Leishmania that affects millions of people around the world. Drugs employed in the treatment of leishmaniasis have limited efficacy and induce local and systemic side effects to the patients. Natural products are an interesting alternative to treat leishmaniasis, because some purified molecules are selective toward parasites and not to the host cells. Thus, the aim of the present study was to compare the in vitro antileishmanial activity of the triterpenes betulin (Be), lupeol (Lu), and ursolic acid (UA); analyze the physiology and morphology of affected organelles; analyze the toxicity of selected triterpenes in golden hamsters; and study the therapeutic activity of triterpenes in hamsters infected with L. (L.) infantum as well as the cellular immunity induced by studied molecules. The triterpenes Lu and UA were active on promastigote  and amastigote forms  of L. (L.) infantum, and their selectivity indexes (SI) toward amastigote forms were higher  than SI of miltefosine (2.7). L. (L.) infantum promastigotes treated with Lu and UA showed cytoplasmic degradation, and in some of these areas, cell debris were identified, resembling autophagic vacuoles, and parasite mitochondria were swelled, fragmented, and displayed membrane potential altered over time. Parasite cell membrane was not affected by studied triterpenes. Studies of toxicity in golden hamster showed that Lu did not alter blood biochemical parameters associated with liver and kidney functions; however, a slight increase of aspartate aminotransferase level in animals treated with 2.5 mg/kg of UA was detected. Lu and UA triterpenes eliminated amastigote forms in the spleen  and liver of infected hamster ; and UA showed similar activity at eliminating amastigote forms in the spleen and liver than amphotericin B (99.2 and 99.8% of reduction). The therapeutic activity of both triterpenes was associated with the elevation of IFN-γ and/or iNOS expression in infected treated animals. This is the first comparative work showing the in vitro activity, toxicity, and therapeutic activity of Lu and UA in the chronic model of visceral leishmaniasis caused by L. (L.) infantum; additionally, both triterpenes activated cellular immune response in the hamster model of visceral leishmaniasis.Leishmaniasis is a neglected tropical disease caused by the flagellated protozoa of the genus  L. (L.) chagasi or L. (L.) infantum [Leishmaniasis is a neglected tropical disease and a public health problem worldwide, affecting vulnerable people in 98 countries, with 12 million cases detected worldwide per year; additionally, 1 billion people live in areas at risk of transmission [infantum .The therapeutic arsenal available for the treatment of leishmaniasis is scarce, and it is based on the use of the first-line drug, pentavalent antimonial . Second-Based on the aforementioned comments, it becomes clear the importance of identifying new leishmanicidal drugs. According to DNDi (Drugs for Neglected Diseases initiative), new prototype drugs need to be safe and affordable. In this sense, different studies have shown that natural molecules have these characteristics and can be considered prototype antileishmanial drugs . FurtherTriterpenes are the most representative group of phytochemicals, comprising more than 20,000 recognized compounds, and are biosynthesized in plants through the cyclization of squalene . Due to L. (L.) infantum [Leishmania sp., the present study was aimed at analyzing the activity and selectivity of Be, Lu, and UA on an American strain of L. (L.) infantum, as well as the possible target organelles. Additionally, the toxicity, therapeutic, and immunomodulatory activities of these triterpenes were studied in the experimental model of chronic visceral leishmaniasis.Betulin (Be), lupeol (Lu), and ursolic acid (UA) are pentacyclic triterpenoids widely distributed in nature and exhibit important pharmacological effects such as antioxidant, antiallergic, antipruritic, antiangiogenic, and antimicrobial agents –18. Addiinfantum , 21 and 1H and 13C nuclear magnetic resonance (NMR) spectra were recorded, respectively, at 300 and 75 MHz in a DPX-300 spectrometer  using deuterochloroform (CDCl3), deuterated dimethyl sulfoxide (DMSO-d6), or deuterated methanol (CD3OD) as solvents and internal standard . Elemental analysis was obtained in an Elemental Analyzer 2400 CHN .Be, Lu, and UA triterpenes were purchased from Cayman Chemicals (USA). Mesocricetus auratus), 8 weeks old, were obtained from Anilab . This study was carried out in strict accordance with the recommendations of the guide for the Care and Use of Laboratory Animals of the Brazilian National Council of Animal Experimentation (http://www.cobea.org.br). The protocol was approved by the Ethics Committee of Animal Experiments of the Institutional Committee of Animal Care and Use at the Medical School of São Paulo University (056/16). Hamsters were housed in the Animal Experimental Institute of Tropical Medicine of São Paulo (IMT-USP), according to the standards of the Committee of Animal Welfare, and allowed access to food and water ad libitum throughout the study under a 12 h light cycle. The animals were anesthetized with intraperitoneal sodium thiopental at 1 mg/200 μL .Golden hamsters (Mesocricetus auratus) (106 macrophages/well) were cultured in 96-well plates in RPMI medium supplemented with 10% of fetal bovine serum , 2 mM L-glutamine , 10 mM Hepes , 1 mM sodium pyruvate, 1% v/v nonessential amino acid solution , 10 μg/mL of gentamicin , and 1000 U/mL of penicillin  (R10) along with triterpenes Be, Lu, and UA or the standard drug miltefosine (2.0 to 240 μM). The plates were incubated at 37°C, 5% CO2, for 24 h and then centrifuged at 400 × g for 10 min at 4°C and washed 3 times, followed by addition of 9.6 μM of 3--2,5-diphenyltetrazolium bromide (MTT) . Four hours later, 50 μL of 10% sodium dodecyl sulfate (SDS) was added to each well. The plates were further incubated for 18 h and read in an ELISA reader at 595 nm. Cytotoxic concentration 50% (CC50) was estimated using the nonlinear regression test with GraphPad Prism 5.0 software. The index of selectivity (SI) was estimated according to Passero and collaborators [50 and IC50.Peritoneal macrophages from golden hamsters  infantum (MHOM/BR/72/46) were incubated in a 96-well culture plate in Schneider's medium  supplemented with 10% heat-inactivated fetal bovine serum and 50,000 IU/mL penicillin and 50 mg/mL streptomycin (S10), at 2 × 106 promastigotes/well with the triterpenes Be, Lu, and UA or standard drug miltefosine from 2.0 to 240 μM . The negative control group was cultivated in medium and vehicle solution (PBS plus 1% DMSO). The parasites were incubated for 24 h at 25°C, then washed with 200 μL of PBS three times with centrifugation at 1200 × g, 10 min at 4°C, followed by addition of 9.6 μM of MTT. Four hours later, 50 μL of 10% sodium dodecyl sulfate (SDS) was added to each well [50) was estimated using GraphPad Prism 5.0 software.Promastigote forms of ach well . The pla6 macrophages) were collected and cultured on round coverslips in 24-well plates during 4 h in R10, and then, cells were infected with L. (L.) infantum promastigotes . The plates were incubated overnight at 5% CO2 at 37°C. Then, the triterpenes Be (14 to 113 μM), Lu (12 to 96 μM), and UA (2 to 22 μM) were added to the infected macrophages. Of note, the highest concentration used was always below the values of the respective CC50 values. Miltefosine (18 to 74 μM) was used as the positive control. After 24 hours, macrophage supernatants were collected and stored at -80°C for quantifications of nitric oxide (NO)  and hydrogen peroxide (H2O2)  according to the manufacturer's instructions of the respective kits. The coverslips were dried at room temperature, fixed in methanol, and stained by Giemsa . The number of infected macrophages and of parasites per macrophage was determined at least in 100 cells. The infection index (II) was expressed as the percentage of infected macrophages multiplied by the average number of amastigotes per macrophage according to Passero and collaborators [50) of the infection index was estimated using GraphPad Prism 5.0. As a positive control for NO and H2O2, macrophages were incubated with 100 ng/mL LPS  according to Passero et al. [Peritoneal macrophages from golden hamsters (10 UA 2 to  μM were L. (L.) infantum (2 × 106 promastigotes/well) were incubated in a 24-well culture plate in S10 with Lu and UA at EC50 for 24 h, 25°C. The control group was cultivated with medium and vehicle solution. The plate was centrifuged at 1200 × g, 4°C, for 10 min and washed three times with 200 μL of PBS. Then, the pellets were resuspended in glutaraldehyde 2% , pH 7.2, and incubated at 4°C, during 60 min. Parasites were postfixed in 1% osmium tetroxide , and further incubated with 0.5% aqueous uranyl acetate overnight, and dehydrated in a graded series of ethanol. Then, samples were embedded in a polyester resin, thin sectioned with Reichert ultramicrotome, double stained by uranyl acetate and lead citrate (Ladd Research Industries), and examined with a Jeol 1010  transmission electron microscope (TEM).Promastigote forms of L. (L.) infantum (2 × 106 promastigotes/well) were incubated in 96-well black culture plate  in S10 with the IC50 of triterpenes Lu and UA as well as miltefosine for 0, 10, 20, 30, 40, 50, 60, 120, and 1440 minutes. At each time point, the probes SYTOX Green at 0.5 μM/well  or Rhodamine 123 at 3 μM/well  were added in the parasite culture in order to evaluate cell membrane damage or mitochondrial membrane potential, respectively. Parasites were placed in the dark for 15 minutes at 25°C, then centrifuged at 1200 g, 5 min at 10°C, and washed three times with 200 μL of PBS. Plates containing parasites stained with SYTOX Green were read in a fluorescence reader using 530 nm emission wavelength and 490 nm excitation, and parasites stained with Rhodamine 123 read with 520 nm emission wavelength and 485 nm excitation. Results were normalized in relation to the control, nontreated parasites. Triton X-100 (0.05 μL)  was used as a positive control for cell membrane damage and oligomycin at 0.1 μM/well  as positive control of mitochondrial membrane potential inhibition.Promastigote forms of Healthy golden hamsters (8 weeks old) were divided into four groups containing 5 animals/group. The experimental groups were arranged as follows: groups 1 and 2 were treated with 2.5 mg/kg of UA or Lu, respectively. Group 3 was treated with 5.0 mg/kg of amphotericin B  (Cristál7L. (L.) infantum promastigotes (MHOM/BR/72/46). The noninfected control group was injected with PBS alone. Infected hamsters were divided into 4 groups, with 5 animals each. After 60 days of infection, animals were treated with UA, Lu, or AmB and the last group was constituted by animals that received only the vehicle solution (control). The protocol of the treatment is described in the Golden hamsters were infected intraperitoneally with 2 × 10β-actin (endogenous control). Quantitative