Aeromonas sp. ARM81 isolated from a wastewater treatment plant in Warsaw (Poland) was obtained. Sequence analysis revealed multiple genes conferring resistance to aminoglycosides, β-lactams or tetracycline. Three different β-lactamase genes were identified, including an extended-spectrum β-lactamase gene blaPER-1. The antibiotic susceptibility was experimentally tested. Genome sequencing also allowed us to investigate the plasmidome and transposable mobilome of ARM81. Four plasmids, of which two carry phenotypic modules , and 28 putative transposase genes were identified. The mobility of three insertion sequences  was confirmed using trap plasmids.The draft genome of multidrug-resistant The online version of this article (doi:10.1007/s00203-016-1285-6) contains supplementary material, which is available to authorized users. Aeromonas spp. are frequently recognized as pathogens of poikilothermic animals , but also humans   , 42 plasmids of Aeromonas spp. have been fully sequenced and analyzed. These plasmids are of various sizes  and some are cryptic  , under the following conditions: 35 cycles  preceded by 3-min denaturation at 96 °C, and followed by 5-min extension at 72 °C. Amplified DNA fragments were analyzed by agarose gel electrophoresis and, if necessary, purified using a Gel Out kit (A&A Biotechnology) according to the manufacturer’s instructions.Plasmid DNA isolation by alkaline lysis and agarose gel electrophoresis was carried out according to the protocols described by Sambrook and Russell . All PCRAeromonas sp. ARM81 were determined on Mueller–Hinton agar plates (OXOID) using MIC Test Strips (Liofilchem) containing the following antimicrobial agents: ampicillin, ceftazidime, chloramphenicol, ciprofloxacin, erythromycin, gentamicin, kanamycin, meropenem, rifampicin, streptomycin, tetracycline or trimethoprim. MICs were read after incubation at 30 °C for 24 h.Minimum inhibitory concentrations (MICs) for cI-tetA cassette or a levansucrase-coding gene (sacB), both enabling positive selection of transposition events  from the plasmid pEBB10 . This isolate was randomly picked from bacteria that produced colonies on Luria–Bertani (LB) agar plates supplemented with 50 μg ml−1 kanamycin after overnight incubation at 30 °C. ARM81 was classified by 16S rRNA and rpoB genes analysis as Aeromonas sp., with the highest sequence similarity to Aeromonas caviae (punctata).Aeromonas sp. ARM81 gave 71 scaffolds consisting of 161 contigs with a total length of 4,640,951 bp. The GC content of the ARM81 draft genome was estimated as 61.3 %, which is in agreement with corresponding values for other Aeromonas genomes, ranging between 58.17 %  and 62 % (A. hydrophila 4AK4). Automatic annotation using the RAST server identified 4240 genes with an average length of 925.8 bp . The total length of the predicted genes was 3,925,446 bp, which covers 84.6 % of the genome. Additionally, 94 tRNA genes were identified .Illumina sequencing and assembly of the genome of Aeromonas sp. ARM81 was initially identified on the basis of kanamycin resistance. However, antibiotic susceptibility testing performed using MIC Test Strips revealed that the strain also shows decreased susceptibility to other antimicrobials representing different classes, i.e., β-lactams, aminoglycosides and tetracyclines (Table aph(3′)-VIb, aph(3′′)-Ib and aph(6)-Id, conferring resistance to aminoglycosides (Ramirez and Tolmasky tetA(E) encoding a tetracycline efflux protein -VIb, aph(3′′)-Ib and aph(6)-Id are located within a non-composite transposon (named Tn5393k), which resembles Tn5393d from Alcaligenes faecalis FL-424/98  three different phosphotransferases, acr3 gene conferring resistance to arsenic  thenc Rosen .Aeromonas sp. ARM81, we also attempted to define its mobilome , since MGEs are frequent vehicles of horizontal transfer of various resistance genes, enabling their dissemination through microbiomes , IS5 (IS5 and IS903 groups), IS21, IS200/IS605, IS66, IS630, IS1595, IS1634 and Tn3.Analyzing the draft genome of As12, ISKpn9, both belonging to ISAs1 family, and ISAs26 grouped within IS200/IS605 family.To identify active (mobile) TEs, two trap vectors, pGBG1 and pMAT1CM (enabling positive selection of transposition events), were used. The analysis of 100 random insertional clones revealed only three types of insertion sequences. Sequencing showed that these were isoforms (98–99 % nucleotide sequence identity) of previously identified elements, i.e., ISAmong the obtained contigs, four had much higher coverage than the average. We assumed that these corresponded to plasmids visible in agarose gel electrophoresis of the ARM81 DNA extracted by alkaline lysis procedure (data not shown). PCRs with primers designed on extremities of the contigs were used to close the plasmids. Linear representations of the plasmid genomes are shown in Fig. repB and RNAI [which is known to regulate replication of ColE1  efflux, and therefore, it may confer resistance to zinc  and mobilization (MobA) proteins. The second replicon, pARM81P2 (5281 bp), is another ColE1-like plasmid with Aeromonas spp., e.g., pAhD4-2 and pAH06-06-2  toxin–antitoxin system, while mobilization is apparently mediated by MobA and MobC proteins. Analysis also revealed a gene (gtr) encoding a putative glucosyltransferase, which might alter the structure and properties of lipopolysaccharides produced by the ARM81 strain , shows some sequence similarity to plasmids previously identified in 6-2 Fig. . Stable The largest plasmid, pARM81P4, is 7599 bp. Its nucleotide sequence and predicted genes do not show any significant similarity to sequences deposited in GenBank (NCBI); however, the predicted protein product of the largest gene contains two distinguishable domains: (1) PriCT-2 found in primases and (2) VirE of the virulence-associated proteins, which suggests that it may be a bifunctional protein.Aeromonas spp., e.g., A. aquariorum AAK1  and A. hydrophila AL06-06 . Identification of plasmids and various TEs (including the Tn5393d-like transposon with blaPER-1) in the ARM81 genome suggests that this strain actively participates in horizontal transfer of various genetic information (including antibiotic resistance genes) in the environment.WWTPs are known to be rich in nutrients and a mixture of toxic compounds/selective agents, including antibiotics and heavy metals Supplementary material 2 (DOCX 513 kb)Supplementary material 3 (DOCX 33 kb)Below is the link to the electronic supplementary material.


The mitogen-activated protein kinases (MAPKs), especially p38MAPK, play a pivotal role in chronic pain. Electroacupuncture (EA) relieves inflammatory pain underlying the descending pathway, that is, the periaqueductal gray (PAG), the rostral ventromedial medulla (RVM), and the spinal cord dorsal horn (SCDH). However, whether EA antagonizes inflammatory pain through regulation of p38MAPK in this descending facilitatory pathway is unclear. Complete Freund's adjuvant (CFA) was injected into the hind paw of rats to establish inflammatory pain model. EA was administrated for 30 min at Zusanli and Kunlun acupoints at 0.5, 24.5, 48.5, and 72.5 h, respectively. The paw withdrawal threshold (PWT), paw edema, and Phosphor-p38MAPK-Immunoreactivity (p-p38MAPK-IR) cells were measured before (0 h) and at 1, 3, 5, 7, 25, and 73 h after CFA or saline injection. EA increased PWT at 1, 3, 25, and 73 h and inhibited paw edema at 25 and 73 h after CFA injection. Moreover, the increasing number of p-p38MAPK-IR cells which was induced by CFA was suppressed by EA stimulation in PAG and RVM at 3 and 5 h and in SCDH at 5, 7, 25, and 73 h. These results suggest that EA suppresses inflammation-induced hyperalgesia probably through inhibiting p38MAPK activation in the descending facilitatory pathway. Chronic pain is maintained and modulated through nociceptive processes at spinal and supraspinal level. Electrophysiological and pharmacological researches have confirmed that stimulation of the periaqueductal gray (PAG) or the rostral ventromedial medulla (RVM) can influence spinal nociceptive process via inhibiting or facilitating nociceptive input –4. Moreoα, the activating transcription factor-2) and inflammatory mediators  in the spinal cord could result in pain facilitation [The activation of p38 mitogen-activated protein kinases (p38MAPK) signaling pathway plays a vital role in intracellular signal transduction on chronic pain –15. Ni elitation , 19. Nevlitation , 21, sugElectroacupuncture (EA) is a traditional complementary and alternative medicine approach with the advantages of safety, efficiency, and quantifiability to ameliorate inflammatory pain , 23. EA In the present study, CFA-induced inflammatory pain model was applied to observe the effect of EA on the paw withdrawal threshold (PWT), paw volume, and the expression level of p-p38MAPK in PAG, RVM, and the spinal cord dorsal horn (SCDH). We hypothesize that EA could downregulate p-p38MAPK expression in the descending facilitatory pathway and then exert its inhibition function in the inflammatory pain.Male Sprague-Dawley rats weighing 220 ± 20 g were provided by Hubei Provincial Center for Disease Control and Prevention (number 42000600005600). One hundred and forty-four rats were housed six per cage with food pellets and water ad libitum and maintained on 12 h alternate light–dark cycles (7 a.m. to 7 p.m.). A quiet environment was provided, and the room temperature was maintained at 22 ± 2°C. Experimental animals were accustomed to being approached, acclimatized in individual plastic enclosures on a metal mesh and housing facilities (30 min/day) for one week before experiment. The experimental protocol was approved by the Animal Care Center, College of Veterinary Medicine, Huazhong Agricultural University .μL saline/per rat); (2) the CFA group with CFA injection (100 μL CFA/per rat); (3) the CFA + EA group with CFA injection (100 μL CFA/per rat) and EA treatment; (4) the CFA + sham group with CFA injection (100 μL CFA/per rat) and needle insertion without electrical stimulation. EA treatment was given at 0.5, 24.5, 48.5, and 72.5 h after CFA or saline injection  into the plantar surface of left hind paw. The rats were separated randomly into 4 groups (36 rats/group): (1) the saline group with saline injection (100 njection . To elimnjection . Six ratEA stimulation was conducted (9:00 a.m.), according to a previous report , 31. The3) on a metal mesh stand for 10 min before behavioral testing. The mechanical stimulus was delivered to the plantar surface of the left hind paw from the bottom floor of the plastic enclosures by an electronic von Frey anesthesiometer . A force transduction fitted with a 0.5 mm diameter polypropylene rigid tip was applied perpendicularly against the hind paw with an ever-increasing force from 0 to 50 g for a 20 s period. When the animal withdrew its hind paw, the mechanical stimulus was automatically stopped, and the force at which the animal withdrew its paw was recorded as PWT by the anesthesiometer. The procedure was repeated three times with 5 min interval. The percentage change in PWT was calculated as the following formula: Δ% = (Vn − V0)/V0 × 100%, where Vn is the PTW after CFA or saline injection and V0 is basal PWT.PWT was used to assess the inflammatory pain. Its assessment was conducted immediately before (0 h) and at 1, 3, 5, 7, 25, and 73 h after CFA or saline injection. Rats were first acclimatized in individual plastic enclosures (12 × 22 × 18 cmTn − T0)/T0 × 100%, where Tn is the paw volume after CFA or saline injection and T0 is basal paw volume.The paw volume was assessed immediately after each PWT testing  on left paw. A water displacement plethysmometer  was used to measure the paw volume. The hind paw was immersed in a chamber containing distilled water up to the boundary between hairy and nonhairy skin, and the volume displacement was determined electronically. The percentage change in paw edema was calculated as the following formula: Δ% =  while one was incubated with TBST instead of the antibody as negative control. The remaining experimental procedures of SABC immunohistochemistry were carried out according to the instructions . The nucleus of positive cells was stained as brown yellow.SABC immunohistochemistry was employed to detect the expression level