Then the chromosome slides were incubated for 42–44 h at 37°C. Following hybridization, the slides were washed in 2x SSC for 3 min at 45°C, then in 50% formamide in 2xSSC for 10 min at 45°C, two times in 2x SSC (10 min each) at 45°C, blocked in 1.5% (w/v) BSA/4x SSC/0.1% Tween-20 for 30 min at 37° in a humid chamber. 18S rRNAgene probe was detected with 5μg/ml Avidin-Alexa Fluor 488 (Invitrogen). Detection reaction was performed in 1.5 % BSA/ 4x SSC/0.1% Tween-20 for 1 h at 37°C. Slides were washed three times in 4x SSC/0.02% Tween-20 (10 min each) at 45° and dehydrated through 70/80/96% ethanol at RT. Chromosomes were mounted in a mounting-antifade  and covered with a glass coverslip.Cimex lectularius, Oxycarenus lavaterae and Nabis sp. was isolated using NucleoSpin Tissue Kit  or a standard Phenol/Chloroform nucleic acid extraction protocol. Telomere probes of ciliate (TTTTGGGG)n and (TTGGGG)n, nematode (TTAGGC)n, insect (TTAGG)n, shrimp (TAACC)n, vertebrate (TTAGGG)n and plant (TTTAGGG)n were PCR amplified using primers labeled with biotin and presented in Genomic DNA from PageBreakbranes were washed two times in 2x SSC/0.1% SDS (10 min each) at RT and two times in 0.2x SSC/0.1% SDS (10 min each) at RT (10 min each). Detection procedure was performed according to the Biotin Chromogenic Detection Kit protocol (Fermentas).About 20 ηg of isolated DNA after denaturation was added drop wise to Hybond N+ nylon membranes . Hybridizations were carried out over night in hybridization mixture containing about 100-200 ηg of labeled probe, 50% formamide, 4×SSC, 0.5% (w/v) SDS and 10 µg salmon-sperm DNA at 40 °C. MemChromosome preparations were analyzed under a Leica DM 4000B microscope with a 100x objective. Fluorescence images were taken with a Leica DFC 350 FX camera using Leica Application Suite 2.8.1 software with an Image Overlay module. The preparations were stored partly at Institute of Biodiversity and Ecosystem Research, BAS in Sofia and partly at the Zoological Institute, RAS in St Petersburg.Deraeocoris ruber Linnaeus, 1758, 2n=30+2m+XYFig. 1a–dThe karyotype is described here for the first time. There are 16 bivalents, including a pair of very small and negatively heteropycnotic chromosomes taken as a pair of m-chromosomes, and the univalent X and Y chromosomes which are largest and smallest chromosomes in the set, respectively . DiploteDeraeocoris rutilus , 2n=30+2m+XYFig. 2a, bDeraeocoris ruber. Likewise, at PMI, there are 16 autosomal bivalents and the univalent X and Y chromosomes. One of the bivalents is very small, negatively heteropycnotic pair of m-chromosomes , 2n=30+XYFig. 3a–fin situ hybridization with a (TTAGG)n probe did not reveal positive signals on the telomeres although occasionally gave rise to variable interstitial hybridization signals, sometimes quite bright, on separate chromosomes (From the counts of 23 plates at prometaphase I (PMI), 15 autosomal bivalents and two univalent sex chromosomes, X and Y, were detected suggesting that this species displays 2n=30+XY  in contromosomes . Major romosomes . At MII,omosomes  and segromosomes , whereasomosomes .Cimex lectularius Linnaeus, 1758, 2n=26+X1X2Ynot figuredCimex lectularius display 2n=26+X1X2Y as it was repeatedly reported previously , 2n=14+2m+XYFig. 4a, bOxycareninae , 2n=22+XFig. 5a–fPageBreak autosomes and absent on a sex chromosome body (arrowed). We call attention to difference in signal strength between the homologues , 2n=12+XYFig. 6a–fEurydema oleracea has 2n=12+XY as previously reported by other researchers n probe did not reveal positive signals on chromosomal spreads. Clear 18S rDNAsignals were evident on both sex chromosomes , 2n=12+XYFig. 7a–dGraphosoma lineatum is in accordance with that published previously for other populations of this species n and (TTGGGG)n, nematode (TTAGGC)n, insect (TTAGG)n, shrimp (TAACC)n, vertebrate (TTAGGG)n and plant (TTTAGGG)n. All the experiments provided no hybridizing bands clearly suggesting some other molecular composition of telomeres in true bugs.Dot-blot hybridization of the genomic DNA from Cimicomorpha  and Pentatomomorpha . Our study confirms the previously published information (see Results and Cimex lectularius (Cimicidae) displays 2n=26+X1X2Y; Pyrrhocoris apterus (Pyrrhocoridae) – 2n=22+X; Oxycarenus lavaterae (Ligaeidae) – 2n=14+2m+XY; Eurydema oleraceaand Graphosoma lineatum (Pentatomidae) – 2n=12+XY. On the other hand, Megaloceroea recticornis (Miridae) in England as having 2n=32+XY; however this count was not corroborated by our observations of this species. The karyotype of Megaloceroea recticornis in Bulgaria, as revealed in our work, is 2n=30+XY. We can not explain this incompatibility, especially as Leston provided neither photograph nor drawing of the chromosomal complement. It should be mentioned here that 2n=32+XY is the first whereas 2n=30+XY the second commonest karyotype in the Miridae  known as m-chromosomes has been described in karyotypes of many bug species  and Dicyphus digitalidis Josifov, 1958 . In meiosis of this type, diplotene and diakinesis stages are absent, and no chiasmata are formed between homologous which are however connected with each other, generally only at one site, by thread-like structures, the so-called collochores. The collochores have the function to hold homologous chromosomes together in the absence of chiasmata, and hence ensure their proper orientation and regular segregation at anaphase I.In all the seven species studied in this work, the first meiotic division is reductional for the autosomes and equational for the sex chromosomes, and vice versa – the second division is equational for the autosomes and reductional for the sex chromosomes. Such species . The speomosomes . In commrespect  form a ring with the sex chromosomes  lying in its center . In two atomidae . In anot3) and GC-specific fluorochrome (most commonly by CMA3) staining. However silver treatment stains only active NORs  with rDNA probes directly detects the location of ribosomal RNA genes, regardless of their activity. In eukaryotes, 5S and 18S ribosomal genes (rDNA) are organized into two multigenic families, namely the major rDNA family formed by the 18S, 5.8S, and 28S genes and the minor one composed of 5S genes  and formation of ribosomes take place . Nucleolive NORs  being th5S genes . ChromosHeteroptera, physical location of genes remains very poorly sampled , mainly with sporadic sampling of a few select species. Out of more than 40,000 described species , Lygaeidae (Oxycarenus lavaterae), Pyrrhocoridae (Pyrrhocoris apterus), and Pentatomidae . Data on all the species (as well as those on the whole families Miridae and Lygaeidae) were obtained for the first time bringing thus the total number of the species and genera studied to 30 and 18, respectively. The species were shown to exhibit different patterns of rDNA location. Some of the species showed their ribosomic cistrons located on sex chromosome, either on the X (Deraeocoris rutilus) or on both X and Y whereas in other species  they were located on a pair of autosomes.In this study we have characterized the chromosomal locations of 18S RNA genes in seven species from six genera of the families Deraeocoris, where Deraeocoris rutilus displayed rDNA clusters concentrated on the X, but Deraeocoris ruber on both X and Y chromosomes. These findings give no way of any inferences especially as a wide variation of chromosomal location for the major rDNA has been observed in different bug taxa, including variations between the co-generic species. For example, the genus Triatoma Laporte, 1832 from the subfamily Triatominae (Reduviidae), which is one of the most studied bug groups, clearly shows the interspecific variation  (Pentatomidae) studied using Southern hybridization ) and Pyrrhocoris apterus (Pyrrhocoridae) subjected both to Southern hybridization and FISH , Cimex lectularius (Cimicidae), Eurydema oleracea and Graphosoma lineatum (Pentatomidae). All these species were shown to lack the insect consensus sequence. Although in both mirid species a number of prominent hybridization signals could be seen on separate chromosomes, these signals most likely did not indicate the telomeres. The presence of signals suggests a sequence related to TTAGG but it seems to have no target specificity in the bug chromosomes.The pentanucleotide sequence (TTAGG)elomeres . Howeverelomeres . The truand FISH . Comparan telomeric repeat in the phylogenetically distant groups within the Heteroptera strengthens thus the view (The absence of (TTAGG)the view  that it n and (TTGGGG)n, nematode (TTAGGC)n, insect (TTAGG)n, shrimp (TAACC)n, vertebrate (TTAGGG)n and plant (TTTAGGG)n, yielded negative results and did not provide hence any answer of the question which is the telomere repeat sequence in bug chromosomes.Dot-blot hybridization of the genomic DNA from bug species with seven types of telomeric probes, ciliate (TTTTGGGG)


