Plasmodium are higher in males than females, suggesting that sexual hormones influence both the pathogenesis of and immune response in CM. However, no studies on inflammation mediators in the brains of both sexes have been reported. In this work, the mRNA expression levels of the proinflammatory cytokines IL-1β, IFN-γ, TNF-α, and IL-2 were measured in the preoptic area, hypothalamus, hippocampus, olfactory bulb, frontal cortex, and lateral cortex regions of gonadectomized female and male CBA/Ca mice infected with P. berghei ANKA . Our findings demonstrate that both infection with P. berghei ANKA and gonadectomy trigger a cerebral sex dimorphic mRNA expression pattern of the cytokines IL-1β, TNF-α, IFN-γ, and IL-2. This dimorphic cytokine pattern was different in each brain region analysed. In most cases, infected males exhibited higher mRNA expression levels than females, suggesting that sexual hormones differentially regulate the mRNA expression of proinflammatory cytokines in the brain and the potential use of gonadal steroids or their derivates in the immunomodulation of cerebral malaria.Cerebral malaria (CM) is the major complication associated with death in malaria patients, and its pathogenesis is associated with excessive proinflammatory cytokine production. Notably, the severity and mortality of natural infections with  However, whether sexual hormones are involved in the pathogenesis of CM remains unclear.Notably, the severity and mortality of natural infections with  females –15. In e females , 17. Furpression , 18, 19.nfection . These sPlasmodium berghei ANKA have many characteristics in common with human diseases and serve as the best CM model available. As in humans, parasitised red blood cells accumulate in the brains of susceptible mice during infection, and numerous leukocytes are present in the brain blood vessels of these mice [CBA/Ca mice infected with ese mice , 22.P. berghei ANKA [α, IFN-γ, and IL-1β [P. berghei ANKA.In previous studies, we have shown sexual dimorphism in the systemic immune responses of CBA/Ca mice infected with hei ANKA . Sex stehei ANKA . In addind IL-1β , 24, andnd IL-1β . Furthernd IL-1β , 27. Howβ, IL-2, TNF-α, and IFN-γ in the brain, may at least partially explain sexual dimorphism in CM.To our knowledge, this work is the first report documenting the dimorphic and sex steroid-regulated expression of proinflammatory cytokines in delimited areas of the brain during CM. The dimorphic mRNA expression of IL-1CBA/Ca mice were originally donated by Dr. William Jarra . Animal care and experiments were conducted at the FES Zaragoza, Universidad Nacional Autónoma de México (UNAM). All experimental procedures in the animals were approved by the Institutional Care and Animal Use Committee, permit number 28/04/SO/3.4.1, and adhered to Mexican regulation NOM-062-ZOO-1999 for the use and care of laboratory animals. Euthanasia of the experimental animals was performed humanely by cervical dislocation after anaesthesia with 5% sevoflurane .P. berghei ANKA parasites were also kindly donated by Dr. Jarra and were cryopreserved in liquid nitrogen. The parasites were thawed and injected into one 4-week-old mouse. When parasitaemia reached 20%, parasitised red blood cells were used to infect the experimental CBA/Ca mice. All the infected animals were intravenously inoculated with 1 × 103P. berghei ANKA-infected red blood cells.P. berghei ANKA. Mice were sacrificed 9 days after infection; the absence of ovaries was confirmed by visual inspection. The complete design was repeated two times using 5 female mice per treatment in each individual experiment (n = 10).Ovariectomies were performed as we previously reported . Brieflyn = 10).Four-week-old male mice were anaesthetised, and their testes were removed via scrotal incisions as we previously reported . Brieflyn = 10) for each sex: (1) intact mice (n = 10), (2) bilaterally gonadectomized (GX) (n = 10) mice, and (3) mice that underwent surgery without gonadectomy (sham operated) (n = 10). The mice were allowed to recover from surgery for 4 weeks prior to parasite infection. The mice were sacrificed by cervical dislocation on day 9 postinfection, and their blood and brains were collected to assess parasitaemia and proinflammatory cytokine mRNA expression, respectively.Mice were allocated into three groups  in Giemsa buffer. Enumeration of the parasite load was performed under a 100x oil immersion lens using a Zeiss Standard 20 microscope . Parasitaemia levels of 0.5% or greater were evaluated by counting the number of parasitised erythrocytes that were present among 200 red blood cells. Lower levels of parasitaemia were determined by counting the number of parasitised red blood cells that were present in 50 microscope fields. The data are presented as the geometric mean of the percentage of parasitaemia. The complete experiment was repeated 2 times .The Mouse Brain in Stereotaxic Coordinates. Briefly, the POA and HT regions were dissected by making a razor cut to the anterior commissure just rostral to the optic chiasm, the anteroventral limit of which was the nucleus of the diagonal band of the lateral Broca. The HC was obtained by making a razor cut underneath the FC, and the FC was excised directly from the frontal lobes; the LC was excised directly from the lateral lobes.The preoptic area (POA), hypothalamus (HT), olfactory bulb (OB), hippocampus (HC), frontal cortex (FC), and lateral cortex (LC) regions from all mice were extracted according to n = 10).Brain tissues from 5 animals were pooled by region to increase the sample quantity. All experiments were performed in duplicate  were then PCR-amplified from the resulting cDNAs.Total RNA was isolated from the POA, HT, HC, OB, FC, and LC sections of control and infected mice using TRIzol . Briefly, each tissue sample was removed and disrupted immediately in TRIzol (1 mL/0.1 g tissue); 0.2 mL of chloroform was then added per 1 mL of TRIzol. The aqueous phase was recovered after 10 minutes of centrifugation at 14,000 ×g. RNA was precipitated with isopropyl alcohol, washed with 75% ethanol, and dissolved in RNase-free water. RNA concentrations were measured, based on absorbance at 260 nm, and RNA purity was verified after electrophoresis on 1.0% denaturing agarose gel in the presence of 2.2 M formaldehyde. Total RNA from all extracted tissues was reverse transcribed, and IL-1μg of total RNA from each tissue was incubated for 1 h at 37°C with 400 units of M-MLV reverse transcriptase  in a 50 μL reaction, containing 50 mM of each dNTP and 0.05 μg of oligo (dT) primer (Gibco). The 50 μL PCR reaction contained 10 μL of cDNA, 5 μL of 10x PCR buffer , 1 mM of MgCl2, 0.2 mM of each dNTP, 0.05 μM of each primer, and 2.5 units of Taq DNA polymerase (Biotecnologías Universitarias).The sequences of primers used for amplification have been previously published . BrieflyTwenty microliters of each PCR reaction was electrophoresed on a 2% agarose gel and visualized with ethidium bromide. A single band was detected in all cases, as expected. To determine whether all reactions were in the exponential phase of amplification and ensure that the changes in expression were not artefacts , we constructed RNA, cycling, and temperature curves for each gene.PCR bands were quantified by the densitometric scanning of several autoradiograms at various exposures and expressed as the ratio of the target gene (cytokines) signal to that of 18S rRNA.μL of serum from each mouse was incubated with 5 μL of nitrate reductase  and 15 μL of NADPH  for 15 min at room temperature; 100 μL of Griess reagent (Sigma-Aldrich) and 100 μL of trichloroacetic acid (10% aqueous solution) were added. The protein precipitate was then removed by centrifugation at 14,000 rpm for 5 min, and 100 μL of each supernatant was transferred to a 96-well flat-bottom plate. Concentrations were evaluated by an ELISA reader  at 540 nm using a standard curve comprising sodium nitrate (Sigma-Aldrich) diluted in pooled serum from uninfected control mice treated in the same manner as the experimental samples.Serum nitrate concentrations were evaluated according to the Griess method as described previously . Brieflyβ, IL-2, IFN-γ, and TNF-α in each tissue sample, expressed as the ratio of the optical density (OD) of the corresponding gel to that of 18S rRNA. Additionally, we measured nitric oxide levels in plasma.We designed the study as a 3-factor experiment. The independent variables were infection , sex , and gonadectomy . The dependent variables were the number of parasites and the mRNA expression levels of IL-1n = 10) were infected with P. berghei ANKA, and the other half served as uninfected controls. Statistical analysis of variance was performed using Prism 7 software (GraphPad Software Inc.). All data are expressed as the mean ± SD of two independent experiments. When applied, post hoc individual contrasts of group means were analysed by Tukey's test using the sum of residual and 3-factor interaction variances to test for significant differences. A P value < 0.05 indicated a significant difference.The complete design was repeated 2 times using 20 mice per group . Half of the mice in each group  than their female counterparts in the same condition (We measured the relative mRNA expression levels of the proinflammatory cytokines IL-1ondition .P < 0.05) . Interestingly, gonadectomy increased TNF-α mRNA expression in the uninfected mice of both sexes (P < 0.01), and infection in GX mice significantly downregulated its expression (P > 0.01)   . Infecti > 0.01) . Finally < 0.01) .β, IL-2, TNF-α, and IFN-γ were detected in the OBs of mice of both genders and in control intact and infected mice. IL-1β mRNA expression was constant in intact female and male mice. Infection induced a dimorphic pattern, particularly in sham-infected animals; females exhibited significantly decreased IL-1β expression compared with that in sham, intact, and uninfected mice, and the opposite effect was detected in males (P < 0.001) (P < 0.05)   (IL-1< 0.001) . IL-2 mR < 0.05) . Infecti < 0.05) . Finally < 0.05) .