of p-p38MAPK. Rats were deeply anesthetized with sodium pentobarbital  and were perfused transcardially with 100 mL of saline followed by 500 mL of 4% paraformaldehyde in 0.1 mol/L phosphate buffer at pH 7.4. Then, the heads of the rats were severed from the bodies and mounted in a stereotaxic instrument with the mouth bar set at the atlas standard (−3.3 mm) for blocking. According to the brain atlas of rats , four stThe gigantocellular reticular nucleus (Gi), the gigantocellular reticular nucleus pars alpha (GiA), and the nucleus raphe magnus (NRM) are main part of RVM. GiA and NRM were counted together because of the difficulty to distinguish them. Therefore, p-p38MAPK-IR cells of Gi and GiA + NRM were counted to represent those of RVM. Optical images of the stained nuclei were obtained under a microscope  connected to a video-based and computer-linked system . Three slides of vlPAG, Gi, GiA + NRM, and SCDH were observed with 200x magnification and their locations were shown in p < 0.05 was considered statistically significant.PWT, paw volume, and the number of p-p38MAPK-IR cells were assessed or quantified by a skilled person who was blinded to the rat assignments. The results of PWT and paw volume were presented as the % changes ± SD. The results of the number of p-p38MAPK-IR cells were presented as the mean ± SD. The data analysis was performed with SPSS 21.0 software . The percentage changes in PWT and paw volume were analyzed with repeated measures ANOVA. The number of p-p38MAPK-IR cells was assessed using one-way ANOVA. Bonferroni's post hoc test was applied when significant differences were found. p < 0.01) than those of saline group at 1, 3, 5, 7, 25, and 73 after CFA injection. However, PWTs of rats in CFA + EA group increased (p < 0.01) as compared with CFA at 1, 3, 25, and 73 h. No differences (p = 1.000) were found between the CFA and the CFA + sham groups in PWTs  than that in saline group during the experiment. The paw volume of CFA + EA group was higher (p < 0.01) than that in saline group within 3–73 h. The paw edema in CFA + EA group decreased (p < 0.05) at 25 and 73 h as compared with CFA group. No differences (p = 1.000) were detected between CFA and CFA + sham groups in the paw edema , while CFA + EA group induced an increase of p-p38MAPK-IR cells at 1 and 5 h (p < 0.05). After EA stimulation, p-p38MAPK-IR cells in CFA + EA group decreased (p < 0.01) as compared with CFA group at 3 and 5 h. There was no difference (p = 1.000) in the number of p-p38MAPK-IR cells between CFA and CFA + sham groups.Phosphor-p38MAPK-IR cells were observed in the vlPAG . Comparep < 0.01) as compared with saline group in Gi at 1–7 h or GiA + NRM at 1–5 h. In CFA + EA group, p-p38MAPK cells increased (p < 0.01) more than saline group in Gi and GiA + NRM at 1–5 h. After EA stimulation, p-p38MAPK cells in CFA + EA group decreased (p < 0.01) as compared with CFA group in GI at 3 and 5 h or GiA + NRM at 1–5 h. There was no difference (p = 1.000) in the number of p-p38-MAPK-IR cells between the CFA and CFA + sham groups.The rostroventromedial medulla represented both Gi and GiA + NRM . Phosphop < 0.01) in the number of p-p38MAPK-IR cells in the measured points. In CFA + EA group, p-p38MAPK cells increased (p < 0.05) more than that in saline group at 1, 3, 7, 25, and 73 h. After EA stimulation, p-p38MAPK-IR cells in CFA + EA group decreased (p < 0.05) as compared with CFA group at 5–73 h. No differences (p = 1.000) were detected between the CFA and the CFA + sham groups in the number of p-p38MAPK-IR cells.Phosphor-p38MAPK-IR cells were observed in the SCDH . CompareCFA inflammation model was widely used for investigating the pain therapy. After the injection of CFA into paw, rats showed mechanical hyperalgesia and allodynia in 2 min and persisted for 7–14 d , 35, whiμ-, δ-, and κ-opioid receptors was reported to induce more effective analgesia than a single frequency  in rats [p < 0.05), indicating that EA exerts some therapeutic effect on inflammation.Acupuncture therapy, especially EA, can induce potent analgesia to relieve persistent pain. Han  reported in rats . Acupoin in rats , 41, 42. in rats . Fang et in rats  also rep in rats . In the Numerous studies indicated that p38MAPK participated in inflammatory response in central nerve system. In CFA model, the number of p-p38MAPK-IR cells was significantly increased in NRM for 0.5–1 h and in GiA at 0.5, 3, and 5 h . Fang etβ (IL-1β) could lead to the activation of IL-1β receptors [β in PAG partly mediated and enhanced pain facilitation [β synthesis was inhibited in PAG, mechanical allodynia and thermal hyperalgesia were significantly attenuated [As activated p38MAPK in spinal and supraspinal sites promotes the production of inflammatory mediators, it is possible that activated p38MAPK in the PAG, RVM, and SCDH can increase descending pain facilitation. Accordingly, disruption of p38MAPK or p38MAPK downstream inflammatory mediators might reverse behavioral hyperalgesia and allodynia. At supraspinal level, p-p38MAPK was localized and increased in PAG after chronic constriction nerve injury  and in Receptors . Previoulitation . Furthertenuated . In a catenuated . Taken tβ, and tumor necrosis factor-α (TNFα) in the spinal cord to enhance central sensitization [β and TNF-α were attenuated following EA treatment [At spinal level, the expression level of p-p38MAPK increased after peripheral inflammation which is induced by CFA , bee-ventization , 45, whetization , 52, 53.tization . CFA-increatment . Taken tEA could significantly inhibit CFA-induced decline of PWTs and CFA-induced increase of paw edema. The number of p-p38MAPK-IR cells increased in PAG, RVM, and SCDH after CFA injection, while it reduced after EA application. These results suggested that EA could disrupt descending facilitation in PAG, RVM, and SCDH and exhibits antihyperalgesic effect by inhibiting p38MAPK activation in descending facilitatory pathway.


Piper spp.) have promising leishmanicidal and trypanocidal activities. Recent studies showed that essential oils from different pepper species  or its main constituents linalool and nerolidol. In search for natural compounds against PaEO chemical composition was obtained by GC-MS. Drug activity assays were based on cell counting, MTT data or infection index values. The effect of PaEO on the T. cruzi cell cycle and mitochondrial membrane potential was evaluated by flow cytometry.PaEO was effective against cell-derived (IC50/24 h: 2.8 μg/mL) and metacyclic (IC50/24 h: 12.1 μg/mL) trypomastigotes, as well as intracellular amastigotes (IC50/24 h: 9 μg/mL). At 4ºC - the temperature of red blood cells (RBCs) storage in blood banks - cell-derived trypomastigotes were more sensitive to PaEO (IC50/24 h = 3.8 μg/mL) than to gentian violet (IC50/24 h = 24.7 mg/mL). Cytotoxicity assays using Vero cells (37ºC) and RBCs (4ºC) showed that PaEO has increased selectivity for cell-derived trypomastigotes. Flow cytometry analysis showed that PaEO does not affect the cell cycle of T. cruzi epimastigotes, but decreases their mitochondrial membrane potential. GC-MS data identified nerolidol and linalool as major components of PaEO, and linalool had trypanocidal effect (IC50/24 h: 306 ng/mL) at 4ºC.PaEO is likely due to the presence of linalool, which may represent an interesting candidate for use in the treatment of potentially contaminated RBCs bags at low temperature. The trypanocidal effect of  Trypanosoma cruzi is the etiologic agent of Chagas disease, a neglected tropical illness that is endemic in 21 Latin American countries  is the drug commonly used to treat individuals with Chagas disease . TreatmeT. cruzi the most studied alternatives are of plant origin  form a large genus of plants (> 700 species) widespread in tropical and subtropical regions of the world, and used in traditional medicine as analgesic, antiseptic and antimicrobial agents  between 12.8 and 63.3 µg/mL  when treating L. amazonensis-infected macrophages  is a tropical bush typical from Central and South America. In Brazil it is found naturally in the Amazon and Atlantic Forests. P. aduncum extracts inhibits the proliferation of T. cruzi epimastigotes in vitro, with IC50 concentrations lower than 20 µg/mL  against different developmental forms of T. cruzi, in bioassays for 24 h. Gas chromatography-mass spectrometry (GC-MS) data identified nerolidol and linalool as the major components of this essential oil, and linalool had strong trypanocidal effect (IC50/24 h: 306 ng/mL) at 4ºC. The promising trypanocidal effect of PaEO is likely due to the presence of linalool, which may be used as a lead for further drug development.Here, we analysed the trypanocidal effect of Vero cells - Vero cells (ATCC CCL-81) were grown in RPMI-1640 medium with L-glutamine , supplemented with 5% fetal calf serum , at 37ºC, and in a humidified 5% CO2 atmosphere. For seeding, cell monolayers were washed twice with PBS (pH 7.2), trypsinized and collected by centrifugation at 100 g for 2 min.Parasites - In this work, we used the T. cruzi clone Dm28c. Epimastigote forms were kept at 28ºC in LIT medium supplemented with 10% inactivated FCS, with passages at every three days. For the experiments, parasites obtained from 72-h cultures were used.2 culture flasks containing 8 mL DMEM. After 4 h of interaction, non-internalised parasites were removed by rinsing with phosphate-buffered saline (PBS), new medium was added, and then changed to fresh medium every 24 h. After 96 h of infection, trypomastigotes released into the supernatant were collected by centrifugation at 3000 g for 10 min.To obtain cell-derived trypomastigotes, Vero cells were incubated with cell-derived trypomastigotes (1:10 ratio of cells to parasites) in 75 cm7 cells/mL) were collected by centrifugation at 7000 g for 5 min at 10ºC, and resuspended in TAU medium , at a concentration of 5 x 108 cells/mL. After incubation for 2 h at 28ºC , parasites were transferred to 25 cm2 flasks containing 5 mL TAU3AAG medium , at a final concentration of 5 x 106 cells/mL. After 72 h, metacyclic trypomastigotes in the supernatant (~80% parasites) were collected by centrifugation and purified by passage through a DEAE-cellulose affinity column, equilibrated in PSG buffer .To obtain metacyclic trypomastigotes, culture epimastigotes in late logarithmic growth phase (five days) were subjected to nutritional stress as described previously . CultureP. aduncum essential oil (PaEO) purification and chemical analysis - P. aduncum L. (matico) leaves were collected on March 2013 in the morning, with no rain, at the Medicinal Plants Garden of the Universidade Federal de Lavras . P. aduncum EO was obtained by distillation in a Clevenger equipment, at the Department of Chemistry, Federal University of Lavras. Prior to use in experiments, PaEO was diluted to 100 mg/mL in DMSO (PaEO stock solution). Final DMSO concentrations in activity assays did not exceed 0.5%. Both undiluted and diluted (stock) PaEO were kept at 4ºC, and in the dark.PaEO chemical analysis was performed in the Department of Chemistry of UFLA, in a GC-17A Shimadzu gas chromatograph coupled to a QP 5000 Shimadzu mass spectrometer, with a selective detector. The column used was of fused silica/bound type , with helium (1 mL/min) as the mobile-phase gas. The following analysis conditions were used: injection at 220ºC, detection at 240ºC; oven temperature between 40ºC and 240ºC, with addition of 3ºC/min; initial column pressure of 100.2 kPa; 1:10 split ratio and injected volume of 1 µL (solutions at 1% v/v) in dichloromethane. Mass spectra of each compound were compared with the Wiley 229 library database, and with the tabulated Kovats index.Single drug activity assays on T. cruzi developmental forms - All experiments were performed in biological and technical triplicates. Incubations were performed for 24 h in all bioassays. The absorbance of untreated cells in culture media containing 0.5% DMSO (control) was used as 100% cell viability, and the percentage of dead cells in each treatment was estimated by comparison with the untreated control.50/24 h values  and dose-response curves were generated using the CompuSyn software , and statistical analysis was performed in Excel. For all assays, the fraction of affected cells (Fa) was calculated relative to the untreated