Spirogyra neglecta extract (SNE) has shown antihyperglycemia and antihyperlipidemia in type 2 diabetic mellitus (T2DM) rats. This study investigated the antioxidant and renoprotective effects of SNE in T2DM rats induced by high-fat diet with low-single dose streptozotocin. T2DM rats were fed daily with SNE  for 12 weeks. Renal morphology, malondialdehyde levels, qPCR, and western blotting were analyzed. Renal cortical slices were used to determine renal transport of organic anions, which are estrone sulfate and para-aminohippurate, mediated through organic anion transporter 3-Oat3. Insulin and PKCζ were known to activate Oat3 function while it was inhibited by PKCα. Compared to T2DM, plasma glucose, triglyceride, insulin resistance, renal morphology, and malondialdehyde levels were significantly improved by SNE supplementation. Reduced glutathione peroxidase and nuclear factor κB expressions were related to antioxidant effect of SNE. Oat3 mRNA and protein were not different among groups, but insulin-stimulated rOat3 followed by anion uptakes was abolished in T2DM. This was restored in the slices from SNE treatment. The mechanism of SNE-improved Oat3 was associated with PKCα and PKCζ expressions and activities. These findings indicate that SNE has beneficial effects on renal transport through antioxidant enzymes and PKCs in T2DM rats. Spirogyra neglecta (SN) has been widely grown in the Nan River, North of Thailand. It is commonly used as an ingredient in several Northern Thai food and contains high amount of nutritional compositions, including basic nutrients, which are carbohydrate, fat, proteins, multivitamins, and mineral substances ES  and [3H]PAH (SA 1 Ci/mmol) were purchased from Perkin Elmer . Unlabeled ES, PAH, streptozotocin (STZ), and CelLytic MT mammalian tissue lysis/extraction reagent were purchased from Sigma Aldrich . Ascorbic acid was obtained from Merck , insulin was obtained from Biocon , and complete protease inhibitor cocktail was purchased from Roche Applied Science . Thiobarbituric acid reactive substances (TBARS) assay was purchased from Cayman Chemical . All other chemicals with high purity were obtained from commercial sources. Polyclonal rabbit anti-rOat3 antibody was obtained from Cosmobio . Polyclonal rabbit anti-PKCSpirogyra neglecta extract was identified for species and kindly provided by Dr. Yuwadee Peerapornpisal, Faculty of Sciences, Chiang Mai University. A voucher specimen (number AARL G047) has been deposited at the herbarium of the Applied Algal Research Laboratory, Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai, Thailand.Dried SNE was weighed and blended thoroughly followed by boiling at 100°C for 1 hr. The extract was then filtered through filter paper  with negative pressure pump . The filtrate was subsequently evaporated using lyophilizer . Lyophilized SNE was stored at 4°C prior subsequent experiments. The SNE was standardized by determination of total phenolic content using Folin-Ciocalteu reagent as was recently described by Rattanapot et al. . The quaMale Wistar rats with 120–150 g were obtained from the National Laboratory Animal Center, Mahidol University, Salaya, Thailand. The animal facilities and protocols were approved by the Laboratory Animal Care and Use Committees at Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand. All experimental rats were housed in a room and maintained at 25 ± 1°C on a 12 hr light/dark cycle. Animals were randomized and equally divided into 7 groups: normal diet  (ND), normal diet supplemented with SNE at the dose of 1.0 g/kg BW (ND + SNE1.0), T2DM (DM), T2DM supplemented with SNE at the dose of 0.25, 0.5, and 1.0 g/kg BW, respectively , and T2DM supplemented with antioxidant compound, ascorbic acid (vitamin C), at the dose of 0.2 g/kg BW (DM + vitamin C) similar to that of the treatment previously described by Owu et al. . These DµU)/mL) × fasting plasma glucose (mmol/L)/(22.5).To characterize T2DM rat model, the quantitative total plasma glucose and triglyceride were determined by commercial enzymatic colorimetric assays . The plasma insulin concentration was determined using a Sandwich ELISA assay kit obtained from LINCO research . Insulin resistance index was estimated using the homeostatic model assessment of insulin resistance (HOMA index) that was calculated by the following formula: fasting plasma insulin  levels in renal cortical tissues was carried out. Briefly, the rats were euthanized by pentobarbital and sacrificed. Kidneys were excised and renal cortical tissues were cut and suspended in CelLytic MT mammalian tissue lysis/extraction reagent  containing 1% complete protease inhibitor cocktail  according to the manufacturer's protocol. The tissues were then homogenized and centrifuged at 1,600 g for 10 min at 4°C. The supernatants were collected for determination of MDA concentrations using commercial TBARS assay kit purchased from Cayman Chemical . Each sample was expressed as total MDA level to total protein concentration (nmol/mg protein). μm thick sections and subsequently stained by hematoxylin and eosin (H&E) and the lesions were confirmed by periodic acid-Schiff base (PAS), which are appropriate standard methods for renal biopsy as previously described ES or 5 μM [3H]PAH for 30 min at room temperature. For upregulation of rOat3 function by insulin stimulation, the slices were preincubated in 0.5 mL of buffer in the absence or presence of 30 μg/mL insulin for 30 min and subsequently incubated in 0.25 mL of buffer containing either 50 nM [3H]ES or 5 μM [3H]PAH for 30 min at room temperature. Uptake was stopped by the addition of ice-cold buffer. Slices were rinsed, blotted, weighed, and dissolved in 0.5 mL of 1 N NaOH and neutralized with 0.5 mL of 1 N HCl. The radioactivity was measured using a liquid scintillation analyzer . Transport of ES and PAH was calculated as tissue to medium (T/M) ratio (dpm/g tissue ÷ dpm/mL medium). It has been suggested that Oat3 is highly expressed in kidneys, liver, and choroid plexus and has a major impact on the secretion and excretion of anionic drugs and xenobiotics for maintaining systemic homeostasis . These ot-test. Statistical analyses were conducted using Statistical Package for the Social Sciences (SPSS) version 11.5 . Differences were considered to be significant when P < 0.05. Data were expressed as mean ± S.E.M. Statistical differences were assessed using one-way analysis of variance, followed by Tukey-Kramer test. Insulin stimulation experiment was analyzed using unpaired, two-tailed Student's  As it was suggested that SNE has antidiabetic effects in T2DM rat model , the effTo determine the effect of SNE on renal structural changes in T2DM rat model, renal morphological analysis was semiquantitatively assessed using standard methods for renal biopsy, hematoxylin and eosin H&E; , and perTo determine renal oxidative stress and evaluate antioxidant effect of SNE, the total MDA level was determined in renal cortical tissues using TBARS assay. As shown in 3H]ES, specific substrate of Oat3, and [3H]PAH, a typical substrate of OATs, in renal cortical slices. As shown in 3H]ES uptake mediated by rOat3 was not significantly different among experimental groups. The similar result was also seen in rOat3 transport mediated by [3H]PAH uptake . These enzymes and nuclear factor were quantitatively analyzed using qPCR and western blotting, respectively. As shown in Since the earlier experiment has shown that the effect of SNE was not involved with rOat3 expression and the study by Peerapornpisal et al.  demonstrescribed . These iκB has been proposed to be a common stress response factor under hyperglycemia, elevated free fatty acid (FFA), and oxidative stress [κB is present in the cytosol. Once it is activated by either hyperglycemia, elevated FFA, or ROS, p65 subunit of NFκB (p65NFκB) would be translocated into the nucleus [κB expression in subcellular fractions extracted from renal cortical tissues using western blotting analysis. The detection of p65NFκB bands was analyzed by densitometry. The amount of p65NFκB expression in each sample was normalized by the amount of β-actin present in each respective fraction as shown in κB expression in whole cell and cytosolic fractions among experimental groups. However, NFκB activation (p65NFκB) was significantly increased in nuclei fraction of T2DM compared to that of control, whereas this activation was markedly decreased in DM + SNE1.0 relative to T2DM rat kidneys. This result indicated that SNE improved oxidative stress in T2DM by preventing the activation and translocation of NFκB. Increased NFe stress . During  nucleus , 33. Sucα activation downregulated Oat3 function [α (p-PKCα) increased at the plasma membrane of renal cortical tissues in STZ-induced diabetic rats when compared with normal rats [ζ activation. The latter study, however, investigated only PKCζ activity and had not referred to its localization after activation. Therefore, whether SNE has any effect on specific protein kinases, PKCα and PKCζ, regulated rOat3 function in T2DM was identified in subcellular fractions extracted from renal cortical tissues using western blotting analysis. The detection of PKCα, p-PKCα, PKCζ, and p-PKCζ proteins was analyzed by densitometry and the amount of each target protein in each sample was normalized by the amount of β-actin present in each respective fraction as shown in Figures +K+-ATPase was detected in only membrane samples of each experimental group. As shown in α was not significantly different in whole cell lysate, membrane, and cytosolic fractions among experimental groups. However, activated PKCα (p-PKCα) was markedly increased in both whole cell and membrane fractions in T2DM rat kidneys while it was significantly reduced in cytosolic fraction, indicating that oxidative stress in T2DM resulted in the activation and translocation of PKCα to the plasma membrane  in whole cell lysate, membrane, and cytosolic fractions was significantly increased in T2DM relative to control, suggesting that diabetic condition also influenced PKCζ activity  Kützing (SN) or commonly known in Thai as “Tao” has been widely grown in the Nan River, Northern Thailand. In vivo studies indicated that this species has several beneficial effects, including antigastric ulcer, anti-inflammatory, antihyperglycemic, and antihyperlipidemic actions [in vitro [ actions , 5. Previn vitro  and its in vitro . These fin vitro , 35. To As shown in At the present, several studies have suggested the diagnostic protocols for diabetic-induced renal dysfunction , 30. Suc1C, and this factor was positively correlated with the accumulation of MDA levels in cerebral, liver, and kidney tissues [Recently, the role of oxidative stress in diabetic complication has been extensively studied , 10. Per tissues . ApparenAs mentioned above, organic anion transporter 3 (Oat3) is a membrane transporter that is highly expressed in the renal tubular epithelium, liver, and choroid plexus, where it plays a crucial role in secretion of anionic drugs and toxins and controlling systemic homeostasis of organic anions. The function of