β expression were detected between uninfected intact mice of either gender, a pattern that was not altered by infection or gonadectomy. However, in the sham groups, IL-1β expression was altered and became sex related; sham uninfected mice exhibited increased IL-1β expression, and the expression of this gene was higher in females compared to that in their male counterparts. P. berghei ANKA infection decreased IL-1β expression in sham females and did not alter its expression in males (P < 0.05)   (No differences in IL-1 < 0.05) . IL-2 mR < 0.05) . TNF-α m< 0.001) . IFN-γ e< 0.001) .β mRNA expression did not differ between sexes and did not change with infection. However, in the sham-infected groups, a clear sexual dimorphism was detected; IL-1β expression was higher in infected males than in infected females (P < 0.001)   (IL-1< 0.001) . IL-2 mR< 0.001) . TNF-α m< 0.001) . IFN-γ e< 0.001) .β mRNA expression was dimorphic in intact infected and sham-infected groups; males exhibited higher IL-1β mRNA expression than females in the same condition. IL-2 mRNA expression exhibited a dimorphic pattern; it was upregulated in intact infected and sham-infected males compared with their female counterparts. Interestingly, infection downregulated IL-2 mRNA expression in intact females compared with that in males in the same condition and also in GX-infected mice of both sexes (P < 0.001)   (IL-1< 0.001) . TNF-α e< 0.001) . IFN-γ m< 0.001) .β was not dimorphic in any group analysed except for GX-infected animals, which clearly showed a dimorphic pattern; IL-1 β mRNA expression was significantly higher in males than in females in the same condition (P < 0.05) (P < 0.001)   (IL-1 < 0.05) . IL-2 mR< 0.001) . TNF-α m< 0.001) . IFN-γ m< 0.001) .Importantly, in all the experimental groups analysed, IL-4, IL-10, IL-6, and IL-12 were undetectable in every area of the brain analysed. In the positive control (spleens of intact infected mice), these cytokines were always detected (results not shown), suggesting that these findings are not artefacts.in vivo [Nitric oxide is promptly oxidized to the stable inorganic nitrogen oxides nitrite and nitrate in vivo , 31, andin vivo . Nitric P. berghei ANKA and gonadectomy triggered a sexually dimorphic cerebral mRNA expression pattern of the cytokines IL-1β, TNF-α, IFN-γ, and IL-2. This dimorphic cytokine pattern was different in each brain region analysed . In most cases, infected males exhibited higher mRNA expression levels than females.In this work, we report that both infection with P. berghei ANKA is lethal to CBA/Ca mice; all female and male infected mice died by day 13 postinfection. Gonadectomy, which substantially decreases sex hormone levels, decreases survival in infected female mice by one day compared with intact female mice. In contrast, gonadectomy in male mice increased survival by one day relative to the intact male group. Interestingly, gonadectomy increased the splenic index only in intact infected male mice, suggesting that gonadal male sex hormones negatively regulate the proliferation of spleen cells in mice infected with P. berghei ANKA [Previously, we have documented that infection with hei ANKA . These fhei ANKA . The splγ, TNF-α, and IL-1β, contribute to CM pathogenesis in humans [α mRNA expression was upregulated in the POA, HT, LC, FC, and HC of male infected mice compared to that in their female counterparts. Contrary to our expectations, infection downregulated IL-1β, TNF-α, and IFN-γ mRNA expression in some cerebral regions, such as the OB and HC, particularly in the sham and GX mice, suggesting that strong regulatory mechanisms prevent inflammatory immune responses in these specific brain regions. In contrast, gonadectomy upregulated IL-1β expression in the POA region, TNF-α expression in the POA, HT, OB, and FC regions, and IFN-γ expression in the HT region. These findings suggest that gonadal hormones negatively modulate the expression of IL-1β, TNF-α, and IFN-γ in these brain regions. Interestingly, infection triggered dimorphic mRNA expression of proinflammatory cytokines; in general, males exhibited higher expression levels than females. IFN-γ was upregulated in the POA, OB, HC, FC, and LC regions of infected male animals compared with that in their female counterparts. These findings are consistent with those of Hunt et al., who reported that IFN-γ mRNA is strongly expressed in the cortex of C57Bl/6 mice infected with P. berghei ANKA [γ mRNA expression between sexes and performed even less extensive comparisons in GX mice. In our experiments, P. berghei ANKA infection in GX mice upregulated IFN-γ mRNA expression in the HT and HC, suggesting that gonadal steroids downregulate IFN-γ expression in these specific brain regions. In fact, 17β-oestradiol, produced primarily by the ovaries in females, is recognized as a neuroprotector in both acute and chronic neurodegenerative disorders, such as cerebral ischaemia, traumatic brain injury, and Alzheimer's disease [γ and TNF-α mRNA expression was upregulated in the cortex, HC, and HT regions in C57Bl/6 mice infected with P. berghei ANKA [γ receptor is widely expressed in cells of the central nervous system and provides many targets for this cytokine during CM, such as endothelial and astrocyte activation, parasitised red blood cell sequestration, blood brain barrier permeabilization, and CD8+ T cell accumulation [γ is a key immune mediator that controls the accumulation of P. berghei-infected red blood cells in brain tissues [α and IFN-γ [Proinflammatory cytokines, such as IFN-n humans  and roden humans . In thishei ANKA . However disease –39. Our hei ANKA . Our finmulation , 41. Int tissues . Infectend IFN-γ . These cnd IFN-γ ; therefoβ that we detected in the HC, FC, and LC of GX-infected female mice suggests that female gonadal steroids are involved in regulating inflammatory responses in these specific brain regions. Our results contrast with those of Miranda et al. [P. berghei ANKA-infected intact female mice. These differences are most likely due to two factors. First, Miranda et al. used the C57Bl/6 mouse strain, and we used CBA/Ca mice. Second, Miranda's group collected brain samples on day 5 postinfection, while we performed the experiments 9 days after infection; the mRNA expression of cytokines could be altered after 4 days. These brain areas are involved in cognitive and behavioural alterations during the acute phase of experimental CM [The downregulation of IL-1a et al.  who founental CM .γ and TNF-α mRNA expression in blood and quantified the concentration of both cytokines in the serum of intact, sham-, and GX-infected mice. Infected females exhibited higher levels of both cytokines than their male counterparts [γ and TNF-α in different brain areas because these cytokines are involved in both parasite elimination and development of cerebral malaria. In most of the brain areas assessed, the levels of IFN-γ and TNF-α were higher in intact infected males than in intact infected females. We also assessed the possibility that IL-10 downregulates the mRNA expression levels of both IFN-γ and TNF-α in POA, HT, HC, OB, FC, and LC; however, we were unable to detect IL-10 mRNA expression in the brain areas evaluated. These findings suggest that mechanisms that regulate the expression of both cytokines are different in blood and brain. It is possible that the increased IFN-γ and TNF-α mRNA expression and protein levels detected in blood and serum decreased parasitaemia on day 9 postinfection, and therefore, IFN-γ and TNF-α mRNA expression levels were downregulated in most of the brain areas assessed. These variations between sexes at least partially explain why infected males are more susceptible to developing cerebral malaria than infected female mice.In our previous work, we measured IFN-terparts . In the γ and TNF-α mRNA regulation in CM and the potential use of gonadal steroids or their derivatives in CM immunomodulation.All these findings support the crucial importance of IFN-P. berghei ANKA-infected female mice exhibited higher serum levels of nitric oxide than both intact and GX males, which may at least partially explain the lower severity of P. berghei ANKA infection in females. Our findings are consistent with those of Mwanga-Amumpaire et al. and Hawkes et al. who used nitric oxide inhalation as an adjunctive therapy for severe malaria because it decreases endothelial inflammation and reduces the adhesion of parasitised erythrocytes [On the other hand, nitric oxide is a vasodilator that inhibits platelet aggregation . In CM, hrocytes –50.Gonadal hormones, cytokines, endothelial cells and nitric oxide act together in both systemic immune responses and in the brain in a multifaceted manner, influencing neuroinflammation . In our