PCR (qPCR) reaction was carried out using the GoTaq® 1-Step RT-qPCR System  and 75 nM of primers. The primer sequences  were as follows (5′ to 3′): IFN-γ forward: GACAACCAGGCCATCC and reverse: CAAAACAGCACCGACT; IL-10 forward: TGGACAACATACTACTCACTG and reverse: GATGTCAAATTCATTCATGGC; iNOS forward: CGACGGCACCATCAGAGG and reverse: AGGATCAGAGGCAGCACATC; and β-actin forward: TCCTGTGGCATCCACGAAACTACA and reverse: ACAGCACTGTGTTGGCATAGAGGT. Quantification results are expressed in fold changes of 2ΔCt- over the infected control group. PCR products were electrophoresed on 2% agarose gels to confirm amplification of products with the correct size; one single amplification product of predicted size, according to Lafuse et al. [RNA from hamster spleen fragments (~10 mg) was extracted using the commercial RNeasy Mini Kit  according to the manufacturer's protocol. cDNA was synthesized with the SuperScript®VILO™ cDNA synthesis kit . Amplification consisted of an initial denaturation phase at 95°C for 10 min, followed by 40 amplification cycles consisting of 95°C for 15 s, 61°C for 90s, and 72°C for 30 s, using a thermocycler . Prior to quantification, the efficiency of each reaction was verified using cDNA from the spleens of a healthy animal; it was always above 95%. Expression levels of genes of interest were normalized to e et al. , was alwp value < 0.05.All data obtained have been reported as the mean of three independent assays. Values are expressed as mean ± standard error. Statistical analyses were performed using GraphPad Prism 5.0 software, and the ANOVA test was used to assess the differences between groups. Statistical significance was set at a 1H and 13C) data triterpenes betulin (Be), lupeol (Lu), and ursolic acid (UA) were recorded, and obtained data were compared with those reported in the literature [NMR (terature –31. ThesL. (L.) infantum were incubated with the triterpenes during 24 h, and the inhibitory 50% concentration (IC50) for each compound was estimated. All triterpenes were active against promastigote forms of L. (L.) infantum, being Lu (IC50 = 4.0 ± 0.3 μM) more effective at inhibiting promastigote form growth, followed by UA (IC50 = 8.0 ± 0.2 μM) and Be (IC50 = 133.0 ± 3.0 μM). Miltefosine, used as positive control, showed an IC50 of 13.5 ± 0.9 μM  infantum. In this regard, compound Lu was 58.5 times more selective to promastigote forms than the host macrophages, followed by triterpenes UA and Be . Miltefosine presented a SI of 6.6. These data are summarized in Based on the ratio between ICL. (L.) infantum and treated with triterpenes Lu and UA exhibited significant decrease in the parasitism, as demonstrated by the respective IC50 values. As shown in 50 values of 3.0 ± 0.2 μM and 17.5 ± 0.4 μM. Be was inactive on amastigote forms of L. (L.) infantum. Miltefosine killed amastigote forms with an IC50 of 33.4 ± 2.0 μM. Comparatively, Lu and UA triterpenes were more effective at killing intracellular amastigote forms compared to the standard drug miltefosine. Selectivity indexes of analyzed triterpenes over amastigote forms of L. (L.) infantum were higher than those determined to miltefosine (SI = 2.7).Macrophages infected with L. (L.) infantum and treated with triterpenes Be, Lu, and UA did not produce quantifiable levels of NO. By the other side, macrophages infected and treated with Lu increased the levels of H2O2 in a dose-dependent manner when compared to the control group (p < 0.05), as indicated in 2O2. Noninfected macrophages did not produce measurable levels of H2O2. Macrophages treated with LPS produced high amounts of NO (17.1 ± 2.2 μM) and H2O2 (213.1 ± 24.4 μM).Macrophages infected with Control promastigote forms showed a well-preserved external structure, with fusiform shape and intact cell membrane . The cyt50 for 24 h showed major changes in the morphology (Promastigote forms treated with Lu at the ICrphology . Cell merphology . The comrphology  was obserphology .∗)  infantum treated with miltefosine lost the fusiform morphology, and the cytoplasm was degraded, showing structures resembling autophagic vacuoles (∗). Nuclear membrane detachment was observed (observed . The comobserved  and fragobserved .L. (L.) infantum  in a time-dependent manner at 30 minutes of incubation. Additionally, at 45 minutes, a hyperpolarization was detected in parasites treated with UA and miltefosine. Parasites treated with triterpenes Lu and UA displayed complete inhibition of the mitochondrial membrane potential at 60 and 1440 minutes of incubation , as demonstrated by limiting-dilution assay. In addition, tissue amastigote forms were stained by immunohistochemistry, and it is possible to observe that the treatment carried out with UA or Lu drastically decreased the number of parasites in comparison to the spleen and liver of infected control.Infected hamsters treated with 2.5 mg/kg of UA or Lu showed significant reduction in the splenic and hepatic parasitism  infantum-infected hamsters showed that animals treated with 2.5 mg/kg of UA or Lu expressed higher levels of IFN-γ compared to the infected control (p < 0.05). Animals treated with amphotericin B expressed lower levels of IFN-γ in comparison to the infected control, and it was similar to the healthy control. In respect to iNOS gene expression, only animals treated with lupeol showed significant elevation in the expression of this mediator of inflammation compared to the infected control (p < 0.05). Animals treated with 5 mg/kg of amphotericin B exhibited a significant decrease expression of IL-10 in comparison to the infected control (p < 0.05).Analysis of cellular immune response in the spleen of in vitro leishmanicidal activity on promastigote forms of L. (L.) infantum that were more sensitive to Lu, followed by UA and Be. However, Lu and UA impacted the survival of intracellular amastigote forms, while Be was inactive. Although triterpenes presented similar structures, they also displayed different activities on promastigote and amastigote forms. Possibly, the liposolubility [P) between octanol and water, octanol being assumed to have a similar lipophilicity to cell membranes [P of 7.67 [P values determined, respectively, as 6.43 [The quality of drugs is a fundamental factor to ensure pharmacological activity and minimize the occurrence of unwanted effects resulting from the presence of impurities and/or degradation products. Thus, according to the nuclear magnetic resonance, associated with the elemental analysis, it was possible to confirm the purity degree of all studied triterpene  in comparison to Lu, and such pharmacological activity has been related to the presence of the carboxylic acid at the position C-28 in the structure of compound UA that was not observed in the structure of Lu. By comparing the structure of all studied triterpenes, the presence of the hydroxyl group at the position C-28 found in the structure of Be may determine the low or absent leishmanicidal activity found herein. Additionally, it is important to note that Lu and UA showed higher activity and selectivity toward parasites than miltefosine, and thus, these molecules should be considered to develop new leishmanicidal drugs. Additionally, previous studies have been verified that both Lu and UA are active on promastigote and amastigote forms of different  species , 37–39, 50 above 240 μM that was higher than that of miltefosine. UA showed the highest toxic potential toward macrophage, and it was associated with the presence of carboxylic acid at C-28 and hydroxyl group at C-3 position. In spite of showing some toxicity to the host cell, the selectivity toward amastigote forms (SI~14) suggests that this triterpene is still an important leishmanicidal agent. Moreover, other studies have already shown that these triterpenes have absent or low cytotoxic effects on different cell lineages [Regarding cytotoxic potential of triterpenes on peritoneal macrophages of golden hamsters, it is important to note that Lu had CClineages , suggestLeishmania sp. suppresses respiratory burst in the host macrophages, so compounds able to increase macrophage respiratory activity can aid the parasite elimination. In this regard, it was observed that infected macrophages treated with Lu produced elevated amounts of H2O2 that is able to trigger programmed cell death in Leishmania sp. [During infection, ania sp. , 44, 45.ania sp. .L. (L.) infantum were analyzed by transmission electron microscopy, followed by physiological changes in the plasma and mitochondrial membranes. In general, parasites showed altered morphology, intracellular disorganization, blebs, chromatin condensation, nuclear fragmentation, and different cytoplasmic alterations, mainly related to the cytoplasmic extraction. Additionally, changes in the parasite mitochondria and nucleus suggest that triterpenes act on the parasite's bioenergetics or even inducing programmed cell death [L. (L.) infantum. According to Vannier-Santos and Castro [Leishmania sp. Previous studies have shown that programmed cell death may be a common mechanism of cell death in Trypanosoma sp. [Leishmania [L. (L.) infantum.In order to analyze major morphological changes induced by triterpenes, promastigote forms of ll death –48. In fll death , suggestd Castro , pyknosid Castro , 51, sugsoma sp. –54 and Lishmania , 55 in rishmania  and tritishmania , 42, 43.Trypanosoma cruzi treated with lysophospholipid analogues, such as edelfosine, ilmofosine, and miltefosine [Despite cell protrusions and blebs observed in the cell membrane of promastigote forms treated with Lu or UA, cell membrane lysis was not detected by morphology and physiology studies. Protrusions and blebs observed in the cell membrane of treated parasites can be the effect of the triterpenes on the cytoskeleton of promastigote forms . Similartefosine . In additefosine , 57, 58;in vitro activity and selectivity toward L. (L.) infantum amastigotes, and thus, golden hamsters were injected with Lu or UA to evaluate hepatic and renal functions after treatment. Healthy golden hamsters treated with UA showed a significant increase of aspartate transaminase (AST) compared to the healthy animals. Both ALT and AST are produced by hepatocytes; therefore, increased levels of ALT and AST indicate hepatocellular injury. In the present study, UA-treated hamsters showed elevation only in AST, suggesting an initial hepatocellular injury. Previous studies already showed that low doses of UA are safe for BALB/c mice, and golden hamster treated with 2 mg/kg showed no changes in serum ALT or AST; furthermore, no morphological changes in the liver structure were observed; however, higher doses of UA (150 mg/kg) administered with a diet over 6 weeks can cause hepatic damage [Both triterpenes showed promising c damage . By the c damage . Thus, UGolden hamsters treated with Lu or UA did not change the levels of urea or creatinine, suggesting that both triterpenes are not nephrotoxic. By the other side, animals treated with 5 mg/kg of amphotericin B showed an increase in the creatinine