Oat3 could be regulated by several compounds, including hormones, endogenous/exogenous substances, and also by pathological status , 40. PreWe, next, identified the mechanisms by which SNE completely improved upregulation of rOat3 after insulin stimulation. Since SNE enriched polyphenols, which is known to have antioxidant activity , 25, 35,κB, a common stress response gene, which, in turn, induced proinflammatory cytokines production including TNF-α and IL1-β [κB activation and prevented its translocation into the nucleus (Previously, it has been suggested that hyperglycemia-induced ROS production led to renal dysfunction by activating NFnd IL1-β , 46, 47. nucleus ; this coα activation [ζ activation [α, β, and ε in diabetic nephropathy [α and PKCζ, which resulted in retrieving Oat3 function after insulin stimulation in T2DM. Finally, as shown in Figures tivation  whereas tivation . Similartivation  whereas tivation . Alternahropathy . Taken tκB expression and function. Moreover, our findings indicated that polyphenol-rich SNE acted directly to regulatory proteins of rOat3, PKCα, and PKCζ, resulting in changes in their expressions, localizations, and activities. However, further supporting information in experimental or human studies on whether SNE has a distinct effect on particular PKC isoform is needed. The beneficial information of our findings allows for further development of SNE into either nutraceutical or pharmaceutical products for prevention of diabetic nephropathy. We have demonstrated antidiabetic, antioxidant, and renoprotective effects of SNE in long-term hyperglycemia-induced ROS in T2DM experimental rat model. SNE was able to improve hyperglycemia, hypertriglyceridemia, insulin resistance, as well as renal oxidative stress, and regulation of rOat3 transport function. The mechanism by which SNE improved rOat3 regulation was directly linked with the modulation of antioxidant systems including free radical scavenging enzymes and NF


Niemann-Pick type C1 disease (NPC1) is a rare progressive neurodegenerative disorder caused by mutations in the NPC1 gene. In this lysosomal storage disorder the intracellular transport and sequestration of several lipids like cholesterol is severely impaired, resulting in an accumulation of lipids in late endosomes and lysosomes. The neurological manifestation of the disease is caused by dysfunction and cell death in the central nervous system. Several animal models were used to analyze the impaired pathways. However, the underlying pathogenic mechanisms are still not completely understood and the genetic variability in humans cannot be reflected in these models. Therefore, a human model using patient-specific induced pluripotent stem cells provides a promising approach.We reprogrammed human fibroblasts from a NPC1 patient and a healthy control by retroviral transduction with Oct4, Klf4, Sox2 and c-Myc. The obtained human induced pluripotent stem cells (hiPSCs) were characterized by immunocytochemical analyses. Neural progenitor cells were generated and patch clamp recordings were performed for a functional analysis of derived neuronal cells. Filipin stainings and the Amplex Red assay were used to demonstrate and quantify cholesterol accumulation.The hiPSCs expressed different stem cell markers, e.g. Nanog, Tra-1-81 and SSEA4. Using the embryoid body assay, the cells were differentiated in cells of all three germ layers and induced teratoma in immunodeficient mice, demonstrating their pluripotency. In addition, neural progenitor cells were derived and differentiated into functional neuronal cells. Patch clamp recordings revealed voltage dependent channels, spontaneous action potentials and postsynaptic currents. The accumulation of cholesterol in different tissues is the main hallmark of NPC1. In this study we found an accumulation of cholesterol in fibroblasts of a NPC1 patient, derived hiPSCs, and neural progenitor cells, but not in cells derived from fibroblasts of a healthy individual. These findings were quantified by the Amplex Red assay, demonstrating a significantly elevated cholesterol level in cells derived from fibroblasts of a NPC1 patient.in vitro model to study the pathogenic mechanisms of NPC1 disease.We generated a neuronal model based on induced pluripotent stem cells derived from patient fibroblasts, providing a human  Human induced pluripotent stem cells (hiPSCs) feature three major advantages in the field of stem cell research. First, cells can be obtained by reprogramming different somatic cells -4 withouin vitro. However, no in vitro model for Niemann-Pick disease Type C1 (NPC1) based on hiPS cells is currently available. NPC1 is a rare progressive neurodegenerative disease caused by mutations in the NPC1 gene located on chromosome 18q11 encoding for a 1278-amino acid intracellular membrane glycoprotein [Taken together, these characteristics of the cells are excellent prerequisites to model diseases oprotein -17. It ioprotein . The clioprotein .The pathogenic mechanisms ultimately leading to a massive degeneration and loss of neurons in the CNS, especially Purkinje cells in the cerebellum, are not exactly understood. Most of our knowledge regarding NPC1 is based on cell models like human fibroblasts -23 and aIn our study, we generated patient-specific induced pluripotent stem cells from a NPC1 patient and a healthy individual. The hiPS cell lines were differentiated into neural progenitor cells and subsequently differentiated into functional neurons to gain a human neuronal model of NPC1 disease.2 onto 0.1% gelatine coated wells in fibroblast medium 24 h before hiPS cell split. HiPS cells were cultured on a feeder cell layer in iPS medium containing DMEM/ F12, 20% knockout serum replacement, 1% Penicillin/ Streptomycin, 1% GlutaMAX, 1% MEM non essential amino acids, 0.2% 2-mercaptoethanol, and 10 to 15 ng/ ml hFGF-2 . hiPS cells on matrigel  were cultured in mTESR1 medium . Medium was changed daily and cells were passaged weekly using 10 μM ROCK inhibitor Y-27632  for increased plating efficiency. HiPS cells growing on a feeder cell layer were split mechanically weekly using pulled glass hooks by performing the cut and paste technique. Cells growing on matrigel were harvested enzymatically using 1 mg/ ml dispase  for 7 min and large bore tips to break down large clumps according to manufacturer’s recommendations. HEK293FT cells  used to obtain the viral vectors were cultivated in fibroblast medium without Penicillin/ Streptomycin. All cells were cultivated at 37°C in a saturated humidity atmosphere containing 5% CO2.Human dermal fibroblast cell lines GM18436 and GM05659  were obtained by skin biopsies from one-year old male Caucasian donors. GM18436 exhibits compound heterozygous mutations in the NPC1 gene (c.1628delC and GLU612ASP), representing a frameshift mutation and a missense mutation, respectively. The mutations lead to a non-functional protein as demonstrated by cholesterol esterification assay . Fibrobl6 HEK293FT cells per 10 cm-dish were seeded onto ten dishes and incubated overnight. A solution containing 2.5 μg retroviral vector encoding for GFP and one of the transcription factors  was incubated with 0.25 μg VSV-G and 2.25 μg Gag-Pol in X-tremeGENE9 / DMEM High Glucose mixture (1:4) which was added to each of the dishes. Medium was renewed after 18 h and cells were incubated further for 48 h. Subsequently, the virus-containing medium was collected and passed through a 0.45 μm filter. To concentrate the virus, the medium was centrifuged at 70.000 × g at 4°C for 90 min, resuspended in 0.1 to 1 ml DMEM medium, and stored at −80°C. All four vectors contained a GFP sequence thus enabling titering by determining the percentage of GFP positive HEK293FT cells using FACS. Therefore, 1×105 HEK293FT cells were seeded per 12-well in Penicillin/ Streptomycin free fibroblast medium containing 5 μg/ ml protamine sulfate and concentrated virus in the following volumes: 6.25 μl, 12.5 μl, 25 μl, and 50 μl. After 48 h cells were washed with PBS containing Ca2+/ Mg2+, trypsinized and centrifuged for 5 min at 500 × g. Pellet was resuspended in 100 μl PBS without Ca2+/ Mg2+ and fixed by adding 100 μl of 4% paraformaldehyde for 15 min. Afterwards, the percentage of GFP positive cells was determined via FACS analysis.Retroviral pMIG vectors containing the cDNA of the human genes Oct4, Sox2, Klf4, and c-Myc were used as described recently . Briefly5 fibroblasts were seeded per cavity of a 6-well plate and cultured for 18 h in fibroblast medium without Penicillin/ Streptomycin. Afterwards, fibroblast medium without Penicillin/ Streptomycin was supplemented with a volume of retrovirus of Sox2, Oct4, Klf4 (corresponding to 70 – 80% infection efficiency), and c-Myc (corresponding to 40–50% infection efficiency) in the presence of 5 μg/ ml protamine sulfate. Cells were incubated for 48 h. Subsequently, medium was aspirated and cells were washed twice with PBS containing Ca2+/ Mg2+. Transduced cells were trypsinized and reseeded onto a gelatin coated 6 cm-dish. The next day, medium was replaced with iPS medium supplemented with 0.5 mM valproic acid to further increase the efficiency of reprogramming. Medium was changed daily and valproic acid was omitted after seven days.For transduction, 1×102. After 4 to 7 days of proliferation hiPS colonies were mechanically divided into two to four pieces and further expanded. Within six weeks each single hiPS colony was expanded to obtain different clones.Initial hiPS colonies were routinely observed after three to four weeks. For further cultivation, they were picked using a 100 μl pipette tip and a pulled glass hook. Single colonies were transferred to the cavities of a 24-well plate, coated with 0.1% gelatin and 45.000 feeder cells/ cmKaryotyping was performed by Giemsa Trypsin banding. In short, colonies were incubated with a colcemid solution (10 μg/ ml in HBSS) for three hours to arrest cells in metaphase. Cells were treated with trypsin (0.25%) and the enzymatic reaction was stopped