The results revealed that these semi-IPNs possessed improved mechanical and dielectric properties compared with pure polycyanurate. Notably, the impact strength of semi-IPNs was 47%–320% greater than that of polycyanurate. Meanwhile, semi-IPNs maintained comparable or even slightly higher thermal resistance in comparison with polycyanurate. The favorable processability and material properties make TPI/BADCy blends promising matrix resins for high-performance composites and adhesives.Thermoplastic polyimide (TPI) was synthesized via a traditional one-step method using 2,3,3′,4′-biphenyltetracarboxylic dianhydride , 4,4′-oxydianiline , and 2,2′-bis(trifluoromethyl)benzidine (TFMB) as the monomers. A series of semi-interpenetrating polymer networks (semi-IPNs) were produced by dissolving TPI in bisphenol A dicyanate (BADCy), followed by curing at elevated temperatures. The curing reactions of BADCy were accelerated by TPI in the blends, reflected by lower curing temperatures and shorter gelation time determined by differential scanning calorimetry (DSC) and rheological measurements. As evidenced by scanning electron microscopy (SEM) images, phase separation occurred and continuous TPI phases were formed in semi-IPNs with a TPI content of 15% and 20%. The properties of semi-IPNs were systematically investigated according to their glass transition temperatures ( Our gro of PETI . In addim-aminophenoxy) diphenylsulfone. It was observed that polyimides with low compatibility with CE resins were more efficient for improving the mechanical properties of the resultant thermosets. With the addition of 15% copolyimides, the fracture toughness was increased by 65% without significantly diminishing other thermal and mechanical properties [gT of less than 250 °C because of the presence of a flexible ether linkage, which could downgrade the gT and high-temperature modulus of the resultant thermosets [Blending with high-performance engineering plastics is an alternative methodology for toughening CE resins. Woo et al. investigated the influence of thermoplastic contents on the phase behavior and, consequently, the fracture toughness of modified thermosets derived from bisphenol E dicyanate and engineering plastics (polyethersulfone (PES) and polyetherimide (PEI)). Continuous thermoplastic phases were formed at high PES loading (>10%), and thus, the fracture toughness of the semi-IPNs were remarkably increased . Iijima operties . Polyimioperties . Liang eermosets .gT (360 °C) and excellent solubility (>20% in BADCy) was prepared using 2,3,3′,4′-biphenyltetracarboxylic dianhydride , 4,4′-oxydianiline , and 2,2′-bis(trifluoromethyl)benzidine (TFMB) as the monomers  simultaneously possessing a high monomers . The gooBADCy was provided by Yangzhou Techia Material Co., Ltd. . 3,4′-BPDA, 4,4′-ODA, and TFMB were purchased from Changzhou Sunlight Pharmaceutical Co., Ltd. . All other reagents were obtained from Tianjin Tiantai Fine Chemical Industry Co., Ltd. . All chemicals were used as received unless specified. m-cresol (250 mL) were placed into a 500-mL, three-necked flask equipped with an overhead stirrer, a condenser, and an argon inlet and outlet. The mixture was heated at 80 °C for 10 h and then at 180 °C for 12 h in argon atmosphere. Water, generated as the byproduct of imidization, was depleted from the flask by passing an argon flow. After cooling to room temperature, the polyimide solution was poured into methanol (2 L), collected by filtration, Soxhlet extracted with methanol for 24 h, and dried in vacuum at 150 °C for 48 h to afford an off-white fibrous solid . The number average molecular weight and polydispersity for this polyimide, determined by using gel permeation chromatography (GPC), were 22 kg mol−1 and 2.12, respectively.3,4′-BPDA , 4,4′-ODA , TFMB , and TPI/BADCy blends were formulated by dissolving appropriate amounts of TPI in melting BADCy at 100 °C. The weight percentages of TPI were 3%, 5%, 10%, 15%, and 20%. The blends were accordingly designated as Blend−3, Blend−5, Blend−10, Blend−15, and Blend−20. The homogenous solution was then transferred into a preheated stainless-steel mold and degassed at 100 °C in vacuum for 3 h. The blend was then cured at 140 °C for 1 h, 180 °C for 1 h, 200 °C for 1 h, and 220 °C for 2 h to afford the corresponding semi-IPN. The polymer was then removed from the mold at 150 °C. Semi-IPNs were named by the blend compositions . The specimens of pure TPI were prepared by compression-molding in vacuum. TPI powder was placed into a stainless mold and heated at 300 °C for 20 min on a hot press. Then, pressure (50 MPa) was applied, and the temperature was ramped up to 420 °C in 30 min and held at 420 °C for 40 min. The shapes were cooled to 250 °C under pressure, and the pressure was subsequently released. The samples were released from the mold and stored under ambient conditions prior to characterization. N,N-dimethylformamide (DMF) as the eluent and polystyrene as the standard. Differential scanning calorimetry (DSC) was recorded on a DSC Q2000  in nitrogen. Thermogravimetric analysis (TGA) was carried out on a TGA Q50  from 100 to 800 °C in air, with a heating rate of 10 °C min−1. Dynamic mechanical analysis (DMA) was performed on a Q800 DMA  from 50 to 400 °C with a heating rate of 3 °C min−1 and a frequency of 1.0 Hz. Rheological measurements were conducted on an AR2000ex rheometer . Tensile and flexural properties were determined on an Instron material testing system . Unnotched impact strength was measured by using a cantilever beam impact testing machine . Dielectric constant and loss factor were measured by using an Agilent impedance analyzer . SEM was conducted on a XL−30ESEM FEG microscope ) with an operating voltage of 25 kV. Semi-IPN specimens were fractured at ambient temperature, etched with dichloromethane for 48 h, and coated with a fine gold layer prior to SEM measurements.Fourier transform infrared spectroscopy (FT-IR) was carried out on a Bruker VERTEX 70 spectrometer . GPC was performed on a Waters 1500 GPC apparatus  with a refractive index detector, using gT and favorable mechanical properties. Nonetheless, the gT values of these polymers (200–250 °C) are significantly lower than that of BADCy-based polycyanurate (310 °C), inevitably downgrading the gT and high-temperature mechanical properties of the resultant semi-IPNs. In contrast, the majority of aromatic polyimides possess an extremely high gT (>300 °C); however, they are immiscible with CE monomers and oligomers, which imposes great difficulties in resin formulations and composite fabrications. Engineering plastics, such as PES, PEI, and PESC, are well-known toughening agents for thermosetting resins thanks to their relatively high m-cresol. Successful synthesis of the abovementioned TPI was evidenced by the characteristic absorption bands for asymmetric C=O stretching (1780 cm−1), symmetric C=O stretching (1720 cm−1), and C–N stretching (1370 cm−1) in FT-IR spectra , corresponding to the temperature of maximum cure rate, decreased from 324 to 273 °C with an inclusion of 20% TPI  for BADCy and TPI/BADCy blends spanned a range of 469–704 J·g−1, decreasing with the increase of TPI loading  or completely (high TPI contents) phase-separated morphologies. Another gT, corresponding to the primary segmental relaxation of TPI, should exist at around 360 °C. However, as the temperature exceeded the gT of polycyanurate, the storage modulus of semi-IPNs deteriorated significantly because of segmental relaxation. The dispersed TPI phase could not support itself, accounting for the absence of glass transitions in DMA curves. Generally, the incorporation of TPI did not decrease the gT and, consequently, service temperatures of the resultant thermosets and composites. The DMA results regarding semi-IPNs with low TPI contents (≤10%) seemed inconsistent with the fact that no obvious phase separation was detected in SEM measurements. This could be rationalized by the fact that the TPI domains in Semi-IPN−10 were too tiny to be observed by SEM.The temperature dependence of storage modulus and tan δ for TPI, polycyanurate, and semi-IPNs was determined through DMA, and the results are illustrated in  residue . Pure poT5%) of 422–429 °C . The mechanical and dielectric properties of the resultant thermosets were significantly enhanced by the inclusion of TPI without compromising their thermal resistance. Semi-IPNs displayed


Aspergillus flavus and Aspergillus parasiticus from section Flavi responsible for aflatoxin accumulation in stored cereals. Both aflatoxins and Aspergillus section Flavi were used to demonstrate the duplex real-time RCR method of simultaneously detecting small molecular contaminants and microorganisms. The detection of aflatoxins and Aspergillus section Flavi was carried out depending on the anti-idiotypic nanobody-phage V2–5 and aflatoxin-synthesis related gene nor-1 (=aflD), respectively. The quantitative standard curves for simultaneous detection of aflatoxins and Aspergillus section Flavi were constructed, with detection limits of 0.02 ng/ml and 8 × 102 spores/g, respectively. Naturally contaminated maize samples (n = 25) were analyzed for a further validation. The results were in good agreement between the new developed method and the referential methods .Simultaneous detection technology has become a hot topic in analytical chemistry; however, very few reports on how to simultaneously detect small molecular contaminants and microorganisms have been in place. Aflatoxins are a group of highly toxic and carcinogenic compounds, which are produced mainly by  Simultaneous detection technology has been becoming a hot topic in analytical chemistry. Many methods have been reported for simultaneous detection of multi small molecular contaminants such as mycotoxins , pestici1, B2, G1, and G2 are the most frequent ones in agricultural products and the most toxic member whereby aflatoxin B1 has been classified as group I human carcinogen by the International Agency for Research on Cancer. In addition, main aflatoxigenic species, namely, A. flavus and A. parasiticus that belong to Aspergillus section Flavi  Display Mediated Immuno-polymerase Chain Reaction (PD-IPCR), which helps to determine total aflatoxins, and (2) a conventional real-time PCR (RT-PCR), which serves for determination of the main aflatoxin-producing fungi Aspergillus section Flavi in stored maize. Through the combination of the two PCRs, a new detection platform was developed, which makes it possible to simultaneously detect small molecular contaminants and microorganisms.Currently, a number of quantitative techniques for aflatoxin determination have been developed, mainly including High-Performance Liquid Chromatography (HPLC), Liquid Chromatography tandem Mass Spectrometry (LC-MS), rapid immune-chromatographic assays (ICA) and enzyme-linked immune sorbent assay (ELISA). Methods for quantifying Escherichia coli ER2738 competent cells were purchased from Lucigen Corp. . The Universal Probes Supermix was supplied by Bio-Rad . DNA polymerase (iTaq), Mg2+, dNTPs, 6× loading buffer, and DNA marker were bought from Takara Bio . All the other reagents used were of analytical grade or better.The standard mycotoxin powders, the surfactants Tween-20, and the enzyme stabilizer bovine serum albumin (BSA) were obtained from Sigma-Aldrich . 