level, suggesting that AmB caused renal toxicity in golden hamsters. Previous study already showed that hamsters treated with 5.0 mg/kg/day of AmB during 16 days displayed morphological changes compatible with acute renal toxicity . AmB alsL. (L.) infantum-infected hamsters; however, UA treatment showed higher efficacy at eliminating hepatic parasites than Lu treatment; moreover, UA showed similar efficacy than AmB treatment. Lu, as well as UA, was poorly investigated concerning the leishmanicidal effect, and few available works showed the therapeutic activity of this triterpene only in the murine model of visceral leishmaniasis that mimics the acute phase of infection; thus, currently, this is the first work showing that Lu is able to decrease amastigote forms in the spleen and liver from hamsters in the chronic phase of visceral leishmaniasis.Although the treatment with UA at 2.5 mg/kg induced toxicity to the liver, its efficacy in the model of visceral leishmaniasis was assessed, since only the levels of AST marker increased after treatment. In fact, UA and Lu were able to eliminate amastigotes forms in the spleen and liver of γ) cytokine that can be mainly produced by NK and T cells [γ, classical activation and induction of microbicidal mechanisms can occur, with the participation of inducible nitric oxide synthase enzyme (iNOS), as well as nitric oxide (NO), a mediator of inflammation, that along with other reactive oxygen and nitrogen species can kill intracellular parasites [in vivo and increased IFN-γ expression was observed, suggesting that both these triterpenes may direct the immune response to a Th1 immune response, emphasizing that besides a direct activity on parasites, the therapeutic activity of triterpenes is mediated by immune response activation [In leishmaniasis, resistance to infection has been associated with the development of a Th1 immune response, with remarkable amounts of interferon gamma  infantum. In addition, this is the first work showing the comparative therapeutic activity of both Lu and UA in the experimental model of chronic visceral leishmaniasis that is the most suitable model of natural infection. Taken together, data showed herein demonstrates that UA and Lu triterpenes were able to promote effective and selective antileishmanial activity on promastigote and amastigote forms of L. (L.) infantum, and possibly, the parasite mitochondria may be the target organelle, since impairment of transmembrane potential was observed after 30 minutes of incubation; furthermore, morphological studies showed a complete disorganization of such organelle. Both triterpenes increased the cellular immune response in hamsters with visceral leishmaniasis, and it was associated with amastigote elimination in the spleen and liver, suggesting that the leishmanicidal activity is mediated by immunomodulation of both innate and acquired immune systems.Previous studies already discussed the u and UA , 37, 69;


Giardia duodenalis. The pro-drug metronidazole (MTZ) is the first-line treatment for giardiasis. Parasite’s proteins as pyruvate:ferredoxin oxidoreductase (PFOR), ferredoxin (Fd), nitroreductase-1 (NR-1) and thioredoxin reductase (TrxR) participate in MTZ activation. Here, we showed Giardia trophozoites long-term exposed to MTZ presented higher IC50 than controls, showing the drug influenced the parasite survival. That reduction in MTZ’s susceptibility does not seem to be related to mutations in the genes pfor, fd, nr-1 or trxr. It points that different mechanism as alterations in other metabolic pathways can account for Giardia resistance to MTZ therapy.Giardiasis is an infectious disease caused by  Giardia duodenalis  that colonises the small intestine. The infection is worldwide distributed affecting around 280 million people each year most of them children.,) Although the prevalence of giardiasis is high in low income regions, the transmission is also a concern in developed countries, especially as outbreaks.)G. duodenalis is a zoonotic parasite classified into eight assemblages (A to H).) The assemblages A and B are classically known to infect humans and dogs. In addition, the assemblage E infecting humans was also described in Brazil) and Australia.) The faecal-oral transmission along with the close interaction with pets increase the risk of G. duodenalis infectionGiardiasis is a waterborne disease caused by the protozoan ,G. duodenalis, the proteins pyruvate:ferredoxin oxidoreductase (PFOR) and ferredoxin (Fd) act together to activate MTZ.) However, two other proteins were also associated with MTZ activation: nitroreductase-1 (NR-1)) and thioredoxin reductase (TrxR).) MTZ activation generates a nitro anion radical that causes oxidative damage to several cellular macromolecules, especially DNA and proteins, leading to the parasite’s death.,There are relatively few therapies available for treatment of giardiasis and 5-nitroimidazoles drugs, mainly metronidazole (MTZ), are first-line treatment.,Giardia infection. Moreover, in a Cuban cohort of 456 Giardia-microscopically positive individuals, 46% of them experienced therapeutic failure after the first course of MTZ treatment.) Indiscriminate use of antiparasitic drugs together with repeated treatments can increase the chances for rising MTZ-resistant G. duodenalis strains. These strains can spread into the environment, infect susceptible individuals and, consequently, make the treatment of giardiasis more difficult.The use of 5-nitroimidazoles is well established in clinical practice, the efficacy is high and the estimate cure rate can achieve up to 90% of the giardiasis treated cases.Giardia resistance to 5-nitroimidazoles was extensively documented in vitro, but the mechanisms underlying this resistance is still largely unknown.-in vitro induced MTZ-resistant G. duodenalis strain presents mutations in these genes.The resistance of microorganisms to different classes of drugs is a worldwide concern. Several mechanisms cause resistance to drugs, such as mutation, changes in gene expression and protein abundance, efflux pumps, epigenetic changes and others. G. duodenalis WB strain [ATCC50803] were grown in TYI-S-33 medium as previously described by Keister,Trophozoites of G. duodenalis was continuously exposed to different concentrations of MTZ as follow: 5 µM (MTZ5); 10 µM (MTZ10); 20 µM (MTZ20); and 80 µM (MTZ80). Two groups were used as controls: the group no exposed to MTZ (SMTZ) and the group exposed to 0.05% (V/V) of dimethylsulfoxide  (CDMSO), the vehicle in which MTZ was solubilised. After 16 weeks, the IC50 was evaluated for all groups evaluated.4 parasites from each experimental group were plated in a 96-well flat-bottom plate, in which a serial dilution of MTZ was carried out. The plates were incubated at 37°C for 72 h. Resazurin was added to evaluate the parasite’s viability. After 4 h incubation, the reaction was quantified by fluorimeter (λex 560 nm - λem 590 nm).21 The tests were performed in triplicate and the IC50 results were analysed using the GrafPad Prism 6.0 software (www.grafpad.com/scientific-software/prism/). The results were expressed as IC50 values and resistance factor, i.e, IC50 fold change in relation to control. The ANOVA test was used for comparative analysis of the IC50 of groups.Four groups were defined where TM reagent  according to the manufacturer’s instructions. The DNA was subjected to PCR to amplify gene fragments. The specific primers for fd (orf. GL50803_9662), nr-1 (orf.GL50803_6175) and trxr (orf.GL50803_9827) were designed using the NCBI Pick Primers Tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/). The amplified fragments of nr-1 (953 base pairs), fd (251 base pairs) and tr  comprised the complete sequence of these proteins. The amplified fragment of pfor (342 base pairs) (orf.GL50803_114608) contains the coding region of the catalytic site of the enzyme.) The primers sequences, the concentration of the mix reagents and amplification conditions are described in The DNA from experimental and control groups were extracted using DNAzol® - PCR purification Kit , according to the manufacturer’s instructions. The effectiveness and efficiency of PCR conditions were assessed by electrophoresis in agarose gel 1%. The purified fragments were subjected to sequencing using the BigDye® Terminator v3.1 Cycle Sequencing Kit . The precipitation and electrophoresis steps were performed at Sequencing Service DNA Sequencing Platform - Fundação Oswaldo Cruz.The gene fragments obtained in the PCR reaction were purified using the QIAquickpfor, fd, nr-1 and trxr of G. duodenalis in GenBank using the CLUSTAL W algorithmThe electropherograms were analysed and their quality was verified by the Chromas 2.4 program (http://tecnelysium.com.au/wp/chormas/). The identity percentage was verified by the Basic Local Alignment Search Tool (BLASTn) (https: /blast.ncbi.nlm.nih.gov./Blast.cgi). The obtained sequences were aligned with the nucleotide sequences of G. duodenalis trophozoites were continuously exposed to different concentrations of the MTZ during 16 weeks. Afterwards, the parasite’s susceptibility to the different MTZ concentrations was evaluated by the rate of trophozoite replication determined by the IC50 values and the resistance factor  However, our strategy of continuous MTZ exposition differed from other authors who used intermittent exposure of G. duodenalis to high concentrations of 5-nitroimidazoles or ultraviolet radiation followed by exposure to this drug.) As expected, the results demonstrated that the continuous exposure of G. duodenalis to MTZ decreased the parasite’s susceptibility to this drug in vitro.The G. duodenalis in vitro resistance to MTZ is still unknown. Since parasite’s proteins such as PFOR, Fd, NR-1 and TrxR participate in the activation of MTZ, nucleotide changes in the genes that encode these proteins could prejudice the activation of the drug, leading to a reduction in Giardia susceptibility. This prompted us to evaluate the gene sequences of these proteins in strains of G. duodenalis resistant to MTZ in vitro. Unexpected, we did not find any nucleotide change in these analysed genes.The mechanism responsible for G. duodenalis have shown great variability in gene expression and protein abundance of Fd and PFOR.,,G. duodenalis strains.) We extended previous studies that showed MTZ-resistant strains of G. duodenalis did not present any nucleotide changes in the gene region that encode PFOR catalytic site.pfor mRNA was shown to be higher in the resistant strain of G. duodenalis compared to controls.) These findings evidence the complexity involving MTZ-G. duodenalis resistance process, which may include another mechanisms. Then, nucleotide changes only in PFOR and Fd are insufficient to elucidate the resistance of G. duodenalis to MTZ.Analysis of transcriptome and proteome of resistant strains of G. duodenalis strains were evaluated.-G. duodenalis strains resistant to MTZ,,nonsense mutation in NR-1 transcripts in one of strains.) This fact demonstrates the importance of evaluating the nucleotide sequence of this gene in different in vitro MTZ-resistant G. duodenalis strains. Like Fd and PFOR, gene expression and protein abundance of TrxR showed variable results when MTZ-resistant G. duodenalis to MTZ. In the present study, we demonstrated that the exposure of G. duodenalis to MTZ decreases the parasite’s susceptibility to the drug; however, this decrease could not be attributed to nucleotide changes in the G. duodenalis genes linked to MTZ activation. Our results points that different mechanisms as alterations in other metabolic pathways can account for Giardia resistance to MTZ therapy.Indiscriminate use of antiparasitic drugs, a common practice in health care units, can select resistant strains of several microorganisms. Given the increase in therapeutic failure cases in the treatment of giardiasis with 5-nitroimidazoles, efforts must be made to elucidate which mechanisms may be responsible for the resistance of