with Amniomax solution . Cells were centrifuged at 300 × g for 10 min and the pellet was resuspended in 4 ml hypotonic potassium chloride solution (5.62%). Cells were incubated for 5 min at 37°C and centrifuged at 300 × g for 10 min. The cells were resuspended and fixed in 5 ml glacial acetic acid and methanol (1:3) and subsequently centrifuged for 7 min at 350 × g. This step was repeated once. Finally, most of the supernatant was removed and cells were resuspended. Cell suspension was dropped onto cold slides and dried at 100°C for 1 h. Giemsa solution (5%) was added and incubated for 5 min. Slides were washed in distilled water two times, dried at room temperature and sealed with cover slips.Genomic DNA of fibroblasts, iPS cells grown on matrigel or neural progenitor cells were isolated using AllPrep Kit  according to the manufacturer’s recommendations. Exon regions were amplified using HotStart Taq  as follows: 95°C for 15 min followed by 13 cycles of 94°C for 30 s, 66°C for 30 s with 1.5°C decrease/ cycle and 72°C for 20 s; followed by 8 cycles of 94°C for 30 s, 46.5°C for 30 s with 1°C increase/ cycle and 72°C for 20 s; followed by 13 cycles of 94°C for 30 s, 66°C for 30 s with 1.5°C decrease/ cycle and 72°C for 20 s; followed by 11 cycles of 94°C for 30 s, 54.5°C for 30 s with 1°C increase/ cycle and 72°C for 20 s. Products were purified using ExoSAP Kit  according to manufacturer’s recommendations. Sequence analysis was performed on a 3130XL Genetic Analyzer .HiPSCs were cultivated on a feeder cell layer for five days. Medium was removed, cells were washed with PBS and fixed with ice-cold methanol (100%) for 10 min at −20°C. Methanol was removed and cells were washed with PBS. Subsequently, cells were incubated at room temperature for 15 min with the staining solution: 75% distilled water, 10% sodium chloride solution (1 M), 10% Tris solution , 5% magnesium chloride solution (1 M), and NBT/ BCIP solution . Staining solution was removed and cells were washed with distilled water. Microphotographs were taken using a Nikon Eclipse TS100 .3 at 4°C. Immunocytochemistry was performed for Nanog , Oct4 , SSEA3 , SSEA4 , Tra-1-60 , Tra-1-81 , Smooth muscle actin , alpha fetoprotein , Nestin , MAP2ab , Tuj1  and Sox-2 . Blocking and permeabilization was carried out using 0.3% Triton X-100 and 5% normal goat serum  for 30 min at room temperature. Cells were incubated with primary antibodies for 3 hours at room temperature in 1% normal goat serum, followed by three washing steps with PBS. Alexa Fluor 568 , Alexa Fluor 488 , or Alexa Fluor 488  were used as secondary antibodies, incubated 1 h at room temperature with 1% normal goat serum in PBS. After washing with PBS, cells were stained with DAPI , washed three times and mounted with Mowiol-DABCO mounting medium. Pictures were taken with a Biozero 8000 microscope system .Cells were fixed at room temperature for 15 minutes in 4% paraformaldehyde, washed with PBS and stored in 0.02% NaN2. Medium was changed every second day until EBs were formed. After five to seven days EBs were transferred onto gelatin coated glass cover slips and supplied with differentiation medium. Once EBs were attached, medium was changed every second or third day. After 10 days of random differentiation, spread cells were washed with PBS and fixed with 4% PFA for 15 min. Fixed cells were washed with PBS and immunocytochemical stainings for nestin (ectoderm), smooth muscle actin (mesoderm), and alpha-fetoprotein (endoderm) were performed.To generate embryoid bodies (EBs), whole hiPS colonies were mechanically lifted off the feeder cell layer and transferred into a 15 ml conical tube. Once the colonies settled at the bottom of the conical tube, the medium was removed and 5 ml of differentiation medium, containing knockout DMEM, 20% FBS, 1% MEM non-essential amino acids, 2 mM GlutaMAX, and 0.1 mM beta-mercaptoethanol, was added. Afterwards, colonies were transferred into the cavity of a low attachment 6-well plate and incubated at 37°C/ 5% COImmunodeficient (SCID) hairless mice  were used for the teratoma formation assay. HiPSCs for injection were cultured on feeder cells in 6-well culture plates. For each injection the amount of 3 cavities of a 6-well culture plate were collected mechanically and centrifuged for 2 min at 200 × g. The pellet was resuspended in 1 ml of 0.25% trypsin/ EDTA. After 1 min, the reaction was stopped by adding 2 ml of fibroblast medium and centrifuged again for 2 min at 200 × g. Cells were resuspended in 140 μl of cold DMEM/ F12 and stored on ice. Directly before injection, cell suspension was mixed with 60 μl matrigel. Cells were injected subcutaneously into the flank of the hind limb. After 8–12 weeks, when tumors were clearly visible, the animals were sacrificed and tumors were removed. Tumor tissue was fixed in 4% formalin for 12 to 18 hours and embedded in paraffin for subsequent staining.4 μm thick tumor tissue sections were deparaffinized in xylol for 10 min and a descending ethanol concentration for 5 min each. Afterwards, the sections were washed in distilled water and stained with Mayers hematoxylin  for 1 min. Next, the tissue was washed two times in distilled water and stained with eosin Y  for 2 min. The slides were washed again twice and then dehydrated using an ascending ethanol concentrattion and xylol. Slides were mounted in Mowiol-DABCO. Microphotographs were taken with a Biozero 8000 microscope system .c-chimera , SB431542  and hFGF-2 . Neural rosettes were manually isolated using pulled glass hooks, gently trypsinized, and seeded as single cells on Poly-L-ornithine/ laminin coated dishes in medium consisting of Neurobasal, DMEM/ F12, 1×N2, 1×B27, and GlutaMAX (2 mM) supplemented with hFGF-2 (10 ng/ ml) and hEGF . Neural progenitor cells were seeded at high densities (100–150.000 cells/ cm2) and passaged one day after reaching confluence using Trypsin/ Benzonase. Differentiation was induced by seeding the cells at a density of 50.000 cells/ cm2 and withdrawal of growth factors in the presence of BDNF .To differentiate hiPS colonies into neural direction, the colonies were cut, transferred to Poly-L-ornithine (15 μg/ ml)/ laminin (10 μg/ ml) coated dishes, and cultivated for 10 days in medium consisting of Neurobasal, DMEM/ F12, 1xN2 supplement, 1xB27 supplement, GlutaMAX (2 mM) complemented with mouse recombinant noggin F2×6H2O 1, CaCl2×H2O 2, Mg-ATP 2. pH was adjusted to 7.2. When filled, electrodes had a resistance of 6–8 MΩ. Cell cultures were continuously superfused with an extracellular solution, consisting of (mM): NaCl 125, KCl 2.5, CaCl2×H2O 2, MgCl2×6H2O 1, NaHCO3 26, NaH2PO4×H2O 1.25, glucose×H2O 25. Solution was continuously bubbled with carbogen to maintain a pH of 7.4. Recordings were made in the whole cell configuration with holding potentials (VH) of −60 or −80 mV. Current voltage responses were evoked by applying 100 ms voltage steps from −60 mV to +50 mV in 10 mV increments. Data were filtered at 3 kHz, digitized and stored on-line using Pulse . Na+ and K+ currents were identified via their I-V relationship. Na+ currents were antagonized in some experiments by TTX (1 μM). Current clamp mode was used to apply current steps to induce action potentials or to measure spontaneous action potentials. Postsynaptic currents were measured in the voltage clamp mode at a VH of −60 mV. Mini Analysis 6  was used to analyse recordings of post-synaptic currents. Data are given as mean ± SEM.Patch clamp recordings were performed using an EPC-10 amplifier . Patch pipettes were pulled from borosilicate glass tubing . The internal solution contained (mM): KCl 130, NaCl 10, HEPES 10, EGTA 11, MgClFilipin is a polyene antibiotic which binds to free cholesterol and is widely used to analyze the sequestration of unesterified cholesterol in NPC1-deficient cells. Therefore, cells were fixed with 4% paraformaldehyde (in PBS) for 15 min, washed with PBS and incubated at room temperature for 45 min in the dark with a staining solution containing 100 μg/ ml Filipin  in PBS. Cells were washed twice with PBS for 5 min. Slides were mounted using Mowiol-DABCO and sealed with cover slips. After 12 h of drying in the dark at room temperature, fluorescence pictures were taken with a Biozero 8000 microscope .To quantify the amount of cholesterol we used the Amplex Red cholesterol assay . FibroblAnalysis of the data was carried out with GraphPad Prism 5 . Data are given as mean ± SEM. Unless otherwise stated, unpaired t-tests were used to test for significance, with * = p<0.05 and ** = p<0.01, *** = p<0.001.We reprogrammed fibroblasts originating from a male patient with an early-infantile form of NPC1 characterized by massive accumulation of unesterified cholesterol in lysosomal and late endosomal structures. Cells derived from this donor will be referred to as mutNPC1 and cells derived from age- and sex-matched fibroblasts of a healthy individual will be referred to as wtNPC1.After three to four weeks of cultivation, the first hiPSC colonies appeared characterized by their embryonic stem (ES) cell-like morphology, e.g. round to oval shape with a sharp border and a high nuclear to cytoplasm ratio Figure A-D. Mechin vitro. The induction of teratoma was used as an in vivo pluripotency assay. The hiPSCs induced teratomas in immunodeficent mice, and the analysis of the tumors revealed tissues of all three germ layers  expression. All hiPSCs colonies demonstrated strong AP expression Figure A,B. The s Figure I-N.+ and K+ channels (NaVs and KVs)  Figure I after t) Figure K. Althoun Figure L. In addn Figure N revealeThe hallmark of NPC1 is abnormal cholesterol trafficking resulting in an accumulation of cholesterol. Free cholesterol can be visualized by Filipin. An analysis of the cholesterol distribution in mutNPC1 fibroblasts, iPSCs, and derived neural progenitor cells Figure A,C,E revIn this study we aimed to reprogram fibroblasts originating from a NPC1 patient with an early-infantile form of the disease. Therefore, we used retroviruses expressing Oct4, Klf4, Sox2, and c-Myc in combination with GFP. These factors have been described previously to be efficient in generating hiPSCs . The retin vitro disease model. So far, two human cellular neural models based on NPC1-knockdown have been reported. These include SH-SY5Y neuroblastoma cells [We further differentiated the hiPSCs into neural progenitor cells to generate a suitable ma cells  and humama cells , which rma cells . In contma cells  obtainedma cells .