control (treated/untreated ratio).For all assays, ICPaEO on epimastigote forms, these cells were seeded into 96-well plates (5 x 106 cells/well) in LIT medium, and treated for 24 h at 28ºC with PaEO at the final concentrations of 9, 18, 37, 75, 150 and 300 µg/mL. As a control, BZ  or LIT medium with 0.5% DMSO (negative control) were used. Cell viability was assessed by the MTT assay (see “MTT assay”).To test the activity of PaEO activity assays with cell-derived trypomastigotes were performed at 28ºC and 4ºC. Cell-derived trypomastigotes (5 x 107 cells/well) were seeded in 96-well plates with DMEM, and treated with PaEO or BZ at the final concentrations 1, 10, 50 and 100 µg/mL, or with gentian violet . Untreated cells were used as a negative control. For treatments at 28ºC, plates were incubated at this temperature for 24 h. Then, each well was diluted 1:10 with 10% formaldehyde in PBS and cells were counted in a hemocytometer. For treatment at 4ºC, plates were incubated at this temperature for 24 h and then subjected to the MTT assay (see “MTT assay”). Due to the small size of trypomastigotes, the MTT reagent was used at the concentration of 1 mg/mL (2.5 fold less than that used for epimastigotes), which improved the correlation between the number of viable cells and the optical density (not shown). The major constituents of P. aduncum EO - linalool and nerolidol  - were also tested  against T. cruzi cell-derived trypomastigotes at 4ºC, as described above.PaEO activity on metacyclic trypomastigotes, these cells were plated in 96-well plates (5 x 106 cells/well) with TAU3AAG medium, and treated with EO at the final concentrations of 1, 10, 50 or 100 µg/mL, for 24 h at 28ºC. Then, each well was diluted at 1:10 with 10% formaldehyde in PBS, and cells were counted in a hemocytometer.To test PaEO activity against intracellular amastigotes, Vero cells were seeded in 24-well plates (2 x 104 cells/well) in DMEM (Sigma-Aldrich), and allowed to adhere for 24 h. Then, cell-derived trypomastigotes were added to each well (1:10 ratio of cells to parasites), incubated for 3 h, and non-internalised parasites were removed by washing with PBS. Infected cultures were incubated for 24 h , and then the culture medium was replaced with RPMI-1640 containing PaEO or BZ at final concentrations between 1 and 100 µg/mL , and plates were incubated for 24 h, at 37ºC (5% CO2). Treated cells were fixed with methanol, stained with Giemsa, and the inhibitory effect on intracellular amastigotes was estimated by counting (a) the number of infected cells, and (b) the number of amastigotes per cell, in 100 cells/wells, from random light microscopy images. These data were used to calculate the infection index (II) for each tested concentration (II = % infected cells x number of amastigotes per cell).To test MTT assay - After drug treatments, MTT solution (10 mg/mL in PBS) was added to each well for a final concentration of 2.5 mg/mL (or 1 mg/mL for trypomastigotes). Plates were incubated for 3 h at 28ºC in the dark, centrifuged for 10 min at 475 g and the supernatant was removed . Then, 20 µL SDS was added to each well, followed by incubation for 1 h at 28ºC, after which 80 µL DMSO was added to each well and incubated for a further 1 h at 28ºC. Finally, the residual material was removed with the aid of a toothpick, and sample absorbance (at 550 nm) was read in an EL800 microplate reader . Dose-response curves were produced using the CompuSyn software, which was also used to calculate IC50/24 values.Cytotoxicity - Vero cells were seeded in 96-well plates (2 x 104 cells/well) with RPMI-1640 medium and cultivated for 24 h at 37ºC (5% CO2). Cells were incubated for a further 24 h in the presence of PaEO , linalool/nerolidol , BZ  or gentian violet . Then, plates were subjected to the MTT assay as described above (see “MTT assay”), and MTT data were used to calculate 50% cytotoxicity (CC50/24 h) values. Plates were examined in an inverted microscope every 12 h, to evaluate monolayer integrity (confluence and adhesion).g. The supernatant was discarded and the pellet of RBCs was diluted in saline-glucose, for a final concentration of 6%. Then, RBCs were seeded in 96-well plates (3% RBCs/well) and treated with EO , for 5 h, at 4ºC. Negative control samples represented untreated RBCs diluted in saline-glucose. After treatment, plates were centrifuged at 1027 g for 2 min, and 100 µL of supernatant from each well were transferred to a new plate and analysed (at 550 nm) in an EL800 microplate reader .To analyse cytotoxicity on red blood cells (RBCs) at 4ºC, human erythrocytes were obtained from 10-mL blood samples (O+), from a healthy volunteer donor, as previously described . After c50) values were calculated using Excel, and selectivity index (SI) values were calculated as the ratio between the CC50/24 h (for Vero cells or RBCs) and IC50/24 h values.Cytotoxicity  in LIT medium and treated with PaEO at a concentration corresponding to the IC50/24 h, for 24 h. Negative control cells were kept untreated. Then, cells were washed twice in PBS (with centrifugation at 7000 g for 2 min) and transferred to plastic flow cytometry tubes (300 µL/tube).PaEO on the T. cruzi cell cycle, 1 mL of 10 µg/mL propidium iodide diluted in 5% NP40, with 20 µg/mL RNAse  was added to each tube, and samples were incubated for 20 min at 28ºC.To evaluate the effect of PaEO on T. cruzi mitochondrial membrane potential, rhodamine 123  was added to cultures after PaEO treatment, and cells were incubated for 20 min at 28ºC, before PBS washing and transfer to flow cytometry tubes. Culture treatment with 10 µM CCCP  after the rhodamine incubation was used as a positive control.To evaluate the effect of Flow cytometry samples were analysed in a FACS Canto II  flow cytometer , and data analysis was performed in FlowJo . Statistical analysis was performed by one-way analysis of variance (ANOVA), using GraphPad Prism 5.01 software.Cell-derived T. cruzi trypomastigotes are sensitive to PaEO - The EO of P. aduncum - a pepper species that has already yielded anti-trypanocidal extracts and molecules - had not been tested for its biological activity against T. cruzi. Thus, we tested the purified PaEO against different T. cruzi developmental forms, namely epimastigotes , cell-derived amastigotes and trypomastigotes  and infective metacyclic trypomastigotes  (tigotes) . As a rePaEO (IC50/24 h at 28ºC = 2.8 µg/mL) than to BZ (IC50/24 h = 16.1 µg/mL)  . MetacycT. cruzi epimastigotes were incubated for 24 h at 28ºC with different concentrations of PaEO or BZ, and dose-response curves estimated the IC50/24 h values as 84.7 µg/mL for PaEO and 14.7 µg/mL (45 µM) for BZ  for BZ .PaEO at 4ºC, the temperature used for red blood storage in blood banks. Incubation at 4ºC led to inactivation of BZ, as evidenced by the presence of clumps of crystalline material in the medium (likely representing BZ agglutination), and by the absence of trypanosome morphological damage or motility loss, as assessed by light microscopy observation (not shown). Therefore, for the experiments at 4ºC we used gentian violet as a control, since this drug can be used to treat blood potentially infected with Chagas disease (P. aduncum EO (IC50/24 h = 3.8 µg/mL) than to gentian violet .Blood transfusion is an important route for Chagas disease transmission, due to the presence of infective trypomastigotes in blood from infected donors. Thus, we tested the sensitivity of cell-derived trypomastigotes to  disease . ImportaPaEO cytotoxicity, we performed cytotoxicity assays in uninfected Vero cells at 37ºC  than PaEO (CC5/24 h = 351.6 µg/mL).To estimate  at 37ºC , and als50/24 h and CC50/24 h values - indicates that PaEO is particularly selective towards cell-derived T. cruzi trypomastigotes .Selectivity index (SI) analysis - representing the ratio between ICPaEO inhibits effectively the intracellular survival/replication of T. cruzi amastigotes - To test the effect of PaEO on amastigotes, Vero cells were infected with trypomastigotes and, after 24 h (when trypomastigotes had differentiated into amastigotes), the infected cultures were incubated with different concentrations of PaEO . PaEO at the concentration of 12.5 µg/mL decreased the T. cruzi amastigote infection index by 71.5%, similarly to 10 µg/mL BZ  of 0.8 µg/mL, compared with 9 µg/mL for PaEO (PaEO (data not shown).for PaEO . Also, sPaEO on T. cruzi, epimastigotes were incubated for 24 h at 28ºC with the EO and then subjected to cell cycle analysis by labeling with propidium iodide, or were labelled with Rhodamine-123, to analyse the effect of PaEO on the mitochondrial membrane potential. Although epimastigotes do not have direct medical importance, this life cycle form is ideal for in vitro analysis, because they can be cultivated easily in axenic medium.To examine the mechanism of action of PaEO at the IC50/24 h did not alter significantly the number of cells in the G1 phase of the cell cycle, but there was a significant decrease in the number of cells in G2  and linalool (13.42%)  for the treatment of cell-derived trypomastigotes at 4ºC of 2.69  showed that the main constituents of the (13.42%) . Aside fromising . LinalooL at 4ºC , indicatL at 4ºC . Cytotox of 2.69 .PaEO was due to linalool.In contrast to linalool, nerolidol had no appreciable trypanocidal activity against cell-derived trypomastigotes  at concentrations between 100 ng/mL and 1 µg/mL, as assessed by light microscopy observation and MTT assays (data not shown). Also, nerolidol did not affect the integrity of Vero cell monolayers, after treatment for 24 h with the highest concentration used . These data indicate that the trypanocidal effect of T. cruzi epimastigotes and trypomastigotes (Piper spp.) have promising leishmanicidal   is a promising alternative for further testing on blood/blood cells, under thermal conditions of red blood cells storage (4ºC), in order to obtain derivatives effective at eliminating T. cruzi in blood bank samples.Also, treatment of  = 92.5) . The fir = 92.5) ; thus, PT. cruzi infection in blood banks (In our tests with cell-derived trypomastigotes at 4ºC the use of BZ for 24 h had no effect against the parasite (not shown). The incomplete trypanocidal effect of BZ at 4ºC can lead to Chagas disease transmission via transfusion of contaminated blood . Gentianod banks . Howeverod banks .50 values  and linalool (13.42%) as its main constituents. GC-MS analysis of the essential oil of P. aduncum in other studies showed that it can yield four major constituents: (a) dillapiole, at 79.9-86.9% (P. aduncum EO is 20ºC (for up to six months), without loss of regenerative capacity  ner.9-86.9% ; (c) cin.9-86.9% , and (d).9-86.9% . This vaacity da .PaEO component - failed to affect significantly the cell-derived trypomastigote form of T. cruzi, at 4ºC, even at the concentration of 1 µg/mL. In contrast, linalool had potent trypanocidal effect against this parasite form, with an IC50/24 h of 306 ng/mL. These results show that the major component of an EO may not always be responsible for the lytic activity. Interestingly, incubation of linalool with T. cruzi blood trypomastigotes (Y strain) resulted in an IC50/24 h value of 264 µg/mL  should be tested, and linalool could be used as a lead for further drug development.In conclusion, our data indicate that the