N‐acetylgalactosamine 6‐sulphatase (GALNS). Consequently, partially degraded GAG, chondroitin 6‐sulfate (CS) and keratan sulfate (KS), accumulate in the lysosomes of affected cells, primarily in cartilage resulting in skeletal disease. Excessive urinary excretion of these GAG is often used as the initial biochemical parameter to inform a laboratory diagnosis. Here we present the utility of a CS‐disaccharide with a non‐reducing 6‐sulfated N‐acetylgalactosamine residue (HNAc‐UA (1S))—the enzyme's substrate—for the diagnosis and biochemical monitoring of MPS IVA patients. Following implementation of this method into the diagnostic laboratory, we identified one MPS IVA patient over 3 years of MPS urine screening, with no false positive results from 2050 urines tested. Uniquely, urinary concentrations of HNAc‐UA (1S) are independent of age meaning that age‐related reference ranges are not required. Urinary HNAc‐UA (1S) was also able to identify two MPS IVA siblings who remained misdiagnosed with spondyloepiphyseal dysplasia for 5 years because of normal urinary GAG. HNAc‐UA (1S) could also be used as a biomarker for monitoring response to enzyme replacement therapy (ERT) as there was a drop in urinary concentration following the administration of ERT in all 12 patients and concentrations correlated with urinary KS (R2 = 0.92). In conclusion, HNAc‐UA (1S) is a reliable, sensitive and specific biomarker for the diagnosis of MPS IVA and can be used to biochemically monitor the response to ERT.Mucopolysaccharidosis type IVA (MPS IVA) is an inborn error of glycosaminoglycan (GAG) catabolism characterized by a deficiency of the lysosomal enzyme,  Urinary measurement of a disaccharide derived from chondroitin sulfate can be used for the diagnosis of MPS IVA independent of age and is useful to monitor the pharmacodynamic efficacy of enzyme replacement therapy correlating with keratan sulfate.1N‐acetylgalactosamine 6‐sulphatase (GALNS), is responsible for hydrolysing N‐acetyl‐D‐galactosamine 6‐sulfate residues within chondroitin 6‐sulfate (CS), as well as de‐sulphating galactose 6‐sulfate residues present in keratan sulfate (KS).Mucopolysaccharidosis type IVA  is one of the 11 mucopolysaccharidoses arising due to inherited deficiencies in lysosomal enzymes required for the degradation of glycosaminoglycans (GAG). The biochemical hallmark is lysosomal accumulation of incompletely digested GAG, which leads to the progressive deterioration of cells, tissues and organs.The diagnosis of MPS IVA can be challenging, compared with other MPS types. This is because initial laboratory investigations for MPS most commonly use a simple dye binding assay that measures total urinary GAG excretion, notably elevated in the MPS.N‐acetylgalactosamine 6‐sulfate residue (HNAc‐UA (1S))—the substrate for the enzyme (GALNS)—that does not rely on deconstruction of the polysaccharides. This approach is based on the premise that each lysosomal enzyme involved in the degradation of GAG, exclusively acts on the nonreducing end such that the incompletely degraded GAG that accumulate in each MPS type reflect the substrate specificity of the enzyme deficiency. Therefore, HNAc‐UA (1S) is unique to MPS IVA and shows absolute specificity for MPS IVA making it a useful and practical diagnostic biomarker.The introduction of mass spectrometric approaches into the diagnostic testing arena has improved the sensitivity and specificity for MPS compared with colourimetric and electrophoretic techniques.22.1Urine samples were submitted to our Department for Diagnosis or Biochemical Monitoring of MPS and the Institutional Human Ethics Committee approved the use of these samples in this study (HREC/15/WCHN/69). Clinical information was available for two siblings, one sibling (S1) presented at 7 years of age with symptoms of knee pain and sibling two (S2) presented at 6 years of age with short stature.2.2Total urinary GAG was measured using the dimethylmethylene blue (DMB) method.2.3The CS‐disaccharide (HNAc‐UA (1S)) was quantified in spot urine samples by liquid chromatography electrospray ionization‐tandem mass spectrometry as described previously.2.4Leukocytes were resuspended in 0.1% Triton X‐100 and lysed by five cycles of freeze‐thaw. The protein concentration of the resultant homogenate was determined by the method of Lowry et al2.5GALNS gene. Alignments and variant calls were generated using SoftGenetics NextGene with an allele frequency of <1%, and variant classification was in accordance with the American College of Medical Genetics (ACMG) criteria.Next generation sequencing was performed on an Illumina HiSeq and analysis was restricted to the coding regions and canonical splice sites of the 33.1The CS‐disaccharide (HNAc‐UA (1S)) has previously been shown to be diagnostic for MPS IVA.3.2Radiographs from two siblings showed mild features of dysostosis multiplex suggestive of MPS . Initial3.3For biochemical monitoring of patients receiving ERT it was necessary to demonstrate linearity of HNAc‐UA (1S) to afford accurate quantification. Dilution of MPS IVA patient urine with control urine revealed linearity over the biological range of 0.01‐5HNAc‐UA (1S) mmol/mol creatinine . Interas4N‐acetyl‐D‐galactosamine residue at the non‐reducing end ‐ the substrate for the defective enzyme. However, as the mass spectrometric detection method, which measures the disaccharide based on its mass‐to‐charge ratio, is unable to distinguish stereoisomers nor determine the position of the sulfate moiety, the structure of this disaccharide remains an assumption. This disaccharide is not known to be elevated in any other MPS type, and from 2050 cases with a clinical suspicion of MPS, one patient was correctly identified as MPS IVA with elevated HNAc‐UA (1S); no false positives and no recognized false negatives were identified throughout 3 years of testing. The accuracy of this method was further highlighted through external proficiency testing (ERNDIM) that correctly identified MPS IVA samples with no false positive or negative results. Testament to the diagnostic utility of HNAc‐UA (1S) was the identification of two MPS IVA siblings that were incorrectly diagnosed with SED after initial laboratory testing returned normal results . Larger cohort studies are required to determine whether this CS‐disaccharide is informative for assessing disease burden and predicting symptom onset in MPS IVA patients.Although the measurement of urinary KS following depolymerization of the GAG polymer has provided a reliable laboratory test for MPS IVA,N‐acetyl‐D‐galactosamine 6‐sulfate, which is the substrate for the deficient enzyme. Similar to our study, they reported that the monosaccharide was less affected by age than urinary KS determinations and demonstrated that it is a disease‐specific biomarker for MPS IVA. Although they also observed a reduction in this monosaccharide in response to ERT, differences were noted in some patients when compared to measuring total urinary KS. This may be due, at least in part, to the source of KS measured as circulating levels are related to proteoglycan turnover within cartilage whereas urinary KS is derived from the kidney either directly or passes through the glomerular filtrate. As only KS molecules small enough to transverse the glomerular capillary membrane will end up in the urine, urinary KS determinations will not include larger fragments. Therefore, fragments of CS may indeed present a better biomarker for substrate burden in MPS IVA than KS and the measurement of the CS‐disaccharide, which did correlate with urinary KS, may therefore better reflect CS storage than the monosaccharide, N‐acetyl‐D‐galactosamine6‐sulfate.Recently, Lawrence et al.N‐acetylhexosaminidases on the large polysaccharide chains of CS, producing oligosaccharides that are subsequently degraded from their non‐reducing termini by the action of specific lysosomal exoenzymes that reduce these oligosaccharides to monosaccharides and inorganic sulfate for lysosomal egress. In MPS IVA the absence of GALNS activity produces a series of oligosaccharides with terminal N‐acetyl‐D‐galactosamine 6‐sulfate residues.Further work is needed to explore the relationship between CS, KS and oligosaccharide fragments as disease biomarkers for MPS IVA. HNAc‐UA (1S) is a naturally produced disaccharide generated from the initial action of endo‐β‐5In conclusion, the CS‐disaccharide, HNAc‐UA (1S), is a specific and sensitive biomarker for MPS IVA that is easily measured in urine in a multiplex assay enabling detection of all MPS types. Importantly, age related reference ranges are not required as the concentrations of HNAc‐UA (1S) do not vary with physiological growth. HNAc‐UA (1S) can also be used to monitor the pharmacodynamic efficacy of ERT, analogous to urinary KS, but with the advantage that enzymatic or chemical depolymerization of the GAG is not required.Jennifer Saville, Sharon Chin, Belinda McDermott, and Andreas Zankl declare they have no conflict of interest. Janice Fletcher and Maria Fuller have received travel support for attendance at conferences and educational meetings from BioMarin.Jennifer T. Saville, Sharon J. Chin, Janice M. Fletcher and Maria Fuller designed the study. Jennifer T. Saville, Sharon J. Chin, and Belinda K. McDermott performed the experimentation. Andreas Zankl provided patient and clinical data. Jennifer T. Saville, Sharon J. Chin, and Maria Fuller analyzed the data and wrote the manuscript. All authors reviewed and intellectually contributed to the final version.The use of patient samples in this study was approved by the Women's and Children's Health Network Human Research Ethics Committee (HREC/15/WCHN/69).All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation  and with the Helsinki Declaration of 1975, as revised in 2000. Individual patient consent was not sought as all procedures were in accordance with routine patient care.This article does not contain any studies with animal subjects performed by any of the authors.Appendix S1: Supplementary MaterialClick here for additional data file.