+ and K+ channels, where inward currents were blocked by TTX. Spontaneous action potentials and postsynaptic currents could only be observed in cells differentiated for 7–8 weeks, indicating the maturation time of human neural progenitor cells to functional neurons, as described for interneurons derived from hiPSCs [Our differentiated neural progenitor cells expressed the neuronal markers MAP2 and Tuj1. We did not observe obvious morphological differences between mutNPC1 and wtNPC1 neuronal cells. In contrast, distortion of neuronal shape and extensive growth of ectopic neurites have been reported for multipotent adult stem cells (MASCs) derived cells . Howeverm hiPSCs ,44. The m hiPSCs ,46. Thesm hiPSCs . Thus, oOur neural progenitor cells were analyzed regarding their impaired cholesterol trafficking by Filipin. It visualizes free cholesterol and is routinely used for human dermal fibroblasts in the diagnostics of the NPC1 disease . We founFinally, we used the Amplex Red assay to confirm and quantify the observed cholesterol accumulations in our cells. These experiments revealed significantly increased cholesterol content in mutNPC1 cells in comparison to wtNPC1 cells, which was conserved in fibroblasts, hiPSCs, and derived neural progenitor cells. To our knowledge this is the first analysis of cholesterol distribution using Filipin staining and cholesterol quantification in hiPSCs and neural progenitor cells derived from human NPC1 deficient fibroblasts.in vitro model of NPC1. We are convinced that the here reported hiPSCs and the derived neural progenitor cells are an excellent model to study the influence of the specific mutation on the phenotype, e.g. consequences of a misfolded NPC1 protein. Moreover, the cells provide the opportunity to analyze the consequences of a NPC1 mutation on the patient-specific (epi)genetic background, and will thus serve to elucidate further the pathogenic mechanisms of this fatal lysosomal storage disorder.In this study we generated, for the first time, induced pluripotent stem cells derived from fibroblasts of a NPC1 patient. The cells demonstrated an accumulation of cholesterol, resembling the phenotype of NPC1 deficient cells, and can provide an The authors declare that they have no competing interests.MT: conception and design, collection and/ or assembly of data, data analysis and interpretation, manuscript writing. RH: conception and design, collection and/ or assembly of data, data analysis and interpretation, manuscript writing. PS: collection and/ or assembly of data, manuscript writing. CK: collection and/ or assembly of data, manuscript writing. AR: conception and design, manuscript writing, final approval of manuscript. MJF: conception and design, collection and/ or assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript. All authors read and approved the final manuscript.


Drosophila suzukii Matsumura (Diptera: Drosophilidae), with seven “reported” hosts  in a postharvest scenario were analyzed. Four aspects of SWD-host interaction were examined: attraction to host volatiles; population-level oviposition performance; individual-level oviposition performance; and key developmental factors. Application of HPI methodology indicated that raspberries  have the greatest potential to serve as a postharvest host for SWD relative to the other fruit hosts, with grapes  having the least potential.Novel methodology is presented for indexing the relative potential of hosts to function as resources. A Host Potential Index (HPI) was developed as a practical framework to express relative host potential based on combining results from one or more independent studies, such as those examining host selection, utilization, and physiological development of the organism resourcing the host. Several aspects of the HPI are addressed including: 1) model derivation; 2) influence of experimental design on establishing host rankings for a study type ; and, 3) variable selection and weighting associated with combining multiple studies. To demonstrate application of the HPI, results from the interactions of spotted wing drosophila (SWD),  There is interest in many areas of biology to understand and define relations between a host and the organism it harbors. Host preference and host suitability, as generally termed, are both critically related to the potential of a host to serve as a resource Host preference studies examine behaviors associated with host selection  Host suitability studies evaluate factors affecting the physiological development/performance of the harbored organism and other features of host quality A vs. male B dominance) There are several rating systems capable of producing ranking indices that have not been applied to host preference or host suitability. For example, several vertebrate behavior studies looking at paired outcomes . Accordingly, the Host Potential Index (HPI) was developed and applied to a scenario where understanding the relation between hosts and the harbored organism are of contemporary economic consequence in North AmericaDrosophila suzukii (Matsumura), (Diptera: Drosophilidae) is a polyphagous insect associated with a variety of economically important hosts, including berry crops and stone fruits The spotted wing drosophila (SWD), totHPIβ, for each host.The HPI model is used to calculate relative host potentials based on results from host suitability and/or preference studies that use no choice, two-choice, or multiple-choice experimental designs. A scoring system is applied and host rankings are subsequently established for each host in each study. The rankings of each host are then referenced against an index of tabulated HPI values. When hosts across studies are equivalent, the study-specific HPI values for each host can be pooled and prioritized using an additive model to yield on overall relative potential, Data can be collected from studies that include no-choice B, followed by hosts A and C equivalently. Host B would have a rank of 1, A = 2.5, and C = 2.5.For no-choice studies, hosts are ranked by the overall means of the measured response with the most positive host response ranked first. If data examined by multi-sample analyses (e.g. ANOVA) followed by multiple comparison tests  are used and result in means that are not statistically different, the ranks are averaged together. For example, a study measuring the mean adult mass of newly emerged females developing on three different hosts may find females are largest when developing on host A∶B, B∶C, A∶C) where it is not uncommon for A to be preferred over B, and B to be preferred over C, but where C is preferred over A. To determine which host is most likely preferred in these situations (which cannot be distinguished by ANOVA), a method was developed that incorporates strengths of preference and results in a C-score for each host (refer to Supporting Information S1 for detailed example). The resulting C-scores may then be used to differentiate host preference in pairwise events. Initially, all unexamined hosts begin with a default C-score, C = 900. C-scores change after differences in preference probabilities are evaluated, as described below, for each pairing of potential hosts .Two-choice studies involve three or more potential hosts each matched pairwise against one another . Note that all variables pertaining to Host A are denoted by a subscripted “a,” and Host B by a subscripted “b.”The expected preference probability for Host A, aE, is calculated from bC is the current C-score of host B, and aC is the current C-score of host A. Thus, if the C-scores are equivalent, the expected probability for host A preference is 0.50. If bC−aC>400 the expected preference probability (aE) for host A is limited to 0.08, or an 8% chance of host A being favored; likewise, if b−CaC<−400, aE is capped at 0.92 to reflect the lack of absolute certainty due to chaos inherent in biological systems The 0C is the C-score of host A prior to the pairwise combination, n is the number of pairwise events evaluated between host A and B, K is a constant determined by the K-factor table , and aE is the expected preference probability of host A when paired with B (from Eq. 2). The K-factor term represents the number of points available to host A  for increasing or decreasing its C-score. After calculation, 1C becomes the new C-score for host A and should be used in the subsequent pairwise calculation. Note that the C-score for host B must be calculated separately in the same manner. To prevent scores from attaining a negative value, a floor of C = 100 was established. No maximum value was set for C-scores.Once the actual and expected preference probabilities are calculated, the C-score for each potential host can be derived using a modified Elo-rating formula or table , Aa is toverall actual preference probability, A, (designated as Host 1) was initially paired with the host that had the lowest overall actual preference probability, and Eq. 3 was used to calculate the scores for each host. Subsequently, Host 1 was matched with the host possessing the second lowest actual preference probability (A) and C-scores were calculated for both hosts, until Host 1 was paired with every host in the study . Computation proceeded as above, without Host 1, for the host with the second highest actual preference probability (Host 2). Evaluation continued until the two least preferred hosts were paired. C-scores for each host were then used to assign rank.To limit potential variation in scores due to pairing order, the host with the highest