The aim of this study was to retrospectively evaluate the patient effective dose (ED) for different PET/CT procedures performed with a variety of PET radiopharmaceutical compounds.n = 123), Whole body (WB) (n = 36), Head and Neck Tumor (HNT) (n = 10), and Brain (n = 41) protocols with 18FDG (n = 170), 11C-CHOL (n = 10), 18FDOPA (n = 10), 11C-MET (n = 10), and 18F-florbetapir (n = 10). ED was calculated using conversion factors applied to the radiotracer activity and to the CT dose-length product.PET/CT studies of 210 patients were reviewed including Torso  were 6.4 ± 0.6, 4.6 ± 0.4, 5.2 ± 0.5, and 9.1 ± 0.4 mSv, respectively, and the corresponding CT contributions were 11, 14, 23, and 26%, respectively. In 18FDG PET/CT, variations in scan length and arm position produced significant differences in CT ED (p < 0.01). For dual-time-point imaging, the CT ED (mean ± SD) for the delayed scan was 3.8 ± 1.5 mSv.Total ED (mean ± SD) for Torso-The mean ED for body and brain PET/CT protocols with different radiopharmaceuticals ranged between 4.6 and 20.0 mSv. The major contributor to total ED for body protocols is CT, whereas for brain studies, it is the PET radiopharmaceutical. PET/CT is a dual-modality technique combining positron emission tomography (PET) with x-ray computed tomography (CT) . Wide ra18F as a label for the 2-[18F]fluoro-2-deoxy-D-glucose molecule (18FDG) -choline (11C-CHOL), l-[methyl-11C]-methionine (11C-MET), [18F]fluoro-l-dopa (18FDOPA), or 18F-florbetapir [Torso imaging (Torso) covering from the base of the skull to mid-thigh Whole body (WB) examination from the top of the head to the feet, performed in two steps: the first step covered the head and torso (H&Torso) followed by PET/CT from the middle of the pelvis to the toes (Limbs)Head and neck tumor (HNT) protocol performed in two steps, the head and neck portion (H&N) with the arms down, and a scan from the apex of the lung to the mid-thigh (Trunk) with the arms upBrain examinations covering the head within one bed positionThe PET/CT protocols, with rbetapir , were as18FDOPA, 11C-MET, and 18F-florbetapir), the registration period had to be extended to obtain a sample of 10. Finally, studies of 210 patients were reviewed, including 170 PETs with 18FDG, 10 with 11C-CHOL, 10 with 18FDOPA, 10 with 11C-MET, and 10 with 18F-florbetapir. This study has been approved by the institutional review board (Comité Ético de Investigación Clínica de Navarra). The need for informed consent was waived, and data anonymization provided the privacy guarantee.Patient data were retrospectively collected for a month from hospital files and PACS. For some infrequent brain PET/CT studies (k (mSv/mGy cm) multiplied by the DLP [Γ dose coefficient for each radiopharmaceutical. The Γ coefficients in mSv/MBq were 0.019 for 18FDG [11C-methionine [18FDOPA [11C-choline [11F-florbetapir [For each patient, effective dose was evaluated using standard coefficients referring to a generic reference individual. Thus, effective dose (ED) from the CT exam was calculated using the conversion factors  the DLP , dependior 18FDG , 0.0084 thionine , 0.025 f[18FDOPA , 0.0044 p < 0.05.Statistical analysis was performed using SPSS v20 (IBM Corporation). Results are expressed as mean ± standard deviation . Statistical differences between groups were evaluated using parametric or non-parametric tests depending on the normality of the data, and they were considered significant for The main technical CT parameters used in each protocol are shown in Table vol, and DLP) is shown in Table A total of 210 adult patients 130 men) were included in the study. Patient demographic characteristics (mean ± SD) were age, 61 ± 13  years old; weight, 73 ± 14  kg; and height, 167 ± 9  cm. The number of patients per diagnostic procedure with the corresponding PET scan parameters (administered activity and number of acquired bed positions) and CT exposure parameters  produced 6.1 mSv due to PET radiotracer and 10.3 mSv due to CT.Table p < 0.01); thus, for 18F-florbetapir studies, it was 3.6 times higher than for Brain-18FDOPA studies. CT effective dose was 11, 14, 23, and 26% of the total dose received by the patients in 18FDG, 18FDOPA, 11C-MET, and 18F-florbetapir brain PET/CT studies, respectively. PET ED was also statistically different among radiotracers (ANOVA p < 0.001). In particular, 18FDOPA and 11C-MET PET scans provided lower doses than 18FDG and 18F-florbetapir PET scans .CT ED in brain protocols was statistically different  were found in mAs, CTDIvol, DLP, and CT ED between patients with arms above the head and patients with arms alongside the body. For these groups, the ED due to the CT examination (mean ± SD) was 9.7 ± 6.0  mSv and 17.1 ± 7.8  mSv, respectively.When arm position was considered for the Torso protocol, statistically significant differences , no significant differences in radiotracer activity and PET effective dose were found, but the CT effective dose for the CT portion that included torso or trunk was statistically different . Although CT parameters were the same, scan length was different. Thus, the CT effective dose increased according to the scanned length, with 8.0, 10.4, and 11.9 mSv for the trunk, torso, and H&Torso parts of the HNT, Torso, and WB protocols, with mean scanned patient length of 761, 839, and 926 mm, respectively.In 18FDG protocol, and in 31 patients (18% of the 169 body studies), a delayed PET/CT was performed for different parts of the torso. The frequency of the number of bed positions explored was 25, 66, and 9% for one, two, and three bed positions, respectively. The CT ED (mean ± SD) for the delayed imaging was 3.8 ± 1.5  mSv.Dual time PET/CT was performed in 32 patients. Only one corresponded to a brain-18FDG scans (covering different patient lengths) and brain studies.In this study, we evaluated DRL-related parameters and estimated patient effective dose for different PET/CT procedures performed using a variety of PET radiopharmaceutical compounds, including oncological 18FDG PET/CT study to evaluate the patient effective dose considering three different scanning lengths: torso scan (from base of the skull to mid-thigh), WB scan (from top of the head to the feet), and HNT examinations (from the vertex to the mid-thigh in two steps or scans). This analysis with three different scan lengths may provide an understanding of the differences among previously published results of PET/CT ED where different scan lengths have been considered  was 8.6 mSv. A Japanese national survey [18FDG PET ED for male  patients were 10.1 (9.7) and 4.5 (3.7) mSv, respectively.CTDI10.2 mGy . The widy PET/CT , the avel survey  also obs18FDG PET tumor imaging in our patient cohort, the mean PET effective dose, directly proportional to the administered activity, was approximately 6 mSv, within the range of the published data 4.9 to 9 mSv (from 258 to 450 MBq of 18FDG). In the European Union, only seven countries have DRL, ranging from 200 to 400 MBq [For  400 MBq , with a vol and DLP parameters as well as in the dose conversion factor. Of the evaluated protocols, the H&Torso part of the WB protocol had the highest mean DLP value, and the k factor was slightly lower than the value for Trunk and Torso scans. The Trunk CT part of the HNT-18FDG protocol also had a lower DLP than the Torso CT scans. CT ED for Torso-18FDG and HNT-18FDG protocols were similar (10 mSv) and lower than for WB-18FDG scan (14 mSv). These values, which demonstrate dose dependence on the PET/CT protocol used, lie within the range of published data considering the PET/CT protocol  [Few references exist on patient dosimetry in brain studies, other than those reporting radiopharmaceutical biodistribution and organ dosimetry. This is probably because these protocols are less frequent and produce a lower dose in comparison with oncological 290 MBq) .18FDOPA, 18FDG, 11C-MET, and 18F-florbetapir have been evaluated. The purpose of the CT scan was different in each protocol, and the reference tube current was adjusted according to the required image quality for that specific purpose . In particular, the tube currents for the 11C-MET and 18F-florbetapir protocols were adjusted for diagnostic purposes. Hence, the mean CT ED in a Brain-18F-florbetapir study (2.33 mSv) was 3.6 times higher than the CT ED in a Brain-18FDOPA study (0.65 mSv). Thus, for amyloid brain imaging, it would be desirable to distinguish the relative accumulation of radiotracer binding between gray and white matter, as well as possible pitfalls due to bone deposits and atrophy. CT is also important in neurooncological studies using 11C-MET PET/CT studies, where the CT can help by adding structural information. CT doses reported in this study are similar to those in the study by Kaushik et al. [Patient dosimetry for neurological PET/CT protocols with k et al. , where b18FDOPA and 18FDG brain PET/CT studies [18F-florbetapir corresponds to the dose reported for 370 MBq activity administration to a 70-kg patient [Effective doses due to the radiopharmaceutical compounds in the brain PET/CT protocols have been found to contribute between 77 and 88% of the total dose. Kaushik et al. evaluated a PET effective dose of 3.5 and 5.8 mSv for  studies , respect patient .Finally, further studies are needed in the field of patient dose optimization for each PET/CT protocol. The effective dose due to the PET scan is directly proportional to the administered dose. Hence, reducing the injected activity will provide a direct reduction in the patient dose. In addition, there will be a positive impact on the operating costs of the PET/CT facility and in the medical staff dosimetry performing the examinations. The new generation of PET scanners, which incorporate improved reconstruction methods with point spread function and time of flight information  and imagThe main limitation of this study is the simplified approach used to estimate effective dose, using the same conversion coefficient independently of weight, age, or sex of the patient. Therefore, the estimated doses refer to a generic reference individual. For a more realistic analysis of the actual PET doses received by each patient, additional information about the individual biokinetics and anatomical and physiological properties are required. However, this simple approach is suitable for the aim of this study, which was to estimate and compare population doses for different PET/CT procedures; this approach is also used elsewhere to obtain population PET and CT doses . Another18F-FDG, 11C-choline, 11C-methionine, 18FDOPA, and 18F-florbetapir) ranged between 4.6 and 20.0 mSv. In body protocols, CT is the major contributor to the total effective dose. Moreover, the increase in total scan length, the position of the arms alongside the body instead of above the head, and the delayed PET/CT study in a dual-time protocol significantly increased the contribution of the CT effective dose to the total effective dose. Hence, inclusion of the head and legs in a body protocol, the positioning of the arms alongside the body, and the acquisition of delayed images should be justified for each patient in order to optimize the PET/CT protocol dose and to maintain the dose as low as reasonably achievable without compromising diagnostic purpose. For brain protocols, although the main contribution to the total effective dose was due to the PET radiopharmaceutical, CT can increase the dose by a factor of about 4 if it is used for diagnostic purposes.The mean effective dose for body and brain PET/CT protocols with different radiopharmaceuticals (