2–5 phage displaying nanobody specific for 1C11 were produced by our team  and Vour team . A. flav2–5 phagemids, specific for mAb 1C11, previously transferred to E. coli ER2738 and stored at −70°C, need to be released and amplified from the E. coli, which was carried out as described in VNor-1 gene, catalyzing the transformation from norsolorinic to averantin, is the first key gene in the pathway of aflatoxins synthesis (A. flavus strain 3.4408 was used to obtain nor-1 gene. After the inoculation on Czap ekDox Agar (CDA), the fungus was incubated at 28°C and 90% humidity for 7 days, and then the spores were washed down, counted using a hemocytometer counting chamber, and diluted into 50 ml of potato dextrose broth (PDB) to a final concentration of 1 × 105 spores/ml, followed by a shaking at 180 rpm for 96 h at 28°C using a Thermo Scientific MaxQ 4000 shaker . Finally, the mycelia were washed three times with double-distilled water, filtered through double-filter papers , immediately freeze-dried and stored at −70°C prior to DNA extraction.ynthesis . A. flavnor-1 gene were generated with primers: nor1-F, 5′-ACCGCTACGCCGGCACTCTCGGCAC-3′ and nor1-R, 5′-GTTGGCCGCCAGCTTCGACACTCCG-3′. Then, these larger fragments were purified using E.Z.N.A. TM Gel Extraction Kit  according to the manufacturer’s protocols. Concentration of the purified products was determined by measuring the absorbance of samples at 260 and 280 nm, using a NanoDrop 1000 , and the number of copies was calculated.DNA was extracted using DNeasy Plant Mini Kit  according to the manufacturer’s introductions. After DNA extraction, a conventional PCR was performed essentially as described by 2–5) (nor-1 gene (The primer/probe systems are shown in 2–5) , while Tr-1 gene . The proTM real-time PCR system  was used to perform the real-time PCR assay. The duplex real-time PCR consisted of two single-plex amplification systems that separately used V2–5 phage DNA and nor-1 gene as templates. After the separate optimization, the two single-plex PCRs were combined to form a duplex real-time PCR, with an additional 0.25–1.0 U DNA polymerase (iTaq), Mg+2 (1–2 mM), and dNTPs (200–400 μM).A CFX962–5 phage (2 μl) and nor-1 (l μl) were mixed with the PCR working solution containing two primer/probe systems (2 (2 mM), and dNTPs (400 μM). Double-distilled water was added to make up the total volume to 10 μl. After an initial denaturation at 95°C for 5 min, 40 cycles were at 95°C for 10 s and 60°C for 30 s. No template control was used to verify the quality of amplification. All the assays were carried out in triplicate.Parameters for the optimized system were as follows: V systems , iTaq Un2–5 phage particles were diluted in PBS buffer  to a series of final concentrations ranging from 109 to 102 pfu/ml. The reference nor-1 gene was 10-fold serially diluted in nuclease-free H2O to final concentrations of 108–101 copies/μl. Ct values, corresponding to each dilution, were automatically calculated by the instrument. The efficiency was calculated based on: E = [101/–slope – 1] × 100%, by using logarithm of templates as abscissa and Ct values as ordinate to plot amplification calibration curves.To evaluate the amplification efficiency (E), V2–5 phages (1.0 × 1010 pfu/ml) and the same volume of aflatoxins solution were added into each micro-plate well. After the incubation at 37°C for 1 h, the plate was washed with PBST 10 times. Subsequently, the V2–5 phages captured by mAb 1C11 at the bottom of the plate were eluted by Glycine/HCl buffer  at 37°C for 15 min. Then, the eluent containing the released phages was neutralized using 1 M Tris–base buffer (pH 9.1).A polystyrene microtiter plate (96-well) was coated with 1.0 μg/ml mAb 1C11 at 37°C for 1 h. Then, the plate was washed with PBST [PBS containing 0.05% (v/v) Tween 20] three times and, then, blocked with a buffer [PBST containing 3% (w/v) skimmed milk] at 37°C for 45 min. The plate was washed three times. Then, the mixture (100 μl) containing 50 μl of V1, B2, G1, and G2 occur in natural samples at different ratios  used for total aflatoxin determination were prepared. The standard was threefold serially diluted at serial concentrations (33.3 ng/ml to 1.69 pg/ml). After the immunoreaction as described above, the eluted V2–5 phages solution (2 μl) was used for the duplex real-time PCR system. The standard curve was constructed by plotting Ct values versus Log total aflatoxin concentrations (Log 10) by four parameter logistic regression.Aflatoxins Bt ratios . Accordig for 10 min. After the recovery of the aqueous phase, 1 volume of chloroform:isoamyl alcohol (v:v = 24:1) was added, homogenized, and centrifuged again. The aqueous phase was recovered and 1 volume of chloroform was added. After the centrifugation at 15,000 g for 10 min, the aqueous phase was recovered again, followed by an addition of 0.6 volume of isopropyl alcohol (pre-cooled at −20°C for 2 h). After the centrifugation at 15,000 g for 15 min, the aqueous phase was discarded. The DNA was cleaned with 70% alcohol, suspended in 70 μl of nuclease-free H2O, and stored at −20°C.Ten grams of maize was finely milled into particles < 500 μm diameter using a laboratory mill. Subsequently, 0.2 g of the powder was precisely weighed, transferred into a nuclease-free tube, and smashed using an automatic fast-grinding apparatus Tissuelyser-48 , in the presence of 200 μl of CTAB (hexadecyltrimethyl ammonium bromide) buffer  and two steel beads (1.5 mm diameter). Then, an additional 1.6 ml of CTAB buffer was added into the tube and immediately incubated in a water bath at 63°C for 2 h. Then, 1 volume of phenol:chloroform:isoamyl alcohol  was added, gently mixed, and centrifuged at 15,000 Aspergillus section Flavi determination, 0.2 g of the finely milled blank-maize powder was precisely weighed, transferred into a nuclease-free tube, and inoculated with 200-μl spores (obtained from 6-day-old A. flavus strain 3.4408) to final concentrations of 8 × 102 to 8 × 108 spores/g. After incubation at 28°C for 1 h, samples were used to extract DNA as described above. DNA products (l μl) were used as templates for the duplex real-time PCR system. Log spores/g was used as abscissa and the corresponding Ct values were used as ordinate to plot the standard curve.To generate a standard curve for n = 25) were gathered from Shandong province of China. Samples (10 g) were finely milled into particles < 500 μm diameter, 0.2 g of which was used to extract DNA for Aspergillus section Flavi determination. For aflatoxin extraction, 5.0 g of the milled samples was treated with 15 ml of methanol:water  under a shaking condition at 250 rpm for 1 h. After a centrifugation (5000 g for 10 min at 4°C), samples were filtered with double-filter papers  and diluted sevenfold with 4% BSA/PBS (w/v). Dilutions were used directly in the Section “Immunoreaction for Small Molecular Contaminant Detection.” After the immunoreaction and DNA extraction, the eluted phages DNA and DNA products extracted from maize samples were amplified simultaneously in the optimized duplex real-time PCR system. Ct values were associated to standard curves to calculate concentrations of aflatoxins and aflatoxigenic fungi.The blank maize samples were purchased at a local market and verified as blank using HPLC and conventional plating counts. The naturally contaminated maize samples  were determined using dichloran rose bengal chloramphenicol agar (DRBC) supplemented with 1% NaCl  in parallel. HPLC was carried out as described in  1% NaCl .10, IC50 , and IC20 - IC80 were used to calculate the limit of detection (LOD), sensitivity and linear range, respectively. The statistical analysis and plotting were performed using Microsoft Excel 2007 and OriginPro 9.0 . To assess matrix effects, data were compared according to Student t-test using Graph PadInstat 3.0 .For aflatoxin determination, IC2–5 phages were much higher, which indicated that the amplification of phage DNA was extremely inhibited, whereas the additional Mg+2, dNTPs  buffer and the dilutions of maize extracts to a final concentration of 33.3 ng/ml to 1.69 pg/ml. Maximal and minimal Ct values were obtained at 33.3 ng/ml and 1.69 pg/ml, respectively. As shown in P < 0.001, according to the Student t-test) between 10% methanol/PBS buffer (ΔCt = 10.8) and maize extracts (ΔCt = 6.5), meaning maize matrix had a significant effect on total aflatoxin detection.To assess matrix effects on total aflatoxin determination, 5 g of blank maize samples were treated with 15 ml of methanol/PBS  under a shaking condition (250 rpm for 1 h), centrifuged at 5000 To eliminate matrix effects, the maize extracts were diluted sevenfold with 4% BSA/PBS (w/v), which essentially eliminated the matrix interference. Standard curves for total aflatoxins in 10% methanol/PBS buffer and maize extracts that were diluted with BSA/PBS (w/v) are shown in 50 Total aflatoxins /IC50 analyte) × 100. As shown in 1 (105%) and B2 (93%), lower cross-reactivity toward aflatoxins G1 (42%) and G2 (20%), and no cross-reactivity with zearalenone (ZEN), deoxynivalenol (DON), and fumonisin B1 (FB1) were obtained, indicating that the method was specific for aflatoxins B1, B2, G1, and G2.During assessment of specificity, the cross-reactivity (CR) for common mycotoxins was tested and calculated as: % CR =  and A. flavus  gave PCR amplifications similar to that of A. flavus strain 3.4408 (data not shown), indicating that the new method could detect A. parasiticus and