A might influence selection between hosts B and C, must be taken into consideration. Data from multiple-choice studies was evaluated with a modified DEA formula originally used for voting analysis A, 24, 12, 20, and 18; B, 21, 21, 18, 17; and C, 23, 20, 19, and 16. The tabulated votes of preference would appear as in nd place, which in turn both outrank 3rd place, etc., a weighting factor is assigned to each place, as calculated fromWhen insects choose between several hosts simultaneously, dilution effects, where host jW is the jth place weighting factor, h is the number of hosts in the study, and j is the place . The weighting factor conforms to the rule that 1W≥22W≥33W≥jjW. For the example above, 1W = 0.333, 2W = 0.166, and 3W = 0.055. The M-score is then calculated using the modified DEA formulaiM is the M-score for host i, ijV is the number of jth place “votes of preference” for host i, and iN is the total number of “votes of preference” given to host i. Once M-scores are calculated, hosts are arranged by rank. If similar M-scores exist, ranks of those hosts would be averaged together. In the example . Host B would be ranked 3rd. example , MA = 0.A = 18.5 eggs/trial; Host B = 19.3 eggs/trial; Host C = 19.5 eggs/trial), the results would be markedly different. Furthermore, an ANOVA identifies no differences in rank.It is critical to note the utility of Eq. 5 for distinguishing preference in multiple-choice studies. Given the numerical example above, if the mean eggs per trial were used to establish rank (Host β (vide infra), testHPIβ. The reported value is then followed by the rank of that host followed by the number of hosts examined . Indexed values were derived through an iterative process and bounded such that no value reflected a “zero” potential while still providing a spread of values positively correlated to both rankings and the number of hosts evaluated  . If ties exist in rank order, then ranks of those hosts are averaged together. For example, if 6 hosts were examined and their ranks were 1, 2, 3.5, 3.5, 5 and 6, the HPI for the shared ranks of 3.5 would be mid-way between 271 and 257 (or 264) for both hosts.Once ranks are calculated for each study, the corresponding Host Potential Index value can be determined by referencing the HPI table , which ltotHPIβ), in the form 1β is the weighting coefficient for study 1, 2β the coefficient for the study 2, and nβ is the coefficient for the nth study, and Σxβ = 1.00. The model's multiplicative coefficient terms allow flexibility in prioritizing the contribution of a single study toward the overall evaluation of relative host potential, while the additive function precludes a “near-zero potential” in one study from obscuring results in other studies that support greater relative host potentials.If more than one study is considered when evaluating relative host potential, an overall HPI can be derived as a weighted total, , fltHPIβ2 the Flight Bioassay (two-choice design), popHPIβ3 the Population Level Oviposition Study (multiple-choice design), and indHPIβ4 the Individual Level Oviposition Study (multiple-choice design).The following application of the HPI involves spotted wing drosophila, where s).SWD pupae were originally obtained from the laboratories of Dr. Arytom Kopp (UC Davis) and Dr. Jana Lee (USDA-ARS) and colonies were established at the USDA-ARS-SJVASC  as described in Walse et al. s) and allowed to solidify.Media consisted of a fruit and agar mixture an were prepared as follows. Blueberries, blackberries, cherries, grapes, peaches, raspberries, or strawberries, were quickly dipped into a 35% ethanol bath, removed, and washed thoroughly in DI water. Each fruit type was respectively pureed in a laboratory blender and 400 mL of fruit pulp and juice, as well as 2 mL of formalin fungistat, were added to 400-mL of standard cornmeal-(dextrose or sucrose)-agar-yeast medium 1,15F = 5.610, P = 0.032), grapes , and raspberries  did exhibit density dependent effects on development time, but these were not observed when SWD developed on the fruit-agar media . Additionally, the fruit-agar media provided a surface-area normalized presentation for each fruit type thereby reducing the potential for confounding variables associated with fruit morphology and oviposition behavior.Population density is known to have an effect on development in some insects 3 infestation chamber . Each enclosure housed replicate Petri dishes (two) of blueberry, blackberry, cherry, grape, peach, raspberry, and strawberry agar media. All plates were introduced and lids were removed to initiate exposure to SWD. After a 24-h ovipositional period, Petri dishes were re-covered with lids, removed from the enclosure, labeled, and transferred to a 12.3-m3 incubation unit . Petri dishes were examined daily for adult SWD emergence. All emerging adults (<1-d old) were aspirated off the media, counted, and recorded 2O: 10.93-g NaCl, 1.57-g KCl, 0.83-g CaCl2*2H2O, 0.83-g MgCl2*6H2O) for gravimetric determination of mass. (vide infra).Four nylon-mesh enclosures , containing ∼2000 adult SWD each, were transferred from the rearing chamber to a 14.0-mD, in the form2γ was the kurtosis of the distribution of daily adult emergence, m was the mean mass of newly-eclosed adult females, and t was the mean time for adult emergence. The magnitude of D in Eq. 7 varies directly with host quality Larval performance was quantified, and rank for each host subsequently established, by combining three developmental diagnostics into a developmental score, t) and associated kurtoses, γ2, were calculated from the resulting distributions. A random sub-sample of 10 females was taken from each stored vial of Insect Ringer's solution  and weighed in bulk on an Ohaus Explorer microbalance (model #E04130) and average female mass (m) was determined for each variety of fruit-agar. The 10 females were returned and another sub-sample of 10 females was randomly chosen from the same vial and weighed. This process was repeated five times for each type of fruit. Mean D-scores (Eq. 7) for each fruit-agar variety were calculated and ranked.A histogram of adult emergence was generated for each fruit-agar dish and the mean time for adult emergence (s). Each chamber was fitted with a Dayton blower  powered by a 1118.55-W motor that drew air through the flight chamber and vented the exhaust external to the greenhouse. The air intake end of the flight chambers were furnished with three charcoal filters and one Filtrete® air filter (61×61×2.5 cm) (1900 MPR or 13 MERV rating) which was duplicated on the exhaust end of the chambers. Release platforms consisting of ring stands with adjustable angle flat-clamps were placed centrally 0.4 m from the rear and above the floor of each chamber. Each of two recapture traps were comprised of a 30.5-cm length of L-shaped 5.0-mm (I.D.) Pyrex® glass tubing connected to a 111-mL polystyrene sampling cup by a threaded polyethylene connector placed through the bottom of the sampling cup, thereby orienting the length of the cup horizontally and parallel to the wind direction. The traps on each chamber were located 22.8 cm from the edge of the lateral chamber walls and 25 cm downwind from the air intake. A double-sided yellow sticky card  cut to 38×70 mm was inserted into each trap to immobilized insects. Finally, each trap was sealed with a lid having a 9.5-mm hole drilled centrally to allow passage of the volatile-containing airstreams into the flight chamber and a threshold for test insects.Two Plexiglas flight chambers (0.61×0.61×2.13 m)  were used concurrently. Both flight chambers were oriented north to south in a shaded greenhouse at the USDA-ARS-SJVASC in Parlier, CA maintained at 20–30 °C and 60±5% RH (s) of fruit. Airflow exited each collection tubes through two exit ports (exit ports 3 & 4 of each tube were plugged) and was directed through Teflon® flex-tubing to a trap on each flight chamber. In this manner, each flight chamber simultaneously received equal quantities of fruit volatiles from each collection tube for pairwise comparisons.A modified volatile collection s) to optimize flight conditions for drosophilids To evaluate attraction to fruit volatiles, 80 mated female SWD (4 to 7-d old) were aspirated from the rearing colony and evenly separated into two 55.5-mL polystyrene vials, which were then capped and clamped to the release platforms 1 h prior to connecting the air delivery system to the flight chamber. Wind speed in the flight chambers was adjusted to 0.4±0.31 m/s (A (Eq. 1). For this study, which involved seven hosts, a matrix containing n(n−1) cells was created  along with the 4 replicates of 25 glass jars, each containing a peach sample. Glass jars (peach) and mesh ball cages were both removed from the incubation chamber daily and inspected for adults and fruit quality. If fruit showed signs of desiccation, an additional, uninfested berry or peach slice was added to provide moisture and/or resource. Live adults were removed, counted, and recorded.Four nylon-mesh replicate enclosures, containing ∼2000 adult SWD each, were transferred to an infestation chamber maintained with rearing conditions as described above. Stainless-steel trays (30×30×2 cm) filled with monolayers of mixed fruit types  were introduced into each enclosure. The stainless-steel trays containing infested fruit were removed from enclosures after a 72-hr ovipositional period. Infested berries were transferred to stainless-steel mesh ball cages (5.1-cm diameter) , while peaches were transferred to 473-mL glass jars with fine-screen lids. Mesh ball cages containing berries were grouped by replicate and berry type, placed inside pull-string cloth bags , and transferred to an incubation unit (n = 4) of 25 samples , average emergence from each fruit type was normalized relative to the surface area of the assayed unit . Within each replicate, fruit types were ranked and designated/tallied with 1st, 2nd, 3rd, etc. place “votes of preference.” A steel-mesh ball was estimated to have 