Anthropometric, socioeconomic, and nutritional parameters were measured and growth parameter z-scores were calculated for each child. Seventy-two percent of the children had detectable levels of AFB1-lys, with a mean level of 5.1  pg/mg albumin; and 80% had detectable levels of UFB1, with a mean of 1.3  ng/ml. This cohort had a 75% stunting rate [height-for-age z-scores (HAZ) < −2] for children at 36 months. No associations were found between aflatoxin exposures and growth impairment as measured by stunting, underweight [weight-for-age z-scores (WAZ) < −2], or wasting [weight-for-height z-scores (WHZ) < −2]. However, fumonisin exposure was negatively associated with underweight  in this cohort of children. Relatively low aflatoxin exposure at 24 months was not linked with growth impairment, while fumonisin exposure at 24–36 months based on the UFB1 biomarkers may contribute to the high growth impairment rate among children of Haydom, Tanzania; which may be associated with their breast feeding and weaning practices.Growth impairment is a major public health issue for children in Tanzania. The question remains as to whether dietary mycotoxins play a role in compromising children's growth. We examined children's exposures to dietary aflatoxin and fumonisin and potential impacts on growth in 114 children under 36 months of age in Haydom, Tanzania. Plasma samples collected from the children at 24 months of age (N = 60) were analyzed for aflatoxin B •Growth impairment is one of the key indicators of child malnutrition.•The children (under 36 months) cohort in Haydom, Tanzania had a 75% stunting rate.•Low aflatoxin exposure was not associated with growth impairment in this cohort.•Fumonisin exposure linked to underweight may contribute to child growth impairment. Beyond There are an estimated 130 deaths of children under age five every day in Tanzania . Growth Aspergillus flavus and A. parasiticus, which commonly infect food crops in warm climates worldwide  biomarker concentrations and urine samples collected between 24 and 36 months of age (N = 94) were analyzed for urinary fumonisin B1 (UFB1) concentrations. Eighteen of the participants had both a plasma and a urine sample for the AFB1-lys and UFB1 analysis at the same timepoint of 24 months.IRB approval was obtained from the National Health Research Ethics Committee, which is part of the National Institute for Medical Research of Tanzania, and Michigan State University. In total, plasma samples collected at 24 months of age (N = 60) were utilized for measuring aflatoxin B2.21-lys is a well-established biomarker of long-term dietary aflatoxin exposure during the past 2–3 months. Its concentrations were determined by liquid chromatography isotope dilution mass spectrometry (LC/MS) as described in 1-D4-lys/ml, and pronase and incubated for 18 h at 37 °C. Samples were passed through solid-phase extraction (SPEs) columns and the eluent analyzed using ultra performance liquid chromatography - tandem mass spectrometer (UPLC-MS/MS). The parent ion for AFB1-D4-lys  fragmented to yield a daughter ion at m/z 398.2. The AFB1-lys ion (m/z 457.2) fragmented to yield a daughter ion at m/z 394.1. This methodology had a limit of detection of 0.4 pg AFB1-lys/mg albumin and was run with quality control samples run in triplicate.AFB2.31 (UFB1) has been proposed as an effective biomarker for dietary fumonisin exposure over the past 24 h, and is currently used worldwide for biomonitoring of human fumonisin exposure  were extracted for FB1 with C18-SPE cartridges. The loaded cartridges were eluted using 2 ml of 70% acetonitrile: 30% water made to 0.1% formic acid as previously described  was 30%–70%, and approximately 0.1% formic acid. Quantitation was accomplished by LC/MS as previously described. Normalization of UFB1 concentrations was described in the Supplemental material. The limit of detection for UFB1 is 0.01 ng/ml. The detection limits for FB2 and FB3 are similar  were used to measure weight at the nearest 0.1 kg and length measuring board or non-stretch Teflon synthetic tape (SECA) was used to measure height at the nearest 0.1 cm, respectively. Socioeconomic status (SES) is a conceptualization of an individual's, household's or community's access to resources and can be measured using various methodologies. Prior to initiation of recruitment and consent among participants, the MAL-ED network undertook preliminary research to determine an appropriate methodology to measure SES that could be applicable in a multi-country study. Determination of the critical variables and resources allowed the MAL-ED network to formulate an index of household SES called the Water/sanitation, Assets, Maternal education, and Income (WAMI) to be applied and comparable across multiple countries . ComponeMothers or other adult household members were queried monthly (months 9–36 of the children's lives) to collect the children's 24-hour dietary recall data, which were used to derive food and nutrient intake information. For our analyses, we averaged monthly data points to produce estimated intakes for two age ranges: 16–24 months and 25–36 months. These data were analyzed in relation to mycotoxin biomarker concentrations. Using Haydom's food composition tables, we quantified energy, macronutrient and micronutrient intakes including vitamin A, zinc, iron, folate, protein, animal protein, and protein from milk, meat, fish, poultry, eggs, and insects . These vp < 0.27) and not adjusted for statistical analysis.Grain-based food items measured in the dietary recall questionnaire included rice, maize, wheat, millet, sorghum, and common beans; whereas chickpeas and mung beans are consumed at much lower frequencies. Maize makes up the main part of the children's weaning foods . These d2.51-lys data were not normally distributed, even following lognormal transformation. Therefore, all statistical analyses of AFB1-lys were conducted utilizing tests for non-parametric data. UFB1 was not normally distributed and had a significant portion of non-detectable values, therefore they were log (x + 1) transformed prior to statistical analysis. AFB1-lys, UFB1 and growth indicators were analyzed by univariate analysis with all possible confounding variables . Anthropometric data  were normally distributed and were not altered for statistical analysis. Wilcox-rank sum tests were used to analyze statistical differences between AFB1-lys or UFB1 concentrations and categorical data. Linear regression models were built with each growth indicator - HAZ, WAZ, and WHZ at 36 months of age - as dependent variables, and AFB1-lys or UFB1 concentrations as independent variables. A p value ≤ 0.05 (two-tailed) was considered statistically significant. All statistical analyses were conducted with JMP software version 13.1 . In addition, both AFB1-lys and UFB1 concentrations were categorized by quartile, and assessed by z-scores using ANOVA followed by Tukey HSD tests to compare between quartiles and all other variables.The AFB33.11-lys analysis , the average height and weight of the children was 79.4 ± 3.12 cm and 10.4 ± 1.31 kg at the sampling time (24 months), and averages used in association with the UFB1 analysis  were 83.2 ± 4.19 cm and 11.3 ± 1.43 kg (24–36 months).A total of 114 children were enrolled in the Haydom, Tanzania cohort: 59 boys and 55 girls, with mean birth height and weight of 48.8 ± 2.52 cm and 3.27 ± 0.49 kg, respectively. At 24 months, when blood samples were taken for the AFB1-lys analysis. At 24 months, underweight incidence (WAZ < −2) rate was 17%, and 3% of the children were wasted (WHZ < −2). At 36 months, 75% of the children were stunted, 21% were underweight, and none were wasted.The child growth trajectories for HAZ, WAZ and WHZ scores are listed in p = 0.0392; WAZ, p = 0.0006; WHZ, p = 0.0068 (1-lys (p = 0.12) or UFB1 concentrations (p = 0.28).The average WAMI score, a proxy for household socioeconomic status, was 0.26 [95% CI: 0.24–0.29] . The WAM= 0.0068 a, b, c. 3.21-lys concentrations among children and dietary intake of average protein (p = 0.35), animal protein (p = 0.60), iron (p = 0.60), folate (p = 0.95) and vitamin A (p = 0.09), nor was zinc (Spearman's ρ = 0.64) intake or grain consumption frequency (Spearman's ρ = 0.40). Similarly, none of the dietary intakes of the number of grain-based food (p = 0.058), protein (p = 0.85), animal protein (p = 0.80), vitamin A (Spearman's ρ = 0.66), iron (Spearman's ρ = 0.66), zinc (Spearman's ρ = 0.28), folate (Spearman's ρ = 0.39) significantly correlated with UFB1 concentrations in children of 24–36 months in Haydom, Tanzania.In the univariate analyses, no significant associations were observed between AFBp = 0.028), and folate is significantly associated with improved WAZ (p = 0.025) and WHZ (p = 0.015).With respect to the relationship between intakes of micronutrients and child growth indicators, iron is significantly associated with improved HAZ  pg/mg albumin; 80% had detectable levels of UFB1 with a mean level of 1.3  ng/ml urine at 25–36 months of age. In total, 60 plasma samples were analyzed for AFB1-lys, 94 urine samples for UFB1, and 40 members of the cohort had both blood and urine samples that were analyzed for AFB1-lys (24 months of age) and UFB1 . Of these, 30 children tested above the limit of detection for both AFB1-lys and UFB1. As seen in studies in the USA . By comparison, regression analysis of UFB1 concentrations showed a negative association with WAZ scores   or WHZ scores .Analysis of associations of log-transformed AFB 0.0285) a, b, but1 levels were divided into quartiles (n = 23 or 24/quartile), there was a change in significance; with HAZ scores (p = 0.048) and WAZ scores (p = 0.031) being associated with biomarker concentrations of fumonisin exposure (p = 0.029)  . Those c= 0.029) .Fig. 3As4p = 0.0285; non-detectable samples excluded, p = 0.0053). Similarly, 1 was 1.3 ng/ml and the incidence of stunting was 75%. Therefore, there may be a dose-dependent effect: greater fumonisin exposure resulting in greater stunting risk. In contrast to the studies in Tanzania, fumonisin exposure was low in a cohort of children in Bhaktapur, Nepal, with only one fumonisin positive sample detected among 50 urine samples, along with a lower stunting rate of 19%  and 3.62 (range 0.58–22.7) pg/mg albumin, respectively, for children in Haydom, Tanzania and Bhaktapur, Nepal.In the present study, 72% of the children had detectable levels of AFB of life . The afl1-alb concentrations based on enzyme-linked immunosorbent assay (ELISA) method ranged from 4.7 to 23.5 pg/mg albumin, and aflatoxin exposure was not associated with LAZ (length for height z-score)  . Childrez-score)  had simiz-score) . However albumin .threshold of aflatoxin intake, below which child growth would not be affected; and the child cohorts that showed no association between aflatoxin exposure and growth impairment may have been exposed to low aflatoxin levels overall. However, the sample size of 60 for the analysis of aflatoxin exposure in the present Haydom cohort limits the statistical power of association between AFB1 and child growth impairment observed. Another limitation of our study is the cross-sectional nature of the aflatoxin and fumonisin analyses, based on biospecimens at specific timepoints in the children's development.It is possible that relatively lower aflatoxin exposure levels may not affect child growth, but higher levels do. There is likely a This is partially accounted for in that the biomarker used to measure aflatoxin exposure is for long-term (past 2–3 months). Optimally, biospecimens for the mycotoxin analyses would have been available at multiple timepoints to track with growth markers; however, absent the availability, we present the exposure data that were available at specific timepoints, and observe how they correlate with child growth at those same points. In addition, it has been shown that aflatoxin biomarker levels in African children increase over the first 36 months of life, which is affected by the weaning practices . The med1 levels in urine have recently been shown to positively correlate with elevated levels of sphinganine 1-phosphate and the sphinganine 1-phosphate to sphingosine 1-phosphate ratio in blood in a dose dependent manner in women in Guatemala; a result consistent with fumonisin inhibition of ceramide synthase in humans  are higher than recommended nutrient intakes (RNIs) which is 5.8 and 4.1 mg/day, respectively, based on the moderate bioavailability . HoweverIn a recent nutritional analysis for children aged 6–23 months in rural central Tanzania, deficiency of iron, zinc and calcium in the diets was considered as a main factor for the high prevalence of child stunting in this area, although their diets met requirements for vitamin A, protein and energy . In addiPoverty, education, socioeconomic and nutritional status, vaccinations against diseases, and dietary behaviors are traditionally considered the most important influences on childhood development. Among this cohort of children in Haydom, Tanzania, socioeconomic parameters and dietary fumonisin exposure from contaminated maize were associated with child growth outcomes.51 concentrations) during 24–36 months through contaminated maize may increase the risk of underweight among children in Haydom, Tanzania; which may be associated with their breast feeding and weaning practices. Investigation of the mechanism by which fumonisin may cause growth impairment in children is recommended. Furthermore, practical interventions of fumonisin-reduction strategies are needed for sub-Saharan African populations with high fumonisin exposure through maize-based foods.The relatively low biomarker concentrations of aflatoxin at 24 months of age in this cohort of children were not linked with growth impairment, while fumonisin exposure , the National Institute of Environmental Health Sciences of the National Institutes of Health (NJM), and the United States Department of Agriculture Agricultural Research Service .