The identification of pathologically altered neutrophil granulocyte migration patterns bears strong potential for surveillance and prognostic scoring of diseases. We recently identified a strong correlation between impaired neutrophil motility and the disease stage of myelodysplastic syndrome (MDS). Here, we apply this assay to study quantitively increased neutrophils of a patient suffering from a rare leukemia subtype, atypical chronic myeloid leukemia (aCML).A 69-year-old male was analyzed in this study. Besides routine analyses, we purified the patient’s neutrophils from peripheral whole blood and studied their migration behavior using time-lapse video microscopy in a standardized assay. These live cell migration analyses also allowed for the quantification of cell morphology. Furthermore, the cells were stained for the markers CD15, CD16, fMLPR, CXCR1 and CXCR2.Despite cytoreductive therapy with hydroxyurea, the patient’s WBC and ANC were poorly controlled and severe dysgranulopoiesis with hypogranularity was observed. Neutrophils displayed strongly impaired migration when compared to healthy controls and migrating cells exhibited a more flattened-out morphology than control neutrophils. Because of a detected CSF3R (p.T618I) mutation and constitutional symptoms treatment with ruxolitinib was initiated. Within 1 week of ruxolitinib treatment, the cell shape normalized and remained indistinguishable from healthy control neutrophils. However, neutrophil migration did not improve over the course of ruxolitinib therapy but was strikingly altered shortly before a sinusitis with fever and bleeding from a gastric ulcer. Molecular work-up revealed that under ruxolitinib treatment, the CSF3R clone was depleted, yet the expansion of a NRAS mutated subclone was promoted.These results demonstrate the usefulness of neutrophil migration analyses to uncover corresponding alterations of neutrophil migration in rare myeloid neoplasms. Furthermore, in addition to monitoring migration the determination of morphological features of live neutrophils might represent a useful tool to monitor the effectiveness of therapeutic approaches. Migration assays of neutrophil granulocytes, referred to as neutrophils, bear strong potential as valuable diagnostic and surveillance tools. As neutrophils are among the first cells to counter infections , alteredIn this regard, we recently demonstrated that neutrophil migration strongly correlates with the revised international prognosis scoring system (IPSS-R) in myelodysplastic syndrome , represeAtypical chronic myeloid leukemia (aCML) is a rare subtype of myelodysplastic / myeloproliferative neoplasms (MDS/MPN) characterized by poor prognosis and lack of standardized treatment algorithms , 16. aCMAs the diagnosis of aCML is a complex endeavor and criteria for the monitoring of aCML therapy have not been established, we investigated the applicability of our novel migration assay in this disease setting.Here, we analyzed neutrophil migration of an aCML patient in a longitudinal manner. Neutrophils of the present aCML case displayed severely reduced migration compared to healthy controls. Upon treatment with ruxolitinib, neutrophil migration remained at a low level, even though blood parameters and clinical presentation of the patient improved. Interestingly, before initiating ruxolitinib treatment, aCML neutrophils had a flattened morphology steadily normalizing upon treatment with ruxolitinib.Our findings suggest that analyses of neutrophil migration and morphology might be a valuable diagnostic / monitoring tool for myeloid neoplasms in general. Hence, we conclude that neutrophil migration analyses may be suitable to monitor a spectrum of hematological diseases and should possibly be part of future diagnostic workup strategies and therapy monitoring.n = 11 of the overall analyzed n = 111 blood samples were included to match the presented aCML case in age and gender (n = 6) or age only (n = 5) , University Hospital Essen, Essen, Germany, as part of the Heinz-Nixdorf Recall MultiGeneration (HNRM) study. This study served to extend the Heinz-Nixdorf Recall Study (HNRS), whose objectives and study design were published previously . Both stTo detect somatic, mutational events, a molecular screen was set up analyzing 65 candidate genes in unseparated patient leukocytes derived from peripheral blood samples taken soon after 1st diagnosis, at follow-up-1 after ruxolitinib (sample taken 7 months after start of treatment) and follow-up-2 (sample taken 12 months after start of treatment). DNA of unenriched leukocytes from these consecutive samples was analyzed by next generation gene capture based deep sequencing (NGS) using a custom myeloid gene panel , ARID1A, ASXL1 (E12), ATRX , BCOR, BCORL1, BRAF (E15), CALR (E9), CBL , CLBB , CBLC (E7), CEBPA, CSF3R (E13–17), CSMD1, CSNK1A1 , CUX1, DNMT3A, EED, ETNK1, ETV6, EZH2, FLT3 , GATA1, GATA2, GNAS (E7–9), HRAS, IDH1 (E4), IDH2 (E4), IKZF1, JAK2 (E12–16), JAK3, KIT , KDM6A (syn. UTX), KMT2A (syn. MLL), KRAS, MPL (E4–12), NPM1 (E12), NRAS, PDGFRA , PHF6, PIGA, PRPF40B, PTEN , PTPN11 , RAD21, RUNX1, SETBP1 (E4), SF1, SF3A1, SF3B1 (E13–16), SH2B3 (E2), SMC1A , SMC3, SRSF2 (E1), STAG1, STAG2, STAT3 , SUZ12 (E10–16), TET2, THPO, TP53, U2AF1 , U2AF2, WT1 , ZRSR2, coding exons +/− 20 bp, „E “denotes exon) on an Illumina MiSeq platform. The sequencing runs yielded 2.4 to 3.5 million reads for the samples with totals of 4.7 to 10.6 gigabases in the untrimmed raw data of the sequencing runs, whereof 91.9, 95.6, and 94.7% had quality scores exceeding Q30, resulting in average coverages of 857, 823, and 1042 reads per base, respectively. The LOD for somatic mutations varies depending on mutation type, percentage of neoplastic cells in the sample and copy number of individual loci. Mostly, mutations with VAF > 4% can be detected with our bioinformatics pipeline: Bioinformatics and evaluation of sequence data after cutadapt Version: 1.9.1, bwa Version: 0.7.5a-r405, SAMtools Version: 1.2 (using htslib 1.2.1). Software: Seqnext (JSI) Version 4.3.1; if required for confirmative Sanger: Seqpilot (JSI) Version 4.4.0 Analyzed NGS data after trimming were 100% above QS-cutoff > 30 (mostly ≥38). The ROI were 100% over minimal sequencing deepness of 100. Mutation nomenclature according to HGVS. Reference sequences of genes in which mutations were detected are given in italics: ASXL1_NM_015338_c.1934dup, p.Gly646Trpfs*12; CSF3R_NM_000760_c.1853C > T p.Thr618Ile plus presumably germline variant CSF3R_ NM_000760_c.1795C > A, p.His599Asn; TET2_NM_001127208_c.3320C > G p.Ser1107Ter; TET2_ NM_001127208_c.4222G > T p.Gly1408Ter; CEBPA_NM_04364_c.1004 T > A p.Leu335Gln; EZH2_NM_004456_c.2069G > A p.Arg690His; NRAS_NM_002524_c.35G > A p.Gly12Asp; STAG2_NM_001042749_c.1178 T > A p.Leu393Ter; U2AF1_NM_006758_c.460 T > A p.Cys154Ser.4Cl, 10 mM KHCO3, 0.1 mM EDTA in distilled H2O. After another washing step in sterile PBS, cells were resuspended in sterile hematopoietic progenitor growth medium  and automatically counted using a Cellometer Auto T4 . Since Polymorphprep™ isolation did not reliably separate neutrophils from the aCML patient, isolations of aCML neutrophils after day 14 were carried out using magnetic negative isolation with the MACSxpress® Neutrophil Isolation Kit  according to manufacturer’s instructions. Residual erythrocytes were also magnetically depleted using MACSxpress® Erythrocyte Depletion Kit  according to manufacturer’s instructions. Afterwards, purified neutrophils were washed in sterile PBS, resuspended in sterile HPGM and automatically counted. Comparability of the procedures was ensured by side-by-side measurements of the same sample on day 14  in 198 μl HPGM supplemented with serum replacement 3 . Neutrophils were stimulated with 2 μl fMLP , 2 μl human recombinant CXCL1 , or 2 μl human recombinant CXCL8 . As all stimuli were reconstituted in sterile PBS, the addition of 2 μl PBS alone served as a vehicle control. The plates were centrifuged and incubated at 37 °C, 5% CO2 for 20 min before microscopy.For all healthy controls, neutrophils were isolated from 3 ml EDTA-supplemented blood via density centrifugation using Polymorphprep™  as previously described . In shor2. The generated movies were exported as *.mov files. These files were analyzed with the Automated Cellular Analysis System . The evaluation interval was set to 30 s, the minimum track duration to 60 s, the movement threshold to 8 μm and the microscopy resolution to 0.458716 pixel/μm.All samples were imaged in a Leica DMI6000 B  coupled to a workstation running Leica Application Suite X  with a motorized stage with a HC PL FLUOTAR L 20x/0.40 DRY objective  at an imaging rate of one frame every 8 s for 1 h at 37 °C, without COOne hundred thousand purified neutrophils were stained with the following antibodies: CD15 VioBlue , CD16 FITC , fMLP receptor Alexa Fluor 647 , CXCR1 PE , and CXCR2 PE-Vio770 . After an incubation step of 15 min in the dark at 4 °C, the suspensions were diluted 1:1 with PBS and analyzed on a MACSQuant VYB (Miltenyi Biotec).https://imagej.nih.gov/ij/, 1997–2017.). For that, the first image of every video was exported as *.tif from the LASX software and imported to ImageJ. Subsequently, the outer cell margins were manually marked as regions of interest (ROI) and the occupied area was computed by ImageJ’s ROI manager. Results were given in μm2. Additionally, five age- and gender-matched probands were quantified as controls.To quantify the changes in cellular morphology of aCML neutrophils and neutrophils from healthy donors, the size of cells in the respective video were manually analyzed using ImageJ . Experimental data were plotted as bar graphs or scatter dot plots. Statistical computation, such as computation of 9/L and an absolute neutrophil count (ANC) of 53 × 109/L  count of 69 × 10The migration of neutrophil in response to fMLP is highly reduced in individuals suffering from severe MDS . To test, left panel). To quantify the number of enlarged cells, we plotted the distribution of the cell size of healthy and aCML neutrophils for all conditions .The shape and level of adherence of a cell crucially influences its migration . In our To elucidate whether defective neutrophil migration might originate from changes in signaling receptor expression, we performed flow cytometry analyses of two neutrophil lineage markers, CD15 and CD16 , and the, upper panel). Cell size of aCML neutrophils upon fMLP treatment was still significantly higher (p = 0.0001). The number of neutrophils with a cell size > 225 μm2 dropped for all stimulation conditions  . In the subsequent weeks, the diagnosis of pulmonary mucormycosis was made. The patient died despite treatment with voriconazole, liposomal amphotericin B and isavuconazole (day 370).Treatment with ruxolitinib and hydroxyurea caused the WBC to drop from 69 to 16 × 1009/L day 5  and whether there are specific mutations that affect neutrophil migration to distinguish MDS or aCML. Additionally, a study recently reported that ruxolitinib itself showed impairing effects on dendritic cell migration by inhibition of Rho-associated coiled-coil kinase (ROCK) . While w2D and whAn interesting difference between the aCML and the MDS samples, was neutrophil morphology. In aCML the altered morphology normalized in concordance with the therapeutic success of ruxolitinib treatment. aCML neutrophils were significantly enlarged compared to healthy controls before therapy but whether this was the result of an enlarged cytoplasm or increased adherence remains unclear. However, neutrophil size did not correlate with the assessed