A. flavus including aflatoxin producing and non-producing strains, whereas no PCR amplifications were detected for the other tested strains .The specificity of the primer/probe set of ed fungi . Their snor-1 gene as a biomarker for A. flavus and A. parasiticus detection has been demonstrated for several times. nor-1 gene copies was the same as that of A. flavus CFUs in wheat with the prolonged incubation time  between nor-1 copies and CFUs in naturally stored peanut. These results indicated that the PCR system based on nor-1 gene was specific and accurate for A. flavus and A. parasiticus determination, which was in accordance with our finding.Regarding ion time . Additio paprika . PassoneAspergillus section Flavi in stored peanut samples was established, with a detection linear range of 2.5 × 103 to 107cfu/g . Assays were carried out in triplicate on the same day for intra-assay precision evaluation and in five different days for inter-assay precision evaluation. Recoveries for aflatoxins and A. flavus spores were 84–111% and 94–107%, respectively, with variable coefficients (CVs) of 0.47–11.2%  were spiked with total aflatoxin standard  and simultaneously inoculated with fresh spores of 47–11.2% , indicaty = 0.97x – 4.31 and R2 = 0.99; for Aspergillus section Flavi, validation results were also in good agreement, with a linear regression equation: y = 1.06x + 0.38 and R2 = 0.98  in maize was developed as an example to demonstrate it. The entire process for the simultaneous detection requires less than 1 day, thus time saving compared with separate detections. Importantly, this technical platform not only achieved the goal of simultaneous quantifications but also satisfied technical features of high throughput, high sensitivity, and wider linear range. However, the tedious technical procedure might be considered as inefficiency at current stage, especially on the procedure of the DNA isolation. Therefore, simplifying protocols for samples preparation are necessary to be explored, further to save time and improve work efficiency. Overall, this detection platform had a great potential for simultaneous detection of small molecular contaminants and microorganisms, which could, in a significant measure, advance new ideas for the development of detection technologies.In order to provide an analytical technology to detect small molecular contaminants and microorganisms, the simultaneous detection of aflatoxins and its major fungi (The datasets generated for this study are available on request to the corresponding author.PL and QZ conceived the research and acquired the funding. QZ, PL, and XR designed the experiments and analyzed the data. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


MET-loaded PLGA were characterized by mean diameter, particle size, polydispensity index, and entrapment efficiency. Maxillae were scanned using Microcomputed Tomography (µCT) and histopathological and immunohistochemical analysis. IL-1β and TNF-α levels were analyzed by ELISA immunoassay. Quantitative RT-PCR was used . The mean diameter of MET-loaded PLGA nanoparticles was in a range of 457.1 ± 48.9 nm (p < 0.05) with a polydispersity index of 0.285 (p < 0.05), Z potential of 8.16 ± 1.1 mV (p < 0.01), and entrapment efficiency (EE) of 66.7 ± 3.73. Treatment with MET-loaded PLGA 10 mg/kg showed low inflammatory cells, weak staining by RANKL, cathepsin K, OPG, and osteocalcin, and levels of IL-1β and TNF-α (p < 0.05), increased AMPK expression gene (p < 0.05) and decreased NF-κB p65, HMGB1, and TAK-1 (p < 0.05). It is concluded that MET-loaded PLGA decreased inflammation and bone loss in periodontitis in diabetic rats.Evidence shows that metformin is an antidiabetic drug, which can exert favorable anti-inflammatory effects and decreased bone loss. The development of nanoparticles for metformin might be useful for increased therapeutic efficacy. The aim of this study was to evaluate the effect of metformin hydrochloride-loaded Poly ( Polymeric nanoparticles are particles with a diameter between 1 and 1000 nm ,3. Nanopco-glycolic acid (PLGA) has been widely used because it is a biocompatible and biodegradable synthetic polymer that has been approved by the United States Food and Drug Administration (USFDA) [Among the polymers studied for nanoparticle preparation, poly lactic- (USFDA) .Polymer composition is the most important factor to determine the hydrophilicity and degradation rate of a delivery matrix. The amount of glycolic acid is a critical parameter in tuning the hydrophilicity of the matrix and therefore the degradation and drug release rate .Biguanides are an important class of oral hypoglycemic agents and act by inhibiting gluconeogenesis in the liver, increasing the density of low and high affinity receptors for insulin, and decreasing resistance to the peripheral effects of insulin . Nanopard,l-Lactide-co-glycolide) (PLGA) in a ligature-induced periodontitis model in diabetic rats.The treatment in diabetes patients with metformin has shown that there is reduced TNF-α expression , with cop < 0.05), with a polydispersity index of 0.285 ± 0.12 (p < 0.05), Z potential of 8.16 ± 1.1 mV (p < 0.01), and entrapment efficiency (EE) of 66.7 ± 3.73 (p > 0.05). The mean particle size of MET-loaded PLGA nanoparticles was slightly larger than that of pure empty PLGA nanoparticles, indicating the presence of MET in the hydrophilic core of the nanoparticles [The well-defined spherical morphology and smooth surface of free-drug PLGA nanoparticles and MET-loaded PLGA nanoparticles can be directly observed in an atomic force microscope (AFM) image . Table 1articles .p < 0.001, Induction of diabetes occurred in the control groups (diabetes mellitus (DM), PLGA, and positive control) and also in all treated experimental groups 1 and 2, and diabetes was confirmed for values greater than 300 mL/dL of blood glucose. Glucose levels: Sham group (unbound group), periodontal disease (PD) (bound), DM (diabetic group without ligation), PLGA , Met 50 (group bound and treated with MET 50 mg/kg), Met 100 (bound and treated group with MET 100 mg/kg), PLGA + 100 mg/kg Met 100 mg/kg + PLGA) and PLGA + 10 mg/kg Met (group bound and treated with MET 10 mg/kg + PLGA). Only treatment with PLGA + 10 mg/kg Met significantly reduced systemic glucose levels in the animals  and positive control (529.9 + 76.78 mg/dL), p < 0.001), p < 0.05), Histopathological data for the Sham and DM control groups showed that infiltration of inflammatory cells was absent or scarce and was restricted to the marginal gingival region, and that the alveolar bone and cement were preserved with scores 0 (0–0) for both groups; the difference was significant when compared to the PD, PLGA control, and positive control groups . The quantification of TNF-α showed a significant increase in the PD when compared to the Sham group (p < 0.001). The PLGA + 10 mg/kg Met treatment significantly reduced the IL-1β and TNF-α levels when compared to the positive control group (p < 0.05), The quantification of inflammatory cytokines showed a significant reduction of IL-1β and TNF-α in the Sham group when compared to the positive control group (NF-κB p65 transcription factor showed a significant reduction for the PLGA + 10 mg/kg Met groups (p < 0.05) compared to the positive control, AMPK) showed a significant increase in this transcription factor for diabetic animals and periodontal disease treated with PLGA + 10 mg/kg Met when compared to the positive control and PLGA + 100 mg/kg Met (p < 0.05) groups, HMGB1 gene expression showed a significant reduction for the PLGA + 10 mg/kg Met groups (p < 0.05) when compared to the positive control, TAK1 gene expression showed a significant reduction for the PLGA + 10 mg/kg Met groups (p < 0.05) when compared to the positive control, Quantification of gene expression for the inflammatory p < 0.05. However, bone loss was reduced when comparing positive control (0.97 ± 0.35 mm) to PLGA 10 mg/kg Met (0.48 ± 0.14 mm) treatment. PLGA + 100 mg/kg Met showed bone loss (0.81 ± 0.28 mm) (Rats with PD + DM (positive control) (0.97 ± 0.35 mm) showed statistically significant more linear bone loss compared to Sham (0.45 ± 0.08 mm), 0.28 mm) .p < 0.001) and the PLGA + 10 mg/kg Met group (p < 0.05). The PLGA + 10 mg/kg Met treatment resulted in low staining of RANKL, OPG, osteocalcin, cathepsin, and osteocalcin  and poly l-lactic acid (PLLA). PLGA is generally an acronym for poly d,l-lactic-co-glycolic acid, where d- and l-lactic acid forms are in equal ratio [Polylactic acid contains an asymmetric α-carbon which is typically described as the D or L form in classical stereochemical terms. The enantiomeric forms of the polymer polylactide (PLA) are poly al ratio . PLGA caal ratio . These pal ratio . It has al ratio . Resultsal ratio . In this3 (positive control group) to 286. 