Canine MuStem cells have demonstrated regenerative efficacy in a dog model of muscular dystrophy, and the recent characterization of human counterparts (hMuStem) has highlighted the therapeutic potential of this muscle-derived stem cell population. To date, these cells have only been generated in research-grade conditions. However, evaluation of the clinical efficacy of any such therapy will require the production of hMuStem cells in compliance with good manufacturing practices (GMPs). Because the current use of fetal bovine serum (FBS) to isolate and expand hMuStem cells raises several ethical, safety, and supply concerns, we assessed the use of two alternative xeno-free blood derivatives: human serum (HS) and a human platelet lysate (hPL).hMuStem cells were isolated and expanded in vitro in either HS-supplemented or hPL-supplemented media and the proliferation rate, clonogenicity, myogenic commitment potential, and oligopotency compared with that observed in FBS-supplemented medium. Flow cytometry and high-throughput 3′-digital gene expression RNA sequencing were used to characterize the phenotype and global gene expression pattern of hMuStem cells cultured with HS or hPL.HS-supplemented and hPL-supplemented media both supported the isolation and long-term proliferation of hMuStem cells. Compared with FBS-based medium, both supplements enhanced clonogenicity and allowed for a reduction in growth factor supplementation. Neither supplement altered the cell lineage pattern of hMuStem cells. In vitro differentiation assays revealed a decrease in myogenic commitment and in the fusion ability of hMuStem cells when cultured with hPL. In return, this reduction of myogenic potential in hPL-supplemented cultures was rapidly reversed by substitution of hPL with HS or fibrinogen-depleted hPL. Moreover, culture of hMuStem cells in hPL hydrogel and fibrinogen-depleted hPL demonstrated that myogenic differentiation potential is maintained in heparin-free hPL derivatives.Our findings indicate that HS and hPL are efficient and viable alternatives to FBS for the preparation of hMuStem cell batches in compliance with GMPs.The online version of this article (10.1186/s13287-018-0852-y) contains supplementary material, which is available to authorized users. In the last decade, the proof of concept of regenerative medicine for muscular dystrophies has been demonstrated using a variety of tissue-resident stem cell populations –8. PreclFetal bovine serum (FBS) has been traditionally used in vitro as a source of growth factors (GFs) and other elements essential for cell adhesion and proliferation . HoweverBased on their initial delayed adhesion properties, we isolated and characterized a population of adult stem cells (named MuStem cells) from healthy dog skeletal muscle, and demonstrated their efficacy following vascular delivery into the clinically relevant dog model of Duchenne muscular dystrophy (DMD), in which we observed persistent clinical stabilization and significant muscle repair –56. MorehMuStem cells were isolated from paravertebral muscle biopsies from 12 patients (aged 13–19 years) who were free of known muscular disease and underwent surgery for acute scoliosis at the Department of Pediatric Surgery of the Centre Hospitalier Universitaire (CHU), Nantes, France , the supernatant was neutralized with F12 HAM medium  containing either 20% FBS  for the research-grade protocol or 5% HS  or 5% hPL  for the GMP-compliant protocol. The pellet was digested  with either 0.125% research-grade Pronase E (Sigma-Aldrich) or with GMP-compliant neutral protease . Digested tissue was then centrifuged  and the supernatant was pooled with that obtained following the first enzymatic digestion step and centrifuged for an additional 15 min at 300×g. MDCs were obtained after sequential filtering through 100-μm, 70-μm, and 40-μm pore-diameter nylon meshes  and resuspended in F12 HAM medium containing either 2% FBS, 10% HS, or 10% hPL to assess cell number and viability using Trypan blue exclusion . hMuStem cells were isolated after 6 days using a modified version of a previously described preplating technique [2) in growth medium (GM) (Macopharma) supplemented with either 10% FBS/1% 10,000 UI/ml penicillin, 10 mg/ml streptomycin, and 25 μg/ml fungizone (amphotericin B) /10 ng/ml human recombinant basic fibroblast growth factor /25 ng/ml human recombinant epidermal growth factor  (hMuStem cellsFBS), 10% HS/1% PSF/10 ng/ml bFGF/2 ng/ml EGF (hMuStem cellsHS), or 10% hPL/1% PSF/2 ng/ml EGF (hMuStem cellshPL). Heparin  was added to hPL-supplemented media to prevent gelation of the medium. Cells were seeded on CELLstart™ substrate (Invitrogen) at 2.5 × 103 cells/cm2 and GM was replaced twice per week.Muscle-derived cells (MDCs) were isolated using either the previously described research-grade protocol  or an adechnique , 57. TheHS and hMuStem cellshPL; Table HS/HS and hMuStem cellsHS/hPL, respectively (Table Three muscle biopsies (donors ♯4–♯6) were divided and hMuStem cells , and the results were analyzed using Magellan™ data analysis software (Tecan).The contents of bFGF (#DFB50), EGF (#DEG00), insulin-like growth factor (IGF)-1 (#DG100), and hepatocyte growth factor  were measured in triplicate in two batches of hPL and two batches of HS by ELISA , following the manufacturer’s instructions. The absorbance was measured using an Infinite3 cells/cm2 and dissociated with TrypLE™  when the first of the three cell layers reached confluence. After centrifugation , cells were resuspended in appropriate supplemented GM and the viable cell number was determined using Trypan blue staining. The number of population doublings (PDs) was calculated at each passage using the following formula: PD = log(nf/n0)/log(2), where n0 and nf are the number of cells initially plated and the number of cells harvested at the end of the passage, respectively [To define the proliferative properties of hMuStem cells cultured in different conditions, cells were seeded in triplicate at 2.5 × 10ectively . The CPDHS and hMuStem cellshPL at P4  in triplicate in gelatin-coated dishes  at low density (< 5 cells/cm2) and in the appropriate supplemented GM. The medium was replaced every 4 days. After 8 days, cell layers were washed with phosphate buffered saline , fixed with 4% paraformaldehyde  for 10 min, and washed twice with PBS. Colonies were stained with May-Grünwald stain (Sigma-Aldrich) for 5 min at room temperature (RT), washed with distilled water, and counterstained with Giemsa . The frequency of CFUs was determined as the mean number of cell clusters containing at least 50 cells divided by the number of cells initially seeded, and expressed as a percentage.To evaluate the clonogenic potential of hMuStem cells, colony forming unit (CFU) assays were performed by plating hMuStem cellsHS and hMuStem cellshPL (P4). Cells were incubated (30 min at 4 °C in darkness) with antibody (Ab) against the appropriate cell-surface marker  coated with CELLstart™ substrate. Twenty-four hours later, cell layers were fixed with 4% PFA , permeabilized with 0.3% Triton X-100 , and incubated  in blocking buffer (5% goat serum in PBS). Cells were then incubated with the appropriate Ab , quantified using a NanoDrop spectrophotometer  after DNase treatment , and converted to cDNA by reverse transcription as described previously . OligonuHS (n = 3) and hMuStem cellshPL (n = 3) at P4 and processed as described earlier. RNA integrity was determined using the Agilent Eukaryote Total RNA Nano kit with the 2100 Bioanalyzer . A high-throughput 3′ digital gene expression RNA-sequencing (DGE-seq) protocol was performed as described previously [hg19, obtained from the UCSC Genome Browser) using bwa version 0.7.4 4 with the nondefault parameter “-l 24”. Reads mapping to several positions in the genome were filtered out from the analysis. Digital gene expression (DGE) profiles were generated by counting the number of unique UMIs associated with each RefSeq gene for each sample.Total mRNA was extracted from hMuStem cellseviously . Brieflyeviously  and can HS and hMuStem cellshPL were identified using a fold-change threshold ≥ 2 and adjusted p < 0.05.Differential expression analysis was performed using the DESeq2 Bioconductor package version 1.14.1 . The dat4 cells/cm2 on 24-well plates and cultured in media supplemented with either 10% HS, 10% hPL, or 10% FBS for 2 weeks, after which HS, hPL, or FBS was replaced with 1% FBS (differentiation medium (DM)). After 4 days, cultures were fixed in 4% PFA, and incubated with 5% Triton X-100 , 20% goat serum in PBS , and finally anti-human sarcomeric myosin heavy chain isoform (sMyHC) Ab  for 60 min at 37 °C. Specific Ab binding was then visualized using AlexaFluor® 488-coupled secondary Ab  and nuclei were counterstained with DRAQ5 . The fusion index (FI) was determined as the ratio of nuclei within sMyHC+ myotubes (≥ 2 nuclei) to the total number of nuclei. Two random fields in each of three replicate wells were analyzed and at least 651 nuclei per well were considered.For myogenic differentiation, hMuStem cells were seeded at 3 × 104 cells/cm2 in Dulbecco’s Modified Eagle Medium /10% FBS/1% PSF for 1 day, after which FBS was replaced with 2% horse serum. After 4 days, multinucleated cells were visualized as described earlier by immunolabeling for sMyHC. Hybrid myotubes were detected using specific human lamin A/C Ab  and combined with AlexaFluor® 555-coupled secondary Ab .The behavior of hMuStem cells was also assessed in coculture experiments with dystrophic cells . After expansion in different culture conditions, hMuStem cells and D7 cells were mixed at a ratio of 5:1 for a final density of 3 × 10g to pellet debris . The protein concentration was determined using a BCA protein assay (Sigma-Aldrich). Fifteen micrograms of proteins from cell homogenate were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 4–12% polyacrylamide gels  and electroblotted onto nitrocellulose membranes  using a Bio-Rad® liquid blotting system at 30 mA for 2 h. The membranes were blocked using 50% blocking buffer  in PBS  and incubated overnight at 4 °C with primary Abs against sMyHC  and GAPDH . After washing with Tween 0.1% in PBS, the blots were incubated with fluorophore-conjugated anti-mouse and anti-rabbit secondary antibody. Equal protein loading was verified through GAPDH