peripheral blood parameters, like myeloblast and metamyelocyte counts (data not shown), ruling out the possibility that neutrophil progenitors in the assay were responsible for these changes. Additionally, neutrophil morphology normalized to healthy levels already 1 week after the initiation of ruxolitinib therapy. In fact, it has been reported that ruxolitinib causes microtubule instability in JAK (V617F) mutant HEL cells by inhibiting JAK2 and STAT3 activity  and mighStrikingly, we observed severely reduced expression levels of CD15 and CD16, as well as of the corresponding receptors to the chemokines used in this assay, fMLPR, CXCR1 and CXCR2. While we found no direct correlation between the levels of receptor expression and neutrophil migration behavior in healthy donors , it is cWith the case presented here, we provide evidence that the routine assessment of neutrophil migration and receptor expression provides a broader perspective on diseased neutrophils and impact of a treatment on the patient’s cells. We found compelling evidence that cell shape and degree of adherence were changed over the course of ruxolitinib treatment and coincided with the disappearance of specific clones. Furthermore, we noted an increase in neutrophil speed on day 67 to 12.5 μm/min upon fMLP stimulation Fig. b, which Additional file 1: Figure S1. Changes in the relative number of neutrophils above cutoff over the course of ruxolitinib therapy. (A) Cell size distribution of neutrophils upon treatment of CXCL1, representative of all stimuli conditions. Neutrophils were grouped according to their measured cell size in μm2 and binned from 25 μm2 to 725 μm2 with a bin width of 50 μm2. Relative frequency was computed by dividing the number of cells in a group by the number of cells in the entire image. The dashed grey line indicates the mean of the age- and gender-matched controls (n = 6), the blue line indicates the aCML neutrophils and the red dashed line indicates the cutoff of 225 μm2. Binning was performed with GraphPad Prism™. (B) Changes in the relative number of neutrophils with a cell size > 225 μm2 under the four stimulation conditions over the course of therapy. Black triangles and black solid lines indicate aCML neutrophils (every timepoint n = 1), while grey dots and grey dashed lines indicate the median and the grey dotted lines indicate the interquartile range of the age- and gender-matched controls (n = 6). On average, 41 and 56 cells per condition were analyzed in age- and gender-matched controls and the aCML patient, respectively. Numbers label the specific percentage of cells > 225 μm2 for the indicated timepoint.Additional file 2: Figure S2. Expression of surface markers of aCML neutrophils over the course of ruxolitinib therapy and correlation with migration patterns. (A) Comparison of CD15 and CD16 expression of aCML neutrophils after 2 weeks of ruxolitinib treatment (2 weeks Ruxo) after two different purification methods: density gradient centrifugation  and negative magnetic isolation via MACSxpress® separation . (B) Changes in CD16, CD15 and fMLPR (top) as well as CXCR1 and CXCR2 (bottom) expression given as mean fluorescent intensity (mfi) of aCML neutrophils over the course of therapy. Black triangles and black solid lines indicate the aCML patient (every timepoint n = 1), while grey dots and grey dashed lines indicate the median and the grey dotted lines indicate the interquartile range of the age-matched controls (n = 5). Numbers label the specific values for receptor expression at the respective timepoint. (C-F) Correlation of receptor expression against migration upon corresponding stimulus treatment. Black triangles indicate the aCML patient (n = 1) and grey dots indicate age-matched controls (n = 5). Correlations were computed using GraphPad Prism™. Spearman r and p-value for the correlation of aCML samples are given. (C) fMLPR expression correlated against migration upon fMLPR treatment. (D) CXCR1 expression correlated against migration upon CXCL8 treatment. (E) CXCR2 expression correlated against CXCL1 treatment. (F) CXCR2 expression correlated against CXCL8 treatment.Additional file 3: Figure S3. Changes in leukocyte parameters during ruxolitinib therapy and correlation of peripheral leukocyte counts with migration patterns. (A) – (E) Time course of chosen peripheral blood parameters of the aCML patient over the course of his disease and therapy. Displayed are the absolute WBC (A), myeloblast (B) and metamyelocyte (C) counts in cells/nl as well as the banded neutrophil granulocyte (D) and myelocyte (E) counts relative to the overall WBC in %. Red dots indicate timepoints where migration and flow cytometry data were acquired. (F) Correlation of peripheral cell counts with migration patterns upon PBS stimulation. From left to right: leukocyte counts with moving cells (PBS), leukocyte counts with speed excluding non-moving cells (PBS), metamyelocytes with speed excluding non-moving cells (PBS) and banded neutrophils with speed excluding non-moving cells (PBS). (G) Correlation of metamyelocyte counts with speed excluding non-moving cells upon CXCL1 treatment. (H) Correlation of metamyelocyte counts with speed excluding non-moving cells upon CXCL8 treatment. (F) - (H) Correlations were computed using GraphPad Prism™. Spearman r and p-values are given for each correlation below the graph.


Giardia duodenalis is an important zoonotic pathogen for both human and animal health. Although there have been reports on G. duodenalis infections in animals all over the world, information regarding the prevalence and genetic characteristics of G. duodenalis in sheep in Inner Mongolia, China, is limited. In this study, 209 sheep fecal specimens were collected in this autonomous region. We established that the prevalence of G. duodenalis was 64.11% (134/209), as determined using nested PCR detection and sequences analysis of the small subunit ribosomal RNA (SSU rRNA) gene. Based on the beta-giardin (bg) locus, the glutamate dehydrogenase (gdh) locus, and the triose phosphate isomerase (tpi) locus to study genetic characteristics, both assemblages A  and E  were found. Five novel nucleotide sequence of assemblage E were detected, two at the bg locus, two at the gdh locus, and one at the tpi locus. Multilocus genotyping yielded four assemblage E and two assemblage A multilocus genotypes (MLGs), including four novel assemblage E MLGs and one novel assemblage A MLG. Results of this study indicated that G. duodenalis was highly prevalent in sheep in Inner Mongolia. This study is the first to use the multilocus genotyping approach to identify G. duodenalis in sheep from this region. Giardia duodenalis  is a common intestinal parasite that is widespread among vertebrate hosts, including humans, livestock, and wildlife, worldwide [Giardia duodenalis infections often remain asymptomatic, but can cause severe diarrhea and chronic disease in humans [G. duodenalis infections in humans are common in China. The large number of epidemiological investigations conducted at the start of this century suggested that the average infection rate was 0.85%  [orldwide , 34. Gian humans , 16, 24./23,098) , with th/23,098) . As shee/23,098) , 25, 31.G. duodenalis is a species complex, whose members show little variation in their morphology, and the major genetic groups are now described as assemblages (may correspond to distinct species) [G. duodenalis can be sub-classified into at least 8 genetically different assemblages (A–H) [G. duodenalis assemblage E, while assemblage A occurred infrequently [Extensive analysis of protein and DNA polymorphisms have long been considered findings indicating that species) . Studieses (A–H) , of whices (A–H) , 26, 37.equently , 27, 31 equently , 23.G. duodenalis in sheep and goats in China was 6.07% (418/6890) [G. duodenalis infections in sheep were caused by assemblages E and A, with assemblage E being particularly prevalent. However, there are few reports on G. duodenalis infection rates in goats and sheep in Inner Mongolia [For the past few years, the reported infection rate with s 6.07% 4/6890 [18Mongolia , 40.bg), glutamate dehydrogenase (gdh) and triose phosphate isomerase (tpi) loci has increasingly been used to characterize G. duodenalis infections in humans and animals [G. duodenalis in sheep using individual loci, and thus far, there are only a few reports on genotyping G. duodenalis from sheep in China using MLG analysis [In recent years, multilocus genotyping (MLG) of the beta-giardin . The research protocol was reviewed and approved by the Research Ethics Committee of Henan Agricultural University . Permission was obtained from the farm owners before fecal sample collection. In this study, all fecal samples were carefully collected from the rectum of each sheep without causing discomfort.The Inner Mongolia Autonomous Region straddles three major regions of northeast China, north China and northwest China. The area has a plateau-type geology, with a complex and diverse temperate continental monsoon climate. Inner Mongolia makes good use of its local geographical and meteorological features, and is the largest grassland pastoral area in China, with animal husbandry making an important economic contribution.G. duodenalis in this animal population, we selected the representative commercial farm at our study site that has the highest intensity of Ordos fine-wool sheep (>3 months old). There were no symptoms of diarrhea in the flock during sample collection. Fresh feces were collected from animals by rectal sampling and stored in a 2.5% (w/v) potassium dichromate solution in clean containers. Stool samples were of normal shape. All fecal specimens were transported to the laboratory with an ice pack at 4 °C immediately after collection. DNA extraction was performed within 48 h.To study the infection rate and aggregation distribution of DNA extraction was performed using commercial E.Z.N.A Stool DNA kits , following the manufacturer’s recommendations. Extracted DNA samples were stored at −20 °C until PCR analysis.SSU rRNA) gene to determine the G. duodenalis infection rate [G. duodenalis isolates detected in this study, all G. duodenalis positive isolates were tested using nested PCR based on the bg [gdh [tpi [G. duodenalis loci were conducted in 25 μL systems: 2.5 μL 10× PCR buffer, 2 μL dNTPs (1.25 mM each), 0.3 μL each primer (25 μM each), 0.2 μL rTaq DNA polymerase (1 unit/μL each) , 2 μL of DNA sample, 17.7 μL double distilled water.The DNA samples were analyzed using nested PCR amplification of the small subunit ribosomal RNA gdh [tpi  loci , observed, photographed, and recorded on a Tanon 3500 Gel Image Analysis System .SSU rRNA, bg, gdh and tpi genes from G. duodenalis-positive samples were bidirectionally sequenced using an ABI PRISM 3730 XL DNA analyzer with the BigDye Terminator v3.1 Cycle Sequencing Kit , owned by the Tsingke Biological Technology Co. Ltd , and no double peaks were detected during chromatogram inspection.All the secondary PCR amplicons of the http://www.technelysium.com.au), and edited using DNAstar Lasergene Editseq 7.1.0 (http://www.dnastar.com/). The upstream and downstream sequencing results are spliced into a consensus sequence, and the obtained genetic variants were analyzed by multiple-sequence alignments with reference sequences downloaded from the GenBank database, using Clustal X 2.1 .The sequences obtained were assembled using ChomasPro 2.64  to understand the genetic characteristics of Giardia; the MLGs of G. duodenalis were also identified using the sequence data of these loci. Sequences from each isolate at the three