5 dL/mL3 (MET 10 + PLGA group), demonstrating that the incorporation of drugs into nanoparticles may improve the drug efficacy [Our data showed that the association of metformin with PLGA was able to reduce glucose levels of 529.9 dL/mLefficacy . A low pefficacy . Once inefficacy . It is interesting to highlight that we used the same formulation in two different dilutions (PLGA + 10 mg/kg MET or PLGA + 100 mg/kg Met groups). Some drawbacks of the enhanced permeability and retention (EPR) effect of nanoparticles corroborate these phenomena. When a large amount of hydrophobic nanoparticles is administered in a short volume of medium (as occurs with a dose of PLGA + 100 mg/kg Met), their accumulation in a specific administration site is possible, reducing the surface area and, consequently, the drug diffusion rate . Their dPLGA is the most successful and most featured polymer for drug-controlled release systems. The effects of the combination of metformin and PLGA can be observed in both the inflammatory process and in the reduced bone loss in periodontal disease. The histopathological findings show a reduction of the inflammatory infiltrate, little destruction of the periodontal ligament, and absence of dental cement impairment in the group treated with metformin at the dose of 10 mg/kg + PLGA.This association showed excellent results in periodontal disease in diabetic animals; clinical and histopathological data corroborate the findings that elucidate the main cellular and molecular mechanisms involved in controlling inflammation and bone loss in periodontal disease.In general, drugs are small enough molecules to cross the endothelium in almost all regions of the body after systemic administration, and can reach both target regions and other healthy regions not affected by any disease, thus causing associated side effects of medication. The use of colloidal nanoparticle systems helps to control these adverse effects and improves therapeutic efficacy. These drugs are encapsulated within nanoparticles of 50–800 nm, which are not able to cross vessel walls of healthy regions of the body (the space between these cells is only 15–30 nm) . NanoforThe RANKL, RANK, and OPG system represents the key molecular regulation of bone remodeling . StudiesAMPK mRNA. Osteocalcin is specifically expressed in osteoblasts, secreted in circulation, and can regulate glucose homeostasis. Metformin stimulates the expression of osteocalcin and the differentiation of osteoblasts via AMPK activation [AMPK gene expression, which led to stimulated osteocalcin expression and, consequently, osteoblast deposition and bone formation. On the other hand, the increase of AMPK contributed to weak immunolabeling of RANKL and, consequently, of the osteoclast activity.AMP-activated protein kinase (AMPK) has emerged as a detection mechanism in regulating cellular energy homeostasis and is an essential mediator of the central and peripheral effects of many hormones in glucose metabolism . It is ativation . In our Lee et al. 2010  demonstrHMGB1 acts by stimulating the differentiation of osteoclast precursors in the presence of RANKL, and has similar proinflammatory properties to cytokines once it enters the extracellular space . Thus, iThis study showed that the PLGA + 10 mg/kg Met association had better results, as it managed to control blood glucose levels below what is considered diabetes, and so this nanotechnology product guaranteed rational release of the drug at the inflammation site, thereby controlling inflammation and bone loss in the experimental periodontal disease model.d,l-PLGA 50:50 (inherent viscosity of 0.63 dL·g−1 at 30 °C) was purchased from Birmingham Polymers Inc. , polyvinyl alcohol (PVA) was purchased from Sigma-Aldrich Co. , and dichloromethane (DCM) from QHEMIS® . Purified water (1.3 µS·cm−1) was prepared from reverse osmosis purification equipment, . All other reagents were of analytical grade.Metformin hydrochloride was purchased from Companhia da Fórmula, PLGA nanoparticles for metformin encapsulation were fabricated by adapting the double emulsion solvent diffusion method  with somv/v) and dropped in a cover slip, dried under a desiccator for 24 h and then analyzed in a Shimadzu SPM-9700 AFM  at room temperature with a cantilever in noncontact mode at 1 Hz scanning. Samples were prepared using one drop of dispersion, which was placed on a washed microscope slide and dried under a desiccator for 24 h, and then analyzed at 25 °C in a cantilever in noncontact mode.The measurements of mean diameter and particle size distribution were assessed by dynamic light scattering in a ZetaPlus device  equipped with a 90Plus/BI-MAS apparatus at a wavelength of 659 nm, with a scattering angle of 90°. Z potential of the particles was measured by laser Doppler anemometry using the same equipment. All analyses were performed at 25 °C. Experimental values were given as the mean ± SD for the experiments and carried out in triplicate for each sample. The shape and surface of drug-free and MET-loaded nanoparticles were observed using AFM images. The dispersions were freshly diluted with purified water at a ratio of 1:25  (PLGA) nanoparticles (NPs) were assessed by an indirect method, in which dispersions were centrifuged at 16,900 RCF (g) per 60 min at 4 °C using an ultra-centrifugal filter . The supernatant was removed and diluted in purified water 1:20 (v/v) and the measurements were carried out in a UV Thermo Fisher Scientific 60S Evolution Spectrophotometer , using previously validated UV spectrophotometry at 232 nm. Entrapment efficiency (EE) was calculated using the following equation: EE% = /total drug × 100.PLGA nanoparticles were used in this experiment to obtain an efficient drug loading corresponding to 6 mg/mL. Metformin hydrochloride-loaded Poly  housed under standard conditions , with ad libitum access to food and water. All animal protocols were approved by the Animal Ethics Committee of the Federal University of Rio Grande do Norte , Brazil. The anesthesia used to induce periodontal disease by intraperitoneal administration was Ketamine 10%  and 2% xylazine . The animals were euthanized with 80 mg/kg thiopental . Control Groups:Twenty animals without diabetes and without periodontal disease (Sham)Twenty animals with periodontal disease and without diabetes (PD)Twenty animals with diabetes and without periodontal disease (DM)Twenty animals with diabetes and with periodontal disease and treatment with vehicle water (Positive Control). This group, called the Positive Control group, was used to compare with all groups, because it has diabetes and periodontal disease (Positive Control group).Six animals with diabetes and with periodontal disease and treatment with vehicle: PLGA (PLGA Control); this group was used to confirm the same results of the Positive Control group by histopathological analysis. We decided to use the Positive Control group as a standard for all other analyses, avoiding excessive use of animals (following the principles of 3Rs).Experimental groups 1: Metformin hydrochlorideTwenty animals with diabetes and with periodontal disease and Metformin 50 mg/kg/day DOSE (Met 50)Twenty animals with diabetes and periodontal disease and Metformin 100 mg/kg/day DOSE (Met 100)Experimental groups 2: Metformin hydrochloride-loaded Poly  (PLGA) nanoparticles (NPs)d,l-Lactide-co-glycolide) (PLGA) nanoparticles (NPs) . The best anti-inflammatory dose of Experimental groups 1 was used and a lower 10× dose of metformin  was used.Using the best histopathological results from experimental groups 1, we defined metformin hydrochloride-loaded Poly .Twenty animals with diabetes and with periodontal disease and 10 mg/kg/day of Metformin-loaded PLGA (PLGA + 10 mg/kg Met).Diabetes was induced by administration of Streptozotocin/STZ (Sigma-Aldrisch)  through the penile vein, dissolved in sodium citrate buffer  at the concentration of 40 mg/kg body weight under general anesthesia, with 3% isoflurane inhalation. Glucose was measured by a glycosometer (one touch select simple) after one week of STZ administration. Upon reaching plasma glucose stability of ≥300 mg/dL, the animals were considered diabetic and selected for later periodontal disease studies. A puncture was made in the initial portion of the animal’s tail using a sterile needle and the blood was collected on a reagent strip for glucose determination.After diabetes confirmation, periodontal disease induction was performed by placing a 3.0 nylon wire on the second left molar of male Wistar rats with the animals under i.p. ketamine (80 mg/kg) and xylazine (10 mg/kg) anesthesia. Oral treatments were performed by gavage 30 min prior to periodontal disease induction, which continued until the 10th day. Euthanasia by thiopental (80 mg/kg) was performed on the 11th day. After sacrificing the animals, the gingival and maxillary tissue samples were sent for analysis. Histological analyzes were independently performed by two calibrated pathologists. All groups  were analyzed by histopathology. This step selected the best results that were used for the subsequent analyses. The sectioning was performed in the morphology laboratory of the UFRN, and the slides were analyzed by light microscopy in the department of Morphology. Five jaws were used per group. Alveolar bone specimens were collected, fixed in 10% buffered neutral formol, and demineralized in 5% nitric acid. The samples were then dehydrated, embedded in paraffin and sectioned along the molars in the mesiodistal plane for hematoxylin and eosin. Sections (4 µm) corresponded to the area between the first and second maxillary molars, where ligation was placed for analysis by light microscopy (40× magnification). Influx of inflammatory cells and alveolar bone integrity and cement were analyzed. A score of 0 indicated that infiltration of inflammatory cells was absent or scarce and was restricted to the marginal gingival region, and that the alveolar process and cement were preserved; a score of 1 indicated moderate cell infiltration throughout the gingival insert, minor alveolar resorption, and intact cement; a score of 2 indicated marked cellular infiltration in the gingiva and periodontal ligament, marked degradation of the alveolar process, and partial destruction of the cement; and 3 indicated marked cellular infiltration, complete reabsorption of the alveolar process, and severe destruction of the cement.n = 5) of the control  and experimental groups were stored at −70 °C. IL-1β levels  and TNF-α  were determined using commercial ELISA kits , as previously described [Gingival tissues (escribed . All samO-phenylenediamine reagent coloring (50 mL) was added 15 min later, and the plates were incubated in the dark at 37 °C for 15–20 min. The absolute quantitative of IL-1β and TNF-α to control (PD and Positive control) and experimental groups was calculated using the comparison with the control group (Sham). The enzymatic reaction was reduced with H2SO4 and the absorbance measured at 490 