12% the surface area of a peach. Results were input into Eq. 5 to generate an M-score for each fruit host.For each replicate (n = 4), 25 mated SWD females (4–7 d) from the colony were placed into individually respective 3.79-L glass cages with mesh lids containing a single blackberry, blueberry, cherry, grape, peach, raspberry, and strawberry and housed in a 12.3-m3 incubation unit . Females were removed after a 72-h ovipositional period and individual fruits were collected, labeled, and placed in mesh ball cages or jars as described above. Incubation and temporal analysis of adult emergence was as described in population level oviposition studies.To investigate potential for oviposition to be impacted by a myriad of possibilities, such as leking or prior oviposition events vide supra) and M-scores were derived using Eq. 5. If no adults emerged from any of the hosts exposed to a single female then that replicate was omitted from calculations since no fruit preference could be assigned for comparisons.Numbers of adults emerging from fruits were used to generate a table of tallied votes of preference  an ANOVA, or if appropriate, a Welch's ANOVA, was used to rank each host based on the mean responses for each host. If the ANOVA was significant a Tukey-Kramer HSD with α = 0.05 was used to establish rank.n term in Eq. 3, variation in C-scores increased directly with number of pairwise assays. A scenario involving 50 pairwise events was used for all calculations and statistical analyses reported. Computer-simulated C-scores and the resulting variation in rank due to pairwise sequencing were examined using both multiple and simple linear regression analyses An algorithm was written in C++  to evaluate pairing order effects on C-scores using equation 3 and dummy data sets for a specified number of host pairings with fixed differences in host preference. The number of hosts in the dummy data set was varied from 2 to 10 and the number of paired comparisons  was varied from 5 to 50. For each combination of hosts and pairwise assays, C-scores for each host were calculated for 100 random iterations. Quantifying the variation in C-scores based on paring order, number of hosts, and number of pairwise assays was used to estimate these effects on the generation of final scores. Due to the multiplicative nature of the β1 to β4) to values between 0.000 and 1.000, while summing to 1.000. HPI values for each host and each study were placed into Eq. 6 for each simulated weighting scenario and corresponding overall totHPIvaried values were calculated for each host. An ANOVA, or if appropriate, a Welch's ANOVA examine possible shifts in overall relative host potential due to the weighting of coefficients. If the ANOVA was significant (P<0.05) a Tukey-Kramer HSD with α = 0.05 was used.The effect of varying weighting coefficients on possible overall HPI values was examined using a four term simplex-lattice design with 231 distinct weighting scenarios F = 2.407; P = 0.035) with a maximum trial mean of 189.2±23.6 (s) for blackberry-agar medium and a minimum of 70.8±26.99 (s) for grape-agar medium. The overall mean time to peak adult emergence for all hosts following oviposition was 10.5±1.38 d (s), which is 0.9 d longer than the mean oviposition to emergence time reported by Kanzawa F = 5.303, P = 0.001). SWD larvae developed quickest on the cherry-agar medium and slowest on the grape-agar medium (12.1±1.93 d) (s) (Adult emergence varied between hosts (ANOVA:  d) (±s) .F = 28.690, P<0.001). Peach- and cherry-agar diets had the highest kurtoses with the bulk of adults emerging over a span of 48-h (α = 0.05), which displayed distributions that were generally long-tailed and spanned several days.The peakedness of adult emergence distributions  varied amongst types of fruit-agar media F = 17.221, P<0.001; Tukey-Kramer HSD, α = 0.05)(s) and the lightest emerged from grape-agar (1.44±0.037 mg) (s). When all three indicators of larval performance were considered in Eq. 7, peach-agar medium had the highest score (D = 1.56±0.22 mg/d) (s), whereas the grape-agar medium had the lowest score (D = 0.41±0.15 mg/d) (s) F = 29.70, P<0.001; Tukey-Kramer HSD, α = 0.05). The D-scores, ranks and lpHPIβ1 for the development study are shown in In α = 0.05). The heaR2 = 0.93, F = 69.95, P<0.001) between recapture and corresponding actual preference probabilities, A (Eq. 1), (s = 7.15) minimized observed variation.A total of 86 flight trials were conducted, in 21 pairwise combinations, with a minimum of 4 replicate trials for each combination. A total of 3440 mated female SWD were released and a total of 1410 were subsequently trapped . A strong correlation ((Eq. 1),  supportsa; strawberry, 12.46a; blackberry, 11.46a,b; peach, 6.92b,c; cherry, 5.25c; blueberry, 5.04c; grape, 3.96c) F = 9.236, P<0.001). Mean SWD recaptured not connected by the same letter were significantly different . Raspberries have the highest actual preference probability (raspA = 0.917), followed by blackberries (bkbA = 0.729), strawberries (strawA = 0.667), peaches (peachA = 0.396), cherries (cherA = 0.333), blueberries (blbA = 0.327), and grapes (grapA = 0.173). The C-scores, ranks, and fltHPIβ2 for the Flight Bioassay are shown in Mean female SWD recaptured in traps differed depending on host . The range of variation, however, did not differ by sequence position , indicating that no specific position provided a scoring advantage over another. These results reinforce the use of a standard pairing order procedure for HPI analysis (highest WLT vs. lowest WLT method [vide supra]).The 50-event simulation yielded C-scores for the 10 hosts that were subsequently indexed and ranked. Pairing order had a statistically significant effect on the resulting C-scores F = 122.74, P<0.001; Tukey-Kramer HSD, α = 0.05) with mean adult SWD emergence (s): strawberries, 26.45±10.78/berry; raspberries, 5.39±1.45/berry; cherries, 7.01±3.06/berry; blackberries, 6.56±3.74/berry; blueberries, 1.90±0.90/berry, grapes, 0.43±0.30/berry, and peaches, 0.04±0.20/fruit, suggesting females have the greatest preference for ovipositing in strawberries. M-scores corroborated strawberries were preferred over all fruit types, with strawberries accounting for 28.1% of all emerging adults when normalized for surface area as described above F = 6.746, P<0.001; Tukey-Kramer HSD, α = 0.05). While not statistically significant, it is interesting to note that no adult SWD emerged from peaches  in the HPI model to prioritize specific host preference or suitability studies.Based on biological consideration(s) and/or precedence, researchers may adjust the weighting coefficients ; strawberries ; blackberries ; cherries ; peaches ; blueberries ; and grapes   .lpβ = 0.05, fltβ = 0.05, popβ = 0.45, indβ = 0.45), the totHPI0.05,0.05,0.45,0.45 would shift peaches to the host with the least potential  while keeping the remaining hosts in order, respectively. On the other hand, if no-choice larval performance on fruit-agar diet was emphasized , peaches would reflect the highest relative potential to serve as SWD hosts . The calculated HPI values could theoretically change by as much as 108 points for peaches depending upon prioritization  and lead to completely opposite conclusions regarding the potential of postharvest peaches to serve as hosts for SWD. Thus, the prioritization of weighting factors should be justified. In this example, no adults emerged from oviposition studies on intact peaches, so it would be questionable to use the development on fruit-agar material as the only HPI-criterion for evaluating the host potential of peaches relative to the other fruit.A single study may be emphasized. For example, both multiple-choice studies indicated a near zero potential for SWD to emerge from peaches, likely due to the inability of the ovipositor to penetrate the skin of commercially harvested fruit, at least fruit sourced from California for export distribution. If oviposition is emphasized (β-coefficients (Eq. 6) were modulated (from 0.00 to 1.00) using the simplex-lattice design (vide supra); this allowed for differences in the relative host potential to be visualized and possible distinctions between hosts to be evaluated  near lower indexed values with relatively small deviation (s = 3.215). Values for raspberries, on the other hand, were localized at the higher end of the scale  and were also characterized by a small deviation (s = 8.393). As discussed above, HPI values for peaches show non-localized distribution  as characterized by a large deviation (s = 22.241). Mean total meanHPIvaried (s) values were calculated and compared for equivalency F = 3572.2, P<0.001): raspberrya (301.8±8.39), strawberryb (296.9±16.29), blackberryc (274.8±6.64), cherryd (267.4±12.15), peache (261.4±22.24), blueberryf (251.4±10.96), and grapeg (232.2±3.21). meanHPIvaried (s) values were then used as a quantifiable metric for distinguishing relative host potential of SWD in a postharvest scenario.Lastly, all weighting possibilities can be considered to account for variation resulting from prioritization. In the current study, valuated . For exaThe HPI enables researchers to quantitatively probe the relationship between a host and the organism it harbors by combining results from disparate studies, having different designs, data ranges, values and units, or similar studies that potentially yield conflicting results Supporting Information S1C-Score Calculations. Step by step application for C-score analysis with example from two-choice study flight bioassay. Eqs. 1–3 are solved in detail.(DOCX)Click here for additional data file.Supporting Information S2M-Score Calculations. Step by step application for M-score analysis with example from multiple-choice population oviposition study. Eqs. 4–5 are solved in detail.(DOCX)Click here for additional data file.Supporting Information S3HPI Table Derivation. Rules and formulae for developing the Host Potential Index are presented and discussed.(DOCX)Click here for additional data file.