analyzed loci were concatenated (bg-tpi-gdh) to form one multilocus sequence for each isolate.Positive samples at the http://www.megasoftware.net/), based on the Kimura-2 parameter model.Neighbor-joining (NJ) analysis was performed using MEGA 7.0 software  were calculated by the Wald method in SPSS, version 22.0 . Differences in corresponding infection rates among locations were examined by the Chi-square test, and differences were considered significant at MK442896–MK442915.The representative nucleotide sequences generated in this study were submitted to the GenBank database under the accession numbers G. duodenalis-positive fecal samples were identified using the nested PCR analysis of the SSU rRNA genes in this study. The genetic diversity of the G. duodenalis-positive samples was determined by sequencing the bg, gdh and tpi genes, and a total of 39, 72 and 32 sequences, respectively, were obtained for these three genetic loci. Assemblage E (n = 130) and assemblage A (n = 4), were detected, based on the SSU rRNA gene.A total of 134  bg sequences, 7 were identified as assemblage A, and 32 were identified as assemblage E. Sequence A1 (n = 4) was identical to AY655702, and A2 (n = 3) had one single-nucleotide polymorphism (SNP) relative to AY072723 (n = 11), E2 (n = 12), E3 (n = 7), E4 (n = 1), and E5 (n = 1). The E3 and E4 had one SNP each (A170G and C428T) compared to KT922250 and KT922248, respectively and one sequence each was identical to MK610388, KT922250, and KP635098.Of the AY072723 . Assemblgdh sequences, 8 were identified to assemblage A, and 64 were identified to assemblage E. All the 8 assemblage A sequences were identical to the genotype A1 sequence (AY178735) . Using KT369778 as the reference sequence, the intra-assemblage substitutions in assemblage E at the gdh gene can be seen in At Y178735) . Among ttpi locus revealed that 12 successfully amplified isolates were identified as assemblage A, and 20 were assemblage E. A1 was identical to L02120, and A3 had two SNPs (A100G and C363T) relative to EU041754. Among the assemblage E sequences, E4 was identified to be a novel sequence, and the remaining sequences were consistent with KT369763, KT922262, and MF671903, respectively. The intra-assemblage substitutions in assemblage E at the tpi gene can be seen in Sequence analysis of the bg + gdh + tpi gene sequences. Data from the specimens were not included in the MLG analysis when a mixed infection was detected at one of the three loci.Using multilocus sequence typing, 4 assemblage A and 5 assemblage E isolates were successfully sequenced at all three loci . To studMultilocus genotyping yielded two assemblages A MLGs and four assemblage E MLGs. One assemblage A MLG was identical to the AI-1, and the assemblage A MLG was considered a novel MLG (named AI-novel (IM)) which had genetic distance with AI-1 and AI-2; AI-novel (IM) and AI were in the same cluster in the phylogenetic analysis . The MLGGiardia duodenalis is an important intestinal parasite that has a global distribution in humans and a diverse range of other animals [G. duodenalis infection in sheep in various regions of China, including Heilongjiang [G. duodenalis infections in sheep in Inner Mongolia are limited, with only one published report [G. duodenalis infections in sheep in Inner Mongolia, China. animals . There hongjiang , 38, Henongjiang , Jilin, ongjiang , and Qinongjiang . Howeverd report . This stG. duodenalis in sheep was 64.11%, which is considerably higher than previously reported for sheep in Inner Mongolia  [G. duodenalis reported for sheep from other regions of China, such as Heilongjiang  [The results of this study showed that the occurrence rate of  16/375) . It is a 25/539) , Henan ( 25/539) , 33, Jil 25/539) , Qinghai 25/539) , and Qin 25/539) , 40. The 25/539) .G. duodenalis infections in Chinese sheep were all reported to be caused by either assemblage E or assemblage A [G. duodenalis genotype in sheep [G. duodenalis, which is also consistent with previous reports [In previous studies, apart from one study in which two assemblage B isolates were identified in sheep from Heilongjiang province , G. duodmblage A , which iin sheep . In this reports , 38. Ass reports , 12, 37,bg locus. MLG-E1 (IM) were placed in the major cluster of MLGs from Tibetan sheep in Qinghai, whereas MLG-E4 (IM) clustered with MLGs from sheep in Henan Province  and assemblage MLG-E3 (IM) found in this study were genetically distinct from the assemblages found in sheep in Qinghai and Henan Provinces, China , 33. TheProvince .G. duodenalis found in this study have limited zoonotic potential, a threat to public health cannot be ignored.The phylogenetic analysis of the concatenated sequences of the assemblage A MLGs revealed that AI-novel (IM) was a new MLG that belonged to sub-assemblage AI. Sequences obtained from the assemblage A MLG isolates belonged to the sub-assemblage AI, which has been more commonly identified in animals than humans , 35. AltG. duodenalis genotypes in three regions in China. This may be because the Inner Mongolia Autonomous Region is the largest grassland pastoral area in China, and sheep fed here will be distributed to various regions of the country, thus promoting gene exchange of G. duodenalis in various regions. However, there may be specific genotypes in different regions for different breeding environments, and this still needs to be investigated in extensive further research.These MLGs results suggest that there was no significant geographic isolation of G. duodenalis in sheep from Inner Mongolia, in northwest China. Both assemblages A and E were found, with assemblage E being the most prevalent type. Two new bg gene sequences, two new gdh gene sequences, and one new tpi gene sequence was identified. Multilocus genotyping yielded four new assemblage E MLGs and one new sub-assemblage A MLG. In addition, further studies on the zoonotic potential and geographic isolation of G. duodenalis from other regions are required to provide additional data.In conclusion, the results of this study showed that there was a high prevalence of The authors declare that they have no competing interests relevant to this article.


The overall accuracy of HTLV-1/2 LAMP (95% CI 74.8–85.5%) was slightly superior to qPCR (95% CI 69.5–81.1%) and similar to PCR-RFLP (95% CI 79.5–89.3%). The sensitivity of LAMP was greater for HTLV-1 (95% CI 83.2–93.4%) than for HTLV-2 (95% CI 43.2–70.8%). This was also observed in qPCR and PCR-RFLP, which was associated with the commonly lower HTLV-2 proviral load. All molecular assays tested showed better results with samples from HTLV-1/2 mono-infected individuals compared with HIV-coinfected patients, who present lower CD4 T-cell counts. In conclusion, HTLV-1/2 LAMP had similar to superior performance than PCR-based assays, and therefore may represent an attractive alternative for HTLV-1/2 diagnosis due to reduced working time and costs, and the simple infrastructure needed.Laboratory diagnosis of human T-lymphotropic viruses (HTLV) 1 and 2 infection is performed by serological screening and further confirmation with serological or molecular assays. Thus, we developed a loop-mediated isothermal nucleic acid amplification (LAMP) assay for the detection of HTLV-1/2 in blood samples. The sensitivity and accuracy of HTLV-1/2 LAMP were defined with DNA samples from individuals infected with HTLV-1 ( Human T-lymphotropic virus (HTLV) type 1 and 2 are retroviruses transmitted among humans through the parenteral route, unprotected sexual intercourse, and from mother-to-child, particularly by breastfeeding ,4,5,6. HStaphylococcus aureus and/or β-hemolytic Streptococcus  × 100.HTLV-1/2 proviral load was determined by qPCR with TaqMan probes in a Rotor-Gene Q 5-plex HRM Platform . Peripheral blood DNA was quantified by UV spectrophotometry and diluted to 20 ng/µL. Reactions were performed in duplicates with 5 µL of diluted DNA and the Rotor-Gene Probe PCR kit  in a total volume of 25 µL. A 79-bp fragment of the human per cell . Standar2, 0.2 mM of dNTPs and 1.25 U of Platinum™ Taq DNA polymerase. PCR was performed in the following conditions: enzyme heat activation at 95 °C for 3 min, 45 cycles of denaturation at 94 °C for 30 s, hybridization at 60 °C for 30 s and extension at 72 °C for 30 s, followed by a final extension step at 72 °C for 5 min. Subsequently, RFLP was performed by digestion of 20 µL of PCR products with 20 U of TaqI restriction enzyme  at 65 °C for 4 h, following the manufacturer’s instructions. The result was revealed by electrophoresis in 2% agarose gel in 1× TBE buffer with 0.5× GelRed™ at 100 V for 90 min.PCR was performed with the Platinum™ Taq DNA Polymerase  and 15 p-moles of SK43 and SK44 primers. Reactions were carried out with 5 µL of DNA in a total volume of 50 µL containing: 1× PCR buffer, 2 mM of MgClp < 0.05 were tested by post-hoc pairwise McNemar with Bonferroni adjustment. Differences with a p-value of <0.05 were considered significant.The accuracy and sensitivity of HTLV-1/2 LAMP assays were calculated in 2 × 2 contingency tables using the R software version 3.6.1. The definitive diagnosis of HTLV-1/2 infection was determined by a positive result in any of the confirmatory assays performed . Samples with an indeterminate status were considered negative in the analysis. The overall accuracy and sensitivity rates were calculated with results from all patients included, regardless of their original cohort, and also separately for groups of individuals with HTLV-1/2 single infection and coinfected with HIV. The 95% confidence intervals (95% CI) were calculated by the Clopper-Pearson method using the DeskTools package for R software. The agreement between results from the molecular tests was performed using Cochran’s Q-test, and comparisons with tax sequences from the HTLV-1 ATK and the HTLV-2 Mo strains, which belong to the HTLV-1 Cosmopolitan Japanese (1aB) and the HTLV-2a genotypes, respectively. Sequence alignment between HTLV-1 primers and HTLV-1aB isolates showed no nucleotide substitutions within target sequences  had 2 nucleotide polymorphisms, A7819G and A7991G, respectively located within the regions of F2 and B3 primers . Thus, t2+, a DNA polymerase cofactor, and betaine, which reduces the formation of secondary structures in guanine-cytosine (GC)-rich regions. MgSO4 at 6 mM had the best result in LAMP reactions for both HTLV-1 and HTLV-2  and HTLV-2 (n = 19), and with HIV/HTLV-1 (n = 39) and HIV/HTLV-2 (n = 35) coinfections, in addition to HIV carriers coinfected with non-typed HTLV (n = 3) or with HTLV indeterminate status (n = 5). As shown in p < 0.003), possibly as a result of the higher accuracy of LAMP.A total of 226 DNA samples from individuals with laboratory data for the diagnosis of HTLV-1/2 infection from two distinct cohorts were tested by HTLV-1/2 LAMP assays. This included patients mono-infected by HTLV-1 (tax gene regions with low homology (<50%) between HTLV-1 and HTLV-2 to eliminate the possibility of cross-reaction. In addition, conserved regions of the tax gene of each virus were selected, and regions with frequent genetic polymorphisms described for distinct genotypes were avoided [The main objective of this study was to develop a LAMP assay for the confirmatory diagnosis of HTLV-1/2 infection through detection of proviruses in blood DNA samples. Thus, sets of primers were designed for  avoided ,48,49. Tomology <% between avoided .tax sequence recognized by LAMP primers had only one nucleotide substitution between the Cosmopolitan Transcontinental (1aA) and Japanese (1aB) genotypes, which are the most prevalent in Brazil