nm. Values were expressed in pg/mL.First, microtiter plates were coated overnight at 4 °C with rat antibodies against IL-1β and TNF-α. Plates were then blocked, samples and standards added in several dilutions, in duplicate and incubated at 4 °C for 24 h. The plates were washed three times with buffer and the antibodies were added to the wells . Plates were incubated at room temperature for 1 h, washed, and 50 µL of avidin-HRP (1:200) µL were added. Then, The control (sham and positive control) and experimental groups (PLGA + 100 mg/kg Met and PLGA + 10 mg/kg Met) were included in the quantification of expression by RT-PCR. Total RNA from the gingival tissues of the treated groups was extracted using the Trizol reagent  according to the manufacturer’s guidelines and stored at −70 °C.260 unit equivalent to 40 µg/mL RNA). Five micrograms of isolated total RNA were reverse transcribed to cDNA in a reaction mixture containing 4 µL 5× reaction buffer, 2 µL dNTP mixture (10 mM), 20 units of RNase inhibitor, 200 units of avian-myeloblastosis virus (AMV) reverse transcriptase, and 0.5 µg oligo (dT) primer  in a total volume of 20 µL. The reaction mixture was incubated at 42 °C for 60 min, and the reaction was terminated by heating at 70 °C for 10 min. The cDNA was stored at −80 °C until further use. Gene expression was evaluated by PCR amplification using primer pairs based on published rat sequences , mRNA: Forward primer: AGCTCGCAGTGGCTTATCAT, Reverse Primer: GGGGCTGTCTGCTATGAGAG; NF-κB p65-Rattus norvegicus v-rel avian reticuloendotheliosis viral oncogene homolog A (Rela), mRNA Forward primer: 5′-TCTGCTTCCAGGTGACAGTG-3′, Reverse Primer: 5′-ATCTTGAGCTCGGCAGTGTT-3′; HMGB1-Rattus norvegicus high mobility group box 1 (HMGB1), mRNA: Forward primer: 5′-GAGTACCGCCCAAAAATCAA-3′, Reverse Primer: 5′-TTCATCCTCCTCGTCGTCTT-3′; TAK-1 Forward primer: 5′-GTCATCCAGCCCTAGTGTCAGATT-3′, Reverse Primer: 5′-TTCTTTGGAGTTTGGGCACG-3′. Transforming Growth Factor β-activated Kinase 1 TAK-1-Mus musculus, mRNA: Forward primer: 5′-GTCATCCAGCCCTAGTGTCAGAAT-3′, Reverse Primer: 5′-TTCTTTGGAGTTTGGGCACG-3′.RNA concentration was determined from the optical density at a wavelength of 260 nm , and a Step One Plus thermocycler (Applied Biosystems), according to the manufacturer’s instructions. For the 1× PCR master mix, 2.5 µL of each cDNA was added in a final volume of 20 µL. The PCR conditions were as follows: 95 °C for 5 min, 40 cycles of 30 s at 95 °C, 30 s at 52–60 °C (based on the target), and 60 s at 72 °C. The relative quantitative fold change compared with the control (Sham) was calculated using the comparative ® software  for linear bone height analysis. Linear bone height analysis was performed by positioning the second molar cementoenamel junctions (CEJ) parallel to each other in the coronal plane. In the axial plane, the middle of the crown was identified and linear bone distances were recorded on the mesial aspect of the second molar on the sagittal image. Additional measurements were taken 0.3 mm palatal from the middle of the crown, again recording the mesial aspect of the second molar on the sagittal image. The linear measurements were recorded from the CEJ to the alveolar crest (AC). Each second molar received a total of two measurements, and these values were averaged for each group. The images were analyzed using CTAn . A 40-slice volume set at a threshold of 75 in the bifurcation area of the second molar was used as a region of interest for analysis .In this stage, the control groups (Sham and positive control) and experimental groups (PLGA + 100 mg/kg Met and PLGA + 10 mg/kg met) were included. Animals were euthanized at the end of the experiment (10 days after addition of the ligature and first drug treatments); maxillae were dissected and fixed in 10% buffered formalin for 24 h and stored in 70% alcohol. Rat maxillae were scanned using microcomputed tomography   at 20 micrometers resolution. Micro-CT files were converted to Digital Imaging and Communications in Medicine (DICOM) files and imported into DolphinOnly the controls (Sham and positive control) and the experimental group (PLGA + 10 mg/kg met) were included in this step. Fine sections of periodontal tissue (4 µm) (3 mandibles per group) were produced using a microtome and transferred onto gelatin coated slides. Each section was deparaffinized and rehydrated. Gingival and periodontal tissues were washed with 0.3% Triton X-100 in phosphate buffer, then extinguished with peroxidase (3% hydrogen peroxide) and incubated with the following primary antibodies  overnight at 4 °C: RANKL, 1400; OPG, 1400; cathepsin K, 1400; and osteocalcin, 1400, which were washed with phosphate buffer and incubated with streptavidin-HRP-conjugated secondary antibodies  for 30 min, and immunoreactivity for RANK, RANK-L, OPG, cathepsin K, and osteocalcin were visualized using a colorimetric detection kit following the manufacturer’s instructions .t-test. One-way analysis of variance (ANOVA) was applied for multiple comparisons, followed by Tukey’s post hoc test. p < 0.05 was considered statistically significant. Data for the in vivo experiments were analyzed using descriptive and analytical statistics. Parametric tests, such as ANOVA, followed by Bonferroni’s post-test and nonparametric Kruskal–Wallis test were used. A significance level of 5% was considered.Using nanoparticle characterization, pairwise comparisons of the analytical data were performed using the Student’s


Desmondus rotundus) is unstudied in regards to potential vasodilatory mechanisms of their feeding secretions (which are a type of venom). This is despite the intense investigations of their anticoagulant properties which have demonstrated that D. rotundus venom contains strong anticoagulant and proteolytic activities which delay the formation of blood clots and interfere with the blood coagulation cascade. In this study, we identified and tested a compound from D. rotundus venom that is similar in size and amino acid sequence to human calcitonin gene-related peptide (CGRP) which has potent vasodilatory properties. We found that the vampire bat-derived form of CGRP  selectively caused endothelium-independent relaxation of pre-contracted rat small mesenteric arteries. The vasorelaxant efficacy and potency of vCGRP were similar to that of CGRP, in activating CGRP receptors and Kv channels to relax arteriole smooth muscle, which would facilitate blood meal feeding by promoting continual blood flow. Our results provide, for the first time, a detailed investigation into the identification and function of a vasodilatory peptide found in D. rotundus venom, which provides a basis in understanding the convergent pathways and selectivity of hematophagous venoms. These unique peptides also show excellent drug design and development potential, thus highlighting the social and economic value of venomous animals.Animals that specialise in blood feeding have particular challenges in obtaining their meal, whereby they impair blood hemostasis by promoting anticoagulation and vasodilation in order to facilitate feeding. These convergent selection pressures have been studied in a number of lineages, ranging from fleas to leeches. However, the vampire bat ( Desmondus rotundus) are found in Central and South America, and feed exclusively on mammalian blood  at 37 °C and were bubbled with carbogen . In a subset of arteries, the endothelium was gently denuded via insertion of a 40 µm wire inside the lumen and rubbing the vessel walls. The mesenteric arteries were allowed to equilibrate for 30 min under zero force and then a 5 mN resting tension was applied. Changes in isometric tension were recorded using Myograph Interface Model 610 M version 2.2  and PowerLab/835 . Data was recorded with the data acquisition program Chart . Following a 30 min equilibration period at 5 mN, the mesenteric arteries were contracted maximally (Fmax) using a K+ depolarizing solution . The integrity of the endothelium was confirmed by relaxation to acetylcholine  [2 mimetic, U46619 (1 µM) [Male Sprague-Dawley rats (200–250 g) were euthanized via COyographs . Vessels, 10 µM)  in tissuD. rotundus vCGRP (10−12–3 × 10−8 M) or rCGRP (3 × 10−12–10−8 M) [D. rotundus vCGRP and rCGRP were obtained in endothelium-intact mesenteric arteries in the absence or presence of either ODQ (10 µM) [+ [D. rotundus vCGRP was also examined. Sodium nitroprusside  [D. rotundus vCGRP or rCGRP was obtained in each vessel segment [Cumulative concentration-response curves to –10−8 M) ,34,37 we (10 µM) , SQ22536 (10 µM) , L-NAME  (10 µM) , CGRP8-3 (10 µM) ,50, 30mM0 µM) [+ , TEA (100 µM) [+  or glibe0 µM) [+ ,51. All ; 10 µM)  was adde segment ,52.50 values (concentration of agonist resulting in a 50% relaxation) calculated and expressed as –Log mol.L−1. Statistical comparisons between the experimental groups’ mean pEC50 and maximum relaxation (Rmax) values were made using a Student’s unpaired t-test or one-way ANOVA with Bonferroni’s post hoc comparison. Where pEC50 values could not be obtained, concentration-response curves were compared by means of a two-way ANOVA. n = number of artery segments from separate animals. Data represent the mean ± SEM (error bars on graph). Statistical significance was defined as * p < 0.05. All data analysis was performed using GraphPad Prism version 5.02  [Relaxation responses were expressed as a percentage reversal of the U46619 pre-contraction. Individual relaxation curves were fitted to a sigmoidal logistic equation and pECReagents and their sources were U46619 , SQ22356 , ODQ, Glibenclamide, TEA, 4-aminopyridine, L-NAME, SNP, ACh, CGRP8-37 , and CGRP (rat) Peptide Institute, Osaka, Japan. Stock solutions of ODQ (10 mmol/L) and U46619 (1 mM) were dissolved in absolute ethanol. All subsequent dilutions of stock solutions were in distilled water. All other drugs were made up in distilled water and all dilutions were prepared fresh daily.