Recently, MAYV has attracted attention due to its likely urbanization. Currently, there are no licensed drugs against most mosquito-transmitted viruses. Here, we investigated the B. longifolia and chemically identified as quercetin and quercetin glycosides using spectroscopic techniques. Cytotoxicity of purified flavonoids and of EtOAc- and n-BuOH-containing flavonoid mixtures was measured by the dye-uptake assay while their antiviral activity was evaluated by a virus yield inhibition assay.Flavonoids were purified by chromatographic fractionation from leaf extracts of B. longifolia leaves: non-glycosylated quercetin and its glycosides guaijaverin, quercitrin, isoquercitrin, and hyperin. EtOAc and n-BuOH fractions containing these flavonoids demonstrated the highest antiviral activity of all tested substances, while quercetin had the highest antiviral activity amongst purified flavonoids. Quercetin, EtOAc, or n-BuOH fractions inhibited MAYV production by more than 90% at 25 μg/mL, displaying a stronger antiviral effect than the licensed antiviral ribavirin. A mixture of the isomers isoquercitrin and hyperin had a modest antiviral effect (IC90 = 104.9), while guaijaverin and quercitrin did not show significant antiviral activity.The following flavonoids were purified from B. longifolia is a good source of flavonoids with anti-Mayaro virus activity. This is the first report of the activity of quercetin and its derivatives against an alphavirus. Brazil remains extensively covered by tropical forests and other natural ecosystems, despite high deforestation rates. A critical effect of deforestation is the introduction of new viruses into urban areas, providing ideal conditions for the development of several arboviruses. Indeed,Mayaro virus (MAYV), a member of the Togaviridae family (genus Alphavirus), was first isolated in Trinidad in 1954. MAYV isHaemagogus species, although Aedes species might also act as MAYV vectors. Thus, it is possible that, under appropriate conditions, MAYV can be transmitted in urban areas[Antibodies against MAYV are often detected in certain species of monkeys, which are likely to be the main natural reservoir of the virus, while tan areas.In Brazil, MAYV is endemic to the Amazon region, but cases of the disease have been observed elsewhere. Mayaro fever outbreaks occurred in central regions in Brazil, namely Itarumã (Goiás state) and in Peixe (Tocantins state), in 1987in vitro studies such as the cell culture approach in this paper.Currently, about 40 antiviral drugs are available for clinical use, mainly targeting HIV and a small number of other viruses. NeverthBauhinia (Fabaceae) genus are found in tropical regions of Asia, Africa, and Central and South America. In Brazil, these plants are known as ‘pata-de-vaca’ (cow’s foot) because of their leaf shape[B. longifolia and B. forficata leaves is consumed in certain regions of Brazil[Bauhinia genus[Shrubs from the af shape. Tea madf Brazil for its ia genus. Flavonoia genus.Bauhinia longifolia (Bong.) Steud. against MAYV replication in Vero cells. We also determined the selective antiviral activity of purified quercetin and quercetin glycoside derivatives, as well as of leaf extracts rich in these flavonoids.In view of this interesting scenario, in the present study we evaluated the antiviral activity of Bauhinia longifolia (Bong.) Steud. collected in the municipality of Luz, Minas Gerais state (Brazil) were used. Plant species authentication was performed by comparison with herbarium specimens from the Institute of Biological Sciences , where a voucher specimen (BHCB 18778) was deposited.In this study, leaves from wild specimens of 2, in Dulbecco’s modified Eagle’s medium (DMEM)  supplemented with 5% fetal bovine serum , 50 IU/mL of penicillin, and 50 μg/mL of streptomycin . Mayaro viruses  were propagated in Vero cells and viral stocks in 10% glycerol were kept at -70°C. Virus titer was determined by plaque assay .Vero cells  were grown at 37°C with 5% CO2O to a ratio of 9:1, and then extracted successively with n-hexane, CH2Cl2, EtOAc, and n-BuOH.Air-dried and powdered leaves (1.5 Kg) were extracted with MeOH at room temperature for 7 days. Pooled methanol extracts were filtered and concentrated under reduced pressure to produce a crude extract (95 g), which was diluted in MeOH-Hn-BuOH (6 g) fractions were chromatographed separately on amberlite XAD-16 columns (2 m × 8 cm i.d.) (Sigma-Aldrich). Aqueous methanol solutions  were used as the mobile phase, and 11 fractions were collected for each extract . After thin layer chromatography (TLC) analysis, selected fractions were pooled in three groups: F1, including fractions A6–A9 (4.5 g); F2, including fractions A10–A11 (0.9); and F3, including fractions B6–B11 (1.9).EtOAc (7 g) and 2O (1:1) as the mobile phase. F1 chromatography yielded fractions containing guaijaverin (1) and quercitrin (3), F2 fractions contained aglycon quercetin (2), and F3 fractions contained a mixture of the isomers isoquercitrin (4a) and hyperin (4b).F1 and F3 were chromatographed separately on Sephadex LH-20 columns (30 cm × 45 mm) (Sigma-Aldrich) with MeOH-Hn-BuOH fractions described above. The mobile phase of HPLC-DAD analysis consisted of (A) 1% phosphoric acid in water or (B) 1% phosphoric acid in methanol. Gradients used were: 0–15 min 50–70% of B followed by 15–25 min 70–100% of B. The flow rate was 1 mL/min and the injection volume 20 μL. The UV–vis spectra were recorded from 254 to 400 nm, with detection at 254 and 365 nm. TLC was performed on silica gel Plates 60 F254 , using ethyl acetate-methanol–water-acetic acid (8:1:0.5:0.5) as eluent. After elution, TLC plates were observed under 254 nm UV light and then sprayed with solutions of NP (2-aminoethyldiphenylborinate 1% in methanol) and PEG-400 (polyethylene glycol 5% in ethanol)  before examination under 365 nm UV light. The presence of sugars was confirmed by acid hydrolysis of glycosides on TLC plates, and the sugar composition was inferred by comparison with standards of each of the following sugars: arabinose, xylose, galactose, glucose, and rhamnose . Final compound identification was performed by extensive 1D- (1H and 13C) and 2D-  NMR experiments, ESI-MS, UV spectral analysis, and by comparison with literature values.HPLC-DAD (High Performance Liquid Chromatography with Diode Array Detector) and TLC (Thin Layer Chromatography) were used to analyze the chemical composition of EtOAc and B. longifolia-derived flavonoids, EtOAc and n-BuOH fractions, was performed using a dye-uptake method modified from Borenfreund and Puerner[50 and CC90, respectively).Cytotoxicity analysis of  Puerner. Briefly2) with different concentrations (0–100 μg/ml) of flavonoids or extracts diluted in culture medium. After treatment, culture supernatants were recovered and used for titration of extracellular infectious virus particles. For virus titration, confluent cell monolayers in 24-well plates were infected with serial dilutions of recovered supernatants for 1 hour at 37°C, 5% CO2. After inoculum removal, cells were rinsed with PBS and the monolayer was incubated with fresh medium with 2% carboxymethylcellulose  for 48 hours at 37°C, 5% CO2. Finally, cells were fixed with 20% formaldehyde and stained with 0.5% crystal violet in 20% ethanol, and viral plaques were counted. Ribavirin  was used as positive control for MAYV replication inhibition. For each substance or extract, IC50 and IC90 values were calculated and used to obtain a selectivity index (SI), expressed as the ratio CC50/IC50, and to estimate relative potency (RP) as the ratio between ribavirin (reference substance) IC90 and the tested substance’s IC90. Results were presented as mean inhibitory/cytotoxic concentration ± SD, and t-tests were used to evaluate the statistical significance of treatments relative to controls. P-values < 0.05 were considered statistically significant.Antiviral activity was evaluated using a virus yield inhibition assay. Briefly, confluent cell monolayers grown in 24-well plates were infected with MAYV (multiplicity of infection = 0.1) for 1 hour, then rinsed with PBS and treated for 24 hours [2)[O-α-rhamnoside, peak 3)[O-β-glucoside, peak 4a); and hyperin [Using NMR, ESI-MS, and UV spectral analyses, the compounds in profile peaks from Figure  peak 1); quercetk 1)[2); quercit peak 3); isoquerpeak 4b). Figure B. longifolia is revealed by the absence of kaempferol derivatives (commonly found in the genus) and by the identification of quercetin derivatives only. Furthermore, the presence of quercetin 3-O-monoglycosides suggests the effective action of glycosilation enzymes such as 3-O-transferases[The simplicity of the foliar flavonoid metabolism of sferases.n-BuOH fractions inhibited MAYV replication in Vero cells in a dose-dependent manner, with more than 90% inhibition at 25 μg/mL (Table 4a) and hyperin (4b) or the antiviral ribavirin were much less potent inhibitors of MAYV replication, with IC90 values above 100 μg/mL  displayed stronger antiviral activities than those observed for the purified compounds and for the antiviral ribavirin, used as a positive control. Quercetin and the mixture of isoquercitrin and hyperin in identical proportions showed significant anti-MAYV activity while guaijaverin and quercitrin did not.In this study, the antiviral activity of quercetin and quercetin derivatives from et al.[O-rutinoside), kaempferol-3-O-rutinoside, and kaempferol-3-O-robinobioside compared to that of the respective non-glycosylated compounds (quercetin and kaempferol). The difference in the findings may be explained by the fact that MAYV and HSV-1 are viruses with very different replication strategies.Importantly, quercetin displayed better selectivity towards MAYV and improved potency (SI = 94 and RP = 5) when compared to the antiviral ribavirin (SI = 8), clinically approved for the treatment of Hepatitis C virus infection and also experimentally shown to inhibit several other RNA and DNA viruses such as influenza, hepatitis B, poliomyelitis, measles, and smallpox. Also, tet al. reportedO-glycosilation with arabinose and rhamnose is particularly detrimental to the antiviral activity against MAYV. This is an interesting finding already under investigation by our group.The mixture of isoquercitrin and hyperin had anti-MAYV activity similar to that displayed by ribavirin, but the Selectivity Index was lower (4). This was due to the higher toxicity of the isoquercitrin/hyperin mixture if compared to ribavirin. However, the Relative Potency for ribavirin and the isoquercitrin/hyperin were similar, suggesting possible synergism between these isomeric flavonoids. The absence of anti-MAYV activity for guaijaverin and quercitrin in the concentrations tested here suggests that 3-in vitro against a wide array of viruses, including HSP-1, parainfluenza-3, influenza, and dengue virus type-2 (DENV-2)[et al.[According to Gould and Lister, flavono(DENV-2)-43, even)[et al. suggeste)[et al.. Inhibit)[et al..B. longifolia is a valuable source of flavonoids with antiviral activity against the arbovirus MAYV. Although quercetin and its 3-O-glycosides are fairly common in plants, this is the first report of anti-MAYV activity for these flavonoids. Our data are an important step in the evaluation of natural products as sources of novel drugs to be used in combination therapy, to circumvent drug resistance, or to replace currently used antivirals with unwanted cytotoxic effects.Our results show that The authors declare that they have no competing interests.RMK and DF conceived and designed the study. AES collected plant material, performed all phytochemical experiments and wrote the initial draft of the manuscript. KAY and TSS performed cytotoxicity and viral yield inhibition assays. MDFM and RC revised the data and carried out statistical analyses. MRS provided invaluable discussions on the chemical data and antiviral chemistry. All authors read and approved the final manuscript.