and Japan, respectively [The HTLV-1 ectively . In contectively ,53, and ectively ,55. Therectively ,56 to 33ectively , this geectively ,53. Thusectively . In turnpol and rex genes [Previously, Campos et al.  showed tex genes . Moreovetax expression is lost in approximately half of ATLL clones, and this process is associated with 5′-long terminal repeats (LTR) deletion or methylation, or non-sense mutations, deletions or insertions within the tax gene [tax expression in ATLL cells or hinder the detection of such clonal populations by molecular assays targeting tax sequences, interference of this process with the detection of untransformed cells in ATLL patients by LAMP or any PCR-based assay seems unlikely. However, deletions and insertions in tax might indeed be a limitation to molecular assays targeting this gene. Conversely, possible pitfalls of LAMP in the diagnosis of HTLV-1 infection can be improved by performing serological tests in samples with a negative result.It has been shown that tax gene ,64. Howetax gene . Thus, gtax gene . ATLL istax gene . TherefoThe sensitivity of the HTLV-1 LAMP assay was invariably higher than the test for HTLV-2, as also observed in PCR-based tests. This was associated with low proviral load commonly seen in HTLV-2 infection in comparison with HTLV-1 ,68,69, aBst DNA polymerases [Thus, the use of LAMP assays described here prior to confirmatory serological tests would reduce costs of HTLV-1/2 diagnosis. HTLV-1/2 LAMP assays are a reliable alternative to PCR-based tests, reducing the time for diagnosis in comparison with PCR-RFLP, which depends on amplification, digestion with endonucleases, and agarose gel electrophoresis to reveal the result, and simplifying the laboratory infrastructure compared to qPCR. LAMP has the advantage of being carried out in a conventional thermocycler, or even in a water bath or thermal block. In addition, LAMP results can also be revealed by SYBR Green I by pre-loading the dye in the inner side of microtube caps before the reaction, since it inhibits DNA amplification by ymerases , or usinymerases . This ab


Though accounts for 2.5% of all cancers in female, the death rate of ovarian cancer is high, which is the fifth leading cause of cancer death  in female. The 5-year survival rate of ovarian cancer is less than 50%. The oncogenic molecular signaling of ovarian cancer are complicated and remain unclear, and there is a lack of effective targeted therapies for ovarian cancer treatment.In this study, we propose to investigate activated signaling pathways of individual ovarian cancer patients and sub-groups; and identify potential targets and drugs that are able to disrupt the activated signaling pathways. Specifically, we first identify the up-regulated genes of individual cancer patients using Markov chain Monte Carlo (MCMC), and then identify the potential activated transcription factors. After dividing ovarian cancer patients into several sub-groups sharing common transcription factors using K-modes method, we uncover the up-stream signaling pathways of activated transcription factors in each sub-group. Finally, we mapped all FDA approved drugs targeting on the upstream signaling.The 427 ovarian cancer samples were divided into 3 sub-groups  based on the activated TFs . Multiple up-stream signaling pathways, e.g., MYC, WNT, PDGFRA (RTK), PI3K, AKT TP53, and MTOR, are uncovered to activate the discovered TFs. In addition, 66 FDA approved drugs were identified targeting on the uncovered core signaling pathways. Forty-four drugs had been reported in ovarian cancer related reports. The signaling diversity and heterogeneity can be potential therapeutic targets for drug combination discovery.The proposed integrative network analysis could uncover potential core signaling pathways, targets and drugs for ovarian cancer treatment. In United States, ovarian cancer is the fifth leading cause of cancer-related death in female , which aOn the other hand, comprehensive multi-omics data of ovarian cancer patients have been profiled and analyzed , 8. A seIn this study, we aim to systematically investigate potential activated core signaling pathways in ovarian cancer sub-groups by uncovering the up-stream signaling pathways of activated transcription factors (TFs), and identify all available FDA approved drugs targeting on these up-stream signaling and TFs. The combinations of these drugs have the potential to be synergy with standard platinum chemotherapy by disrupting multiple up-stream signaling and their cross-talk. This study will provide a useful reference resource for repositioning effective drugs and drug combinations for ovarian cancer. The rest of the paper is organized as follows. The details of datasets and methods are provided in Section 2. The analysis results are presented in Section 3, followed by a summary in Section 4.We download the gene expression (RNAseq - RSEM expected_count (DESeq2 standardized)) data of 427 ovarian cancer samples (from The Cancer Genome Atlas (TCGA) ), and 88To obtain KEGG signaling pathways, the “Pathview” R package  was emplThis dataset includes screening results of pairwise combinations of 104 FDA-approved anticancer drugs on NCI-60 cancer cell lines (59 cancer cell lines with detailed genomics profiles) . Specifip-value <= 0.05 (using t test) will result in too many up-regulated genes. The Maximum Likelihood Estimate (MLE) method , σ2 : IG.. To get uninformative priors, we set w0 = 0, v0 = +∞, a0 = 0, b0 = 0. Since it is hard to calculate eq. . We take the gene “CENPH” as an example to illustrate this analysis. The PDF generated by the MCMC model is more robust than generated by Maximum Likelihood Estimate (MLE) .We use the conjugate priors for late eq. , we use p-value threshold, 0.05, was used to select the activated TFs.The Fisher’s exact test (using hyper-geometric distribution) was used to identify the activated TFs by comparing the number of up-regulated targets vs. the number of all target genes, with the number of all the up-regulated genes vs. the number of all the genes tested. The p-value to 0–1 using 0.05 as a threshold. For categorical data, we use the k-modes method [We cluster 427 ovarian cancer samples using the identified activated TFs. We transform s method  for the All 282 signaling pathways from KEGG are investigated, and all the signaling cascades from the starting nodes to the activated TFs are extracted using the python package, NetworkX, to extract the up-stream signaling cascades starting from the beginning genes of individual signaling pathways to the given TFs. Then we score each signaling cascades using the average probability of genes . To control the size of up-stream signaling network, the top 3 signaling cascades are kept.gi), is calculated using the impact analysis method [The impact analysis (IA) evaluates both the topology and dynamics of a signaling pathway by considering the gene expression changes, the direction and type of signaling interaction, and the position and role of every gene in a pathway. A perturbation factor for each gene, PF represents the signed normalized measured gene expression change of gene gi. The second term is the sum of perturbation factors of direct upstream genes of target gene gi, normalized by the number of downstream genes of each such gene Nds(gj). The value of βij quantifies the strength of the interaction between genes gj and gi. We use the probability density of gene expression instead of gene expression, which s will be more accurate considering that the standard deviation of different genes is different.The term ΔE based on the activated TFs. For each sub-group, there is a center sample, and we use the center sample to characterize each sub-group. In another word, the activated TFs in the center sample were used as the activated TFs for this sub-group.Group 2 and Group 3 have 26 and 25 TFs respectively. The additional TFs for Group2 are: AR, ETS1, GABPB1, GFI1, HMG, LHX3, NKX6.2, PAX3, PDX1, PITX2, REST(NRSF), S8. The additional TFs for Group 3 are: ARNT_MAX, ETS1, FOXN1, FOXO4, GABPB1, LHX3, NFATC2, NFIL3_ATF2, NKX6.2, PAX3, SREBF1.For visualization purpose, the principal component analysis (PCA) was employed to reduce the 230 TFs to 2 dimensions (see Fig. The up-stream signaling of activated TFs are shown in Figs. see Table To investigate potential drugs that can potentially perturb these up-stream signaling networks, we mapped the FDA approved drugs on the signaling networks (see Figs. Moreover, we investigated the difference of activated core signaling pathways among these 3 sub-groups. The unique TFs appeared in the core signaling pathways in Group 2 and Group 3 are: PDX1, REST and AR; and ROXO4, SREB F1, NFATC2 respectively. In upstream signaling genes, PML, LEF1, MAPK12, FGF22, JUP, AKT3, MAPK10, MAP2K1, TCF7 are the unique genes for Group1. The CLTA, AR, RPS6KB2, AP2S1, AKT1S1, FOXA2, PDPK1, HTT, MAP2K6, TNFRSF1A, TNF, GNB1, MAFA, TRAF2, REST, HHEX, EFNA4, MAP3K5, PDX1 and AP2M1 are unique genes for Group2. The FOXO4, PLCB4, NFATC2, IRS4, KRAS, PRKCI, PTPRF, ICOS, EGLN3, NOTUM, PPP3R1, SREBF1, EPHA2, EGFR, EGF and PRKCZ are the unique genes for Group3. For Group 1, MAPK10, AKT3, FGF22 are in MAPK signaling pathway and RAS signaling pathway, and PML, JUP, LEF1, TCF7 are signaling cascades linking to MYC. For Group2, the signaling cascade from TNF to p38 is an upstream of p53. For Group3, many genes appeared in RAP1 signaling pathway. For the drugs listed in Table Ovarian cancer is the fifth leading cause of cancer-related death among women, and the 5-year survival rate is fewer than one half. Though a set of biomarkers and signaling pathways have been identified to be associated with ovarian cancer, the functional consequence of these biomarkers and signaling pathways remain unclear. Moreover, there is a lack of effective targeted therapies for ovarian cancer, especially for the platinum resistant ovarian cancer. In this study, we analyzed the gene expression data of ovarian cancer samples and ovarian normal tissues via network analysis. We aim to systematically explore the activated signaling pathways of individual ovarian cancer patients and sub-groups, and identify potential targets and drugs that are able to disrupt the core signaling pathways. There are still several limitations of the study. First, in addition to gene expression, mutation, methylation, and copy number variation data should be integrated in the network analysis to uncover the TFs, and up-stream signaling. Second, the signaling cross-talk among these up-streams are not investigated, which might be responsible for drug resistance. In the future, we will also investigate the signaling network and TFs of platinum resistant ovarian cancer samples; and conduct the network-based drug repositioning approaches , 67 to rThe purpose of this study is to systematically uncover potential activated core signaling pathways in ovarian cancer using integrative network analysis. We identified about 37 activated TFs from three sub-groups of ovarian cancer, as well as a set of up-stream signaling pathways linking to these TFs, e.g., WNT, TP53, MYC, AKT, RAS, mTOR, PDGFRA signaling pathways. In addition, 66 FDA approved drugs were identified targeting on the uncovered core signaling pathways. Forty-four drugs had been reported in ovarian cancer related reports. Combinations of these drugs could be potentially synergy to disrupt the cross-talk of multiple activated signaling pathways and TFs for better ovarian therapy. These uncovered signaling networks, TFs and drugs can be used as reference resources to support biomedical studies in ovarian cancer.