labeling and Ponceau red staining of the membranes. Western blot bands were scanned with Odyssey®.For protein extraction, cells were homogenized in RIPA lysis buffer containing 150 mM NaCl, 50 mM Tris–HCl, pH 7.4, 1% Nonidet-P40, 1% glycerol, 1 mM EDTA, and protease inhibitors using the Precellys . Homogenates were centrifuged at 14,000×4 cells/cm2 and cultured in appropriate supplemented GM for 1 day, after which they were incubated in specific adipogenic and osteogenic cell induction media for 14 and 21 days, respectively, as described previously [2, 15 min at 450 nm), as described previously [PPARγ) and the integrin binding sialoprotein (IBSP) genes were determined to assess adipogenic and osteogenic differentiation, respectively. Primers are listed in Additional file hMuStem cells (P4) were seeded in triplicate at 3 × 10eviously . Adipogeeviously . Total Rp < 0.05.Results are presented as the mean ± standard deviation (SD) where technical replicates are presented, and as the mean ± SEM when the number of samples is > 3. Statistical analyses were performed using R software (3.3.2 version). Data were tested for normality and independence before analysis, as described previously . StatistFBS batches cultured in GM supplemented with either 10% HS or 10% hPL, and compared these results with those obtained in GM supplemented with 10% FBS. After 12 days, the number of CPDs recorded was 11.3 ± 0.7 and 10.7 ± 0.9 for hMuStem cellsFBS cultured with HS and hPL, respectively, compared with 9.9 ± 0.9 for those cultured in FBS , while that of cells cultured with hPL was unchanged. No statistical differences were found between hMuStem cellsFBS cultured with HS and hPL, indicating that both human supplements support hMuStem cell expansion. Interestingly, the absence of 10 ng/ml bFGF in HS-supplemented medium negatively affected the proliferation of hMuStem cellsFBS, as evidenced by a 33.7% decrease in CPD, an effect not observed in hMuStem cellsFBS cultured in hPL-supplemented medium   Table . Both ceable FBS . Compareely Fig. . By contons Fig. .Fig. 2InFBS have been described as early myogenic progenitor cells of perivascular origin [HS and hMuStem cellshPL  abundantly expressed (> 99%) the typical MSC markers CD29, CD44, CD73, CD90, and CD105 . In both hMuStem cellsHS and hMuStem cellshPL, two populations were identified among CD56− cells, depending on their expression of CD140b. A significant proportion of CD56−/CD140b+ cells expressed CD146, regardless of culture conditions (data not shown). Culture in HS-GM and hPL-GM poorly affected the expression by CD56+ cells of the well-known perivascular cell markers CD140b and CD146  where only 34% of the cells were positive. In two samples (donors ♯4 and ♯5), CD56+ cells exhibited homogeneous expression of CD146 in both culture conditions, while in one sample (donor ♯6) the percentage of CD146+ cells was 44% in hMuStem cellsHS and 63% in hMuStem cellshPL. Taken together, these results indicate that hMuStem cells cultured in HS-GM or hPL-GM share a similar phenotype, although culture in hPL resulted in a slightly less heterogeneous cell population. The main source of variation was the donor. The myogenic profile of hMuStem cellsHS and hMuStem cellshPL was further analyzed based on their expression of the myogenic regulatory factors (MRFs) Myf5 and MyoD, and of muscle-specific intermediate filament desmin. We found that the proportions of Myf5+, MyoD+, and desmin+ cells were consistently smaller in hMuStem cellshPL versus hMuStem cellsHS, which exhibited decreases of 2.6–3.0-fold, 4.0–15.4-fold, and 1.3–1.4-fold, respectively , few differentially expressed genes were identified , thioredoxin interacting protein (TXNIP), mitogen-activated protein kinase 4 (MAP4K4), laminin subunit alpha 4 (LAMA4), and secretogranin II (SCG2), were upregulated in hMuStem cellshPL versus hMuStem cellsHS. Previous studies have described heparin-induced induction of TXNIP and an interaction between heparin and LAMA4 [F13A1), methylenetetrahydrofolate dehydrogenase 2 (or NADP-dependent methylenetetrahydrofolate dehydrogenase 2-like protein) (MTHFD2), and the myogenic marker myosin light chain 2 (MYL2) were downregulated in hMuStem cellshPL versus hMuStem cellsHS. Together, the results of cytometry, immunocytochemistry, and DGE-seq analysis show that while the mesenchymal/perivascular phenotype is maintained in hMuStem cells expanded in HS-GM or hPL-GM, hMuStem cellshPL tend to display reduced myogenic commitment compared with hMuStem cellsHS.To further evaluate the effects of the nutritive substitutes HS and hPL on global gene expression in hMuStem cells, a DGE-seq protocol was performed. Of the 14,936 genes detected in at least one out of the six cell batches , an effect that may be related to the reduced myogenic commitment of the former.Like canine MuStem cells , in vitrotential . After 4ved Fig. . Thus, aHS and hMuStem cellshPL, confirming the ability of these cells to fuse. Human nuclei located in sMyHC+ myotubes accounted for 33.4–42.3% and 22.6–32.0% of all nuclei in D7 cocultures with hMuStem cellsHS and hMuStem cellshPL, respectively. Moreover, cells cultured with hPL displayed reduced fusion ability compared with those cultured with HS (1.3–1.5-fold change), even though they fused more efficiently when cocultured with the D7 cell line than when cultured alone in DM (FI: 22.6–32.0% versus 2.3–4.9%). Taken together, these results suggest that hMuStem cellshPL are less capable of spontaneously differentiating into myogenic cells than hMuStem cellsHS, which showed a differentiation potential similar to that of hMuStem cellsFBS [hPL were capable of efficiently fusing and generating myotubes when cocultured with the D7 cell line.To further investigate these apparent differences in fusion rates, coculture experiments were performed with the D7 dystrophic mouse cell line. Hybrid myotubes  Fig. , arrow wcellsFBS . NonetheFBS have been described previously as oligopotent cells based on their ability to differentiate into osteogenic and adipogenic lineages as well as the myogenic lineage [HS and hMuStem cellshPL, we next investigated their ability to differentiate into osteogenic and adipogenic lineages. After 14 days in adipogenic induction medium, fluorescent Nile Red staining revealed the presence of cytoplasmic accumulations of small lipid vesicles in cells from all batches, regardless of isolation and culture conditions  were cultured for two passages in either HS-GM or hPL-GM. The experiments described earlier for hMuStem cellsHS and hMuStem cellshPL were repeated for each of these batches   Table .MYF5, MYOD1, and DES were 1.5-fold, 1.9-fold, and 1.7-fold lower, respectively, in hMuStem cellsHS/hPL versus hMuStem cellsHS/HS (p < 0.05). At the protein level, 40.6 ± 3.2%, 20.7 ± 2.9%, and 92.1 ± 2.3% of hMuStem cellsHS/HS were positive for Myf5, MyoD, and desmin, respectively, compared with only 30.7 ± 4.5%, 9.1 ± 1.2%, and 74.0 ± 4.0% of hMuStem cellsHS/hPL . After 4 days in DM, MyoD and myogenin were expressed in 23.5 ± 3.2% and 18.3 ± 1.8% of hMuStem cellsHS/HS, respectively, whereas only 11.6 ± 1.7% and 5.2 ± 0.5% of hMuStem cellsHS/hPL expressed these proteins . In line with our observations in hMuStem cellsHS and hMuStem cellshPL, the FI values recorded for hMuStem cellsHS/HS and hMuStem cellsHS/hPL were 26.5 ± 5.6% and 2.6 ± 1.1%, respectively . In comparison, hMuStem cellsHS/FBS . In D7 coculture experiments performed with hMuStem cellsHS/HS and hMuStem cellsHS/hPL, we detected 34.3 ± 2.3% and 14.8 ± 2.2%, respectively, of human nuclei in sMyHC+ myotubes  . Analysis of adipogenic differentiation potential revealed no significant differences in lipid vesicle staining or PPARγ mRNA expression between culture conditions, confirming the maintenance of adipogenic potential in both hMuStem cellsHS/HS and hMuStem cellsHS/hPL , with the highest dose corresponding to that found in hPL-GM. Culture for 6 days in GM in the presence of heparin resulted in higher population doublings (PDs) that ranged from 5.13 ± 0.34 to 5.25 ± 0.21 compared with 4.86 ± 0.23 in GM without heparin, showing a positive, dose-independent effect of heparin on the proliferation rate of hMuStem cellsHS  did not differ significantly from that observed for the lowest dose   Fig. . We alsoHS were plated on CELLstart™ substrate-coated plates and covered with nutritive gel (hPLgel condition). In this condition, we recorded an FI of 14.9 ± 1.6%, as compared with 5.6 ± 0.4% in the presence of 0.5 IU/ml heparin, indicating a significant inhibitory effect of heparin on myogenic differentiation (p < 0.001) , suggesting that the reduction in myogenic differentiation may not be entirely attributable to heparin. We next investigated the effects of culture in fibrinogen-depleted hPL-GM (hPLfd), produced by the addition of a calcification step to the hPL production process, circumventing the need for heparin supplementation. Myogenic differentiation potential was evaluated after one passage of 6 days in either HS-GM, hPL-GM, or hPLfd-GM , the fusion capacity of hMuStem cellsHS/hPL remained low (FI = 2.3 ± 0.4%), while that of hMuStem cellsHS/hPLfd was significantly higher  but still significantly lower than that of hMuStem cells cultured in HS-GM (p < 0.01). These results indicate that myogenic differentiation potential is maintained in heparin-free hPL, but is lower than that observed in cells cultured in HS. These findings confirm the role of heparin in the suppressive effect of hPL on myogenic differentiation of hMuStem cells, but do not rule out the potential contribution of other factors.We next investigated the myogenic differentiation potential of hMuStem cells in heparin-free hPL-GM. hMuStem cells01) Fig. . Nonethe-GM Fig. . ComparehPL (P3) were cultured for the fourth passage (5 or 6 days) with either hPL, HS, or heparin-free hPL (hPLfd or hPLgel). Myogenic differentiation was assessed at the end of P4 . Interestingly, FI values recorded for hMuStem cellshPL cultured in HS-GM and hPLfd-GM  were higher than those of cells cultured with hPL (p < 0.001)  Fig. . This in-GM Fig. . InteresOver the past 6 years, several studies have demonstrated the clinical efficacy of MuStem cell treatment to repair muscle tissue, highlighting the potential of this muscle-derived stem cell population in cell-based therapies –56. hMuSPotential FBS substitutes evaluated to date include HS and platelet derivatives such as PRP and hPL, which efficiently support and generally promote in-vitro proliferation of MSCs derived from bone marrow , 47, 71,HS and hMuStem cellshPL, the latter contained a lower proportion of cells expressing the myogenic regulatory factors Myf5 and MyoD, and intermediate filament desmin. These observations suggest reduced myogenic commitment capabilities in the presence of hPL, and are supported by the lower FI recorded for hMuStem cellshPL, indicating a reduced ability to differentiate in vitro (when cultured in a myogenesis-promoting medium) into the myogenic cell lineage. By contrast, the rate of differentiation of hMuStem cellsHS was comparable to that described previously for hMuStem cellsFBS [hPL were able to fuse efficiently  when cocultured with the D7 dystrophic cell line, evoking that hMuStem cellshPL may participate in in-vivo muscle regeneration via regeneration signals generated by other cells in a pathological context. Mitogen-activated protein 4 kinase 4 (Map4k4) has been recently shown to negatively regulate skeletal muscle differentiation of C2C12 cells in a Myf5-dependent manner [MAP4K4 and MYF5 genes were upregulated and downregulated, respectively, in hMuStem cellshPL versus hMuStem cellsHS, suggesting that the Map4k4-related signaling pathway may contribute to the suppressive effect of hPL on myogenic differentiation of hMuStem cells.While the expression of classical myogenic (CD56), mesenchymal , and pericyte  cell markers was maintained in the hMuStem cellscellsFBS . NonetheHS, even at the lowest concentration of heparin used to prevent gelation of the medium. The use of fibrin gel as a 3D-nutritive matrix for MSC expansion has been proposed to preclude the need for heparin supplementation [HS in fibrin gel resulted in a drastic reduction in proliferation rate. Another alternative method we tested was to culture hMuStem cellsHS beneath a nutritive fibrin matrix. This produced cells with the spindle-shape morphology typical of hMuStem cells, albeit with a slightly reduced proliferation rate and an intermediate myogenic potential, confirming a suppressive effect of heparin supplementation on myogenic potential. Another strategy to circumvent the need for heparin supplementation is to generate serum-converted hPL by depleting fibrinogen, either by adding CaCl2 or activating thrombin [2 (hPLfd), hMuStem cellsHS/hPLfd displayed an intermediate myogenic potential, highly suggesting that heparin in hPL contributes to the observed reduction in the myogenic potential of hMuStem cells. Interestingly, passage of hMuStem cellshPL in HS-supplemented medium resulted in a rate of differentiation comparable to that observed in hMuStem cellsHS, indicating that the reduction in myogenic commitment capabilities caused by heparin-containing hPL is reversible. Passage of hMuStem cellshPL in hPLfd-GM produced similar results, but only partially restored myogenic potential. The hPLgel condition was the least effective in restoring the myogenic potential of hMuStem cellshPL. Taken together, our results describe an effective two-step alternative for hMuStem cellhPL expansion that produces cells with a myogenic potential comparable to that of hMuStem cellsHS. One possible application of this method is to carry out the isolation procedure and the initial stages of in-vitro expansion using hPL-GM, thereby maintaining MuStem cells in their proliferative state, and subsequently switching to heparin-free hPL-GM (hPLfd) for the final stages of production to increase the myogenic potential. The absence of spontaneous fusion of hMuStem cells during in-vitro expansion could be advantageous in the context of clinical applications, providing greater intra-batch homogeneity in terms of cell phenotype, commitment capabilities, and morphology. Morphology is of particular interest, given that the presence of enlarged differentiated cells may impair biodistribution and tissue dissemination and provoke clotting during in-vivo injection [A key difference between HS and hPL is the presence of plasma coagulation factors such as fibrinogen and other platelet-derived factors , 42 in hentation . Howeverthrombin , or by mthrombin . When thnjection . Furthernjection , 78–80.2, thrombin, or heparin. This increases the likelihood of variability between laboratories and means that a greater level of control is required to ensure adherence to GMP [Key considerations when choosing a culture supplement include its effect on cell features, its compatibility with the large-scale production processes required for clinical applications, and availability. The production of hPL requires more steps than that of HS, including successive freezing/thawing steps, the elimination of debris by centrifugation and/or filtration, and the addition of reagents such as CaCle to GMP . StandarThe present findings indicate that HS and hPL are suitable for the isolation and expansion of hMuStem cells for use in clinical applications. Our findings represent an important step forward in the development of a clinically compliant process for the production of hMuStem cells, and their subsequent testing in future clinical trials.Additional file 1:Table S1. List of antibodies used for hMuStem cell characterization by flow cytometry analysis (PDF 16 kb)Additional file 2:Table S2. List of antibodies used for hMuStem cell characterization by immunocytochemistry analysis (PDF 11 kb)Additional file 3:Table S3. List of primers used for RT-qPCR analysis (PDF 14 kb)Additional file 4:Table S4. Growth factor concentrations (PDF 42 kb)Additional file 5:Table S5. The UMI counts for each gene in each sample normalized by dividing by the sum of UMI counts across all genes in the same sample. Normalized UMI counts expressed in UPM  (XLSX 8238 kb)Additional file 6:Figure S1. Influence of heparin on hMuStem cell proliferation rates. hMuStem cellsHS cultured for 6 days in HS-GM without heparin or with increasing doses of heparin (0.5–5 IU/ml). Population doublings (PDs) determined in three independent cell batches  (PDF 34 kb)


AbstractIoanellamimon are described for the first time parasitizing to Mimoncozumelae from Yucatan, Mexico. Male of Ioanellamimon is characterized by the presence of legs I with the tibia and tarsus fused forming a small complex devoided of apical claws, legs II–IV with two claws, setae vi at level of anterior end of genital plate, genital plate rounded with an anterior projection, all intercoxal setae short; while the tritonymph is characterized by the presence of legs I unequal; legs II–IV with 2-1-1 claws, and posterior region of dorsal idiosoma with 3 pairs of cylindrical and toothed setae. Additionally, we include new locality and host records for Eudusbabekiamimon which was also found on Mimoncozumelae. Both species were described originally in association with Mimonbennettii at Bartica, Guyana.The male and the tritonymph of  Eudusbabekia Jameson, 1971 and Ioanella Dúsbabek & Lukoschus, 1973, include species associated with Phyllostomidae bats. The former is conformed by 32 species  were recorded parasitizing to Mimonbennettii Gray, 1938 from Bartica, Guyana   were removed from bats using fine, sharp forces and fixed and preserved in vials with 96% ethanol. The specimens were cleared in lactophenol and mounted in Hoyer’s medium. Mites were determined taxonomically. Descriptions and nomenclature for idiosomal setation follows SEM), the specimens were dehydrated in 100% ethanol and dried to a critical point with liquid carbon dioxide. The dried specimens were mounted on aluminum specimen stubs, coated with a gold palladium alloy, and examined using a scanning electronic microscope Hitachi Stereoscan Model S–2469 N SEM . Mites were deposited at Colección del Laboratorio de Acarología, Facultad de Ciencias (LAFC), Universidad Nacional Autónoma de México (UNAM). Host were captured under the permission SGPA/DGVS/08257/13 and deposited at Colección de Mamíferos, Museo de Zoología “Alfonso L. Herrera”, Facultad de Ciencias (MZFC), UNAM.A total of five bats were captured inside two hollowness located at carretera Santa Elena-Loltún Km 56, Yucatán, México ; 1♀, 1 PN same data, except Oquedad 2 (LAFC-A02). 1♂ ex Taxon classificationAnimaliaTrombidiformesMyobiidaeMimoncozumelae, Oquedad 1, carretera Santa Elena-Loltún Km 56, Yucatán, México (LAFC-A03); 4 TN, 3 ♀, 2♂, same data, except Oquedad 2 (LAFC-A04).7 TN, 3 ♀, ex Male . Body length 225 (223–228); wide 139 (125–152). Body 1.6 larger than wide. Dorsal idiosoma , sce 28 (26–31), sci 17 (14–17), c2 20 (17–22). Distances between bases of setae: vi-vi: 30 (29–31), ve-ve: 46 (45–47), sce-sce: 53 (52–54), sci-sci: 23 (21–26), c2-c2: 79 (76–82), ve-sce 28 (24–29), sce-c2 68 (65–68), vi-sci 31 (29–33). Genital plate rounded with an anterior projection  but slightly less pronounced.Legs. Tibia and tarsus I fused forming a small complex devoid of apical claws Fig. . Genua ITrytonymph (Based on 4 tritonymphs). Dorsal idiosoma. Posterior region of dorsum with 3 pairs of cylindrical and toothed setae: e1 14 (11–18), e2 15 (14–18), f1 14 (12–15)  Fig. . Setae vhin Fig. . Legs. Thin Fig. ; claspinhin Fig. . Setatiohin Fig. .Ioanella by the presence of legs I with the tibia and tarsus fused forming a small complex devoided of apical claws, legs II–IV with two claws, vi and sci thin and short, all intercoxal setae very short and the lacking of f2 .This work represents the first description of a male of the genus Eudusbabekiamimon and Ioanellamimon are two species of myobiids recorded originally parasitizing to Mimonbennettii, in this work both species are referred for the first time in association with Mimoncozumelae, species formerly included as subspecies of Mimonbennettii  referred by Mimon must include to Mimonbennettii and Mimoncozumelae, and the genus Gardnerycteris (=Anthorhina) to Gardnerycteriscrenulatum  and Gardnerycteriskoepckeae . In this context, Eudusbabekiamimon and Ioanellamimon will be associated with the bat species of the genus Mimon, while Eudusbabekiaanthorhinae Dúsbabek and Lukoschus, 1974 and Ioanellamartae Dúsbabek and Lukoschus, 1973 to the species of the genus Gardnerycteris.On the other hand, PageBreakConsidering of degree of specificity of myobiid mites to genera or groups of species of hosts , the refGuyana (Bartica), Mexico (Yucatan).