Coccidiosis represents a serious threat to the poultry industry, affecting production and causing high morbidity, mortality and significant costs resulting from treatment and prophylaxis. In-feed anticoccidials have been used for decades for managing avian coccidiosis and were very effective until drug resistance emerged. The use of natural remedies has become a promising alternative in combating coccidiosis in chickens. Therefore, the purpose of the present study was to assess the efficiency of a commercial herbal formula (H), as oral liquid preparations, in experimental chicken coccidiosis.Allium sativum and Thymus serpyllum; H2 contained Origanum vulgare, Satureja hortensis and Chelidonium majus; and H3 contained Allium sativum, Urtica dioica, Inula helenium, Glycyrrhiza glabra, Rosmarinus officinalis, Chelidonium majus, Thymus serpyllum, Tanacetum vulgare and Coriandrum sativum. Chickens were divided into five groups for each BE as follows: (i) uninfected untreated control ; (ii) infected untreated control ; (iii) infected treated with amprolium ; and  two experimental groups infected treated with H1 (ITH1) and H2 (ITH2) formulas in the BE1 and with H3 (ITH3-5 and ITH3-10) formula in the BE2. The chickens from infected groups were challenged with 5000 (BE1) and 50,000 (BE2) sporulated oocysts of Eimeria spp. , respectively. The anticoccidial efficacy was assessed by recording the following: oocysts output (OPG), lesion score (LS), weight gain (WG), feed conversion ratio (FCR) and anticoccidial index (ACI). Additionally, polyphenolics and flavonoids  from herb extracts found in H3 formula were determined by the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method.Two independent controlled battery experiments (BE1 and BE2) were designed and the product was tested in 3 different formulas : H1 contained a propylene glycol extract of Eimeria spp., reduced the total lesion score and improved the zootechnical performance (weight gain and feed conversion ratio). According to ACI value, H1 and H2 had no efficacy on Eimeria spp. infection, but H3 had good to marked anticoccidial effect, the ACI being slightly greater in the group ITH3-5. According to the results of LC-MS/MS, the concentration of polyphenols in H3 formula was the highest, the sum of chlorogenic acid and caffeic acid being 914.9 µg/ml.H1 and H2 reduced the WG, and increased the FCR and OPG compared with controls. H1 reduced the duodenal lesions, whilst H2 reduced the caecal lesions, compared with control. H3 decreased the OPG of H3 formula is a promising natural anticoccidial and field trials are recommended in order to validate the obtained data.The online version of this article (10.1186/s13071-019-3595-4) contains supplementary material, which is available to authorized users. Eimeria with different localizations within the intestinal tract of chickens. Eimeria acervulina, E. maxima and E. tenella are the most prevalent species in broilers in the intensive poultry management system [Eimeria strains have gained resistance to all known coccidiostats, and new anticoccidials are unlikely to be developed, mainly because of strict legislative regulations on the use of in-feed drugs and growing concerns in the general population about the chemical residues in poultry products [Artemisia annua and artemisinin [Aloe [Coccidiosis is a parasitic disease caused by seven species of the genus t system , 2. The t system . In-feedproducts –6. Over products . There iproducts . In thisproducts . Numerouemisinin , 11, oreemisinin , garlic emisinin , neem [1emisinin , differein [Aloe , green tin [Aloe , sugar cin [Aloe , turmeriin [Aloe  and manyin [Aloe , 19–21. in [Aloe , 23, or in [Aloe . Furtherin [Aloe , 26. Flain [Aloe .Therefore, the purpose of the present study was to assess the effect of a commercial multi-plant extract compound, in experimental coccidiosis in broiler chickens. The composition of the herbal extract was designed on the basis of a literature search for effective anticoccidial natural compounds.Two independent controlled battery experiments (BE) were designed in order to assess the efficacy of a commercial herbal product (H), in three different formulas of propylene glycol/alcoholic herbal extracts. In the first BE (BE1), the anticoccidial effect of the first (H1) and second (H2) formulas was evaluated. The third formula (H3) was evaluated during the second BE (BE2).n = 15). In both BE the experimental groups were represented by: (i) negative control, uninfected and untreated ; (ii) positive control, infected and untreated ; (iii) treatment control, infected and treated with amprolium ; and  two experimental groups, infected and treated with H1 (ITH1) and H2 (ITH2) formulas in the BE1and with H3 (ITH3-5 and ITH3-10) formula in the BE2.One hundred one-day-old ROSS 308 hybrid broiler chickens were purchased from S.C. VIS AVIS S.A.  for each of the two BE. They were housed in batteries in dedicated facilities at the University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca. At 14-days-old, broiler chickens were randomly divided in five groups, each with three replicates of five chickens/cage  containing E. acervulina, E. tenella and E. maxima. The strains were isolated in 2012 from a broiler farm and the species were identified by PCR [On the same day, the broiler chickens were experimentally infected by the crop-route using insulin syringe, with 1 ml of a mixed suspension of fresh sporulated oocysts  as doses of 2.5 (BE1) and 5 g/l water (BE2), respectively. H1 and H2 herbal formulas were given as doses of 10 ml/l drinking water (BE1) and H3 herbal formula as doses of 5 (ITH3-5) and 10 (ITH3-10) ml/l drinking water (BE2).The control and experimental treatments were given The chickens were fed with standard starter (1–13 days-old) and grower (14–24 days-old) feed free of anticoccidials.The commercial herbal formula was supplied by S.C. PROMEDIVET S.R.L  as oral liquid preparations. The anticoccidial efficacy in chicken coccidiosis was evaluated for three different extracts named H1, H2 and H3. The extracts were obtained from grounded dried plants after maceration in propylene glycol (20 kg of dried plants in 120 l of propylene glycol) for 14 days and cold pressing.Allium sativum (garlic) and leaves of Thymus serpyllum (wild thyme) in equal proportions. H2 was a mixture of extracts from leaves of Origanum vulgare (oregano) 40%, Satureja hortensis (summer savory) 30% and Chelidonium majus (greater celandine) 30%. H3 contained extracts from nine herbs: roots of Urtica dioica (nettle) 10%, Inula helenium (elecampane) 15%, Glycyrrhiza glabra (licorice) 10%, bulbs of Allium sativum 10%, leaves of Rosmarinus officinalis (rosemary) 10%, Chelidonium majus 10%, Thymus serpyllum 15%, flowers of Tanacetum vulgare (tansy) 10% and seeds of Coriandrum sativum (coriander) 10%.H1 contained extracts from bulbs of rpm for 10 min and filtered using nylon micro pore 0.45 µm filters. Solutions were transferred to HPLC vials and 5 μl of the obtained samples were injected into the LC-MS system.The H3 formula was analyzed by liquid chromatography tandem mass spectrometry (LC-MS). One hundred microliters of H3 formula was mixed with 900 µl of purified water, then centrifuged at 10,000× Chromatographic separation of analytes was performed using a NUCLEODUR C18 Gravity, 3 µm, 150 × 3 mm  with a mobile phase consisting of 0.2% formic acid in water and methanol in gradient elution, with a flow rate of 0.6 ml/min. Detection was carried out in multiple reactions monitoring mode (SRM). Ionization of analytes was performed using negative electrospray ionization mode. Ionization parameters used for the ionization source were as follows: spray voltage, 2500 V; vaporizer temperature, 350 °C; ion gas source 1, 25; ion gas source 2, 25; curtain gas, 10; declustering potential, 100; ion release delay, 30; ion release width, 15. Sample run-time was 30 min per sample.Standard solutions of each analyte  (100 µg/ml) were prepared in methanol. For analysis, standard solutions were mixed in methanol to a final concentration of 10 µg/ml for each analyte. Furthermore, the mixture was diluted to the following concentrations: 0.1 , 0.25, 0.5, 1 and 10 µg/ml. The calibration of standard curve solutions was prepared by diluting 100 µl of each standard solution with 900 µl of purified water.The efficacy of the herbal formulas in experimental coccidiosis in broiler chickens was assessed by recording and calculating the number of oocysts shedded per gram of feces (OPG), lesion score (LS), mortality rate (MR), body weight gain (BWG), feed conversion ratio (FCR) and anticoccidial index (ACI).The feces were collected on days 5, 7 and 10, from all 3 cages/group individually and the OPG was determined by duplicate counts of duplicate fecal slurries from each cage by using the McMaster method . The detThe ACI was calculated after the formula: ACI = (%S + %RGW) − (LI + OI) , where %https://www.medcalc.org). The arithmetic mean and standard error were calculated for each assessed parameter and for each group. ANOVA  was used for OPG and body weight gain, and the Kruskal-Wallis test was used for lesion score. Differences were considered statistically significant if the P ≤ 0.05. The experimental groups were compared with both positive and negative control groups and also with group A.The data were processed with MedCalc Software v.18  = 28.13, P < 0.001)  between groups (Table The OPG of groups treated with H1 and H2 formulas was significantly higher compared with the positive control (IU1) or ITA1 groups on day 7 post-infection (p.i.)   = 7.8, P = 0.007)  Table . The low7) Table . The FCR7) Table .Eimeria spp. infection.According to the anticoccidial index, the H1 and H2 formulas had no efficacy on F = 19.7, P < 0.001) at 5 days p.i. On days 7 and 10 p.i., the OPG of chickens treated with H3 decreased under the values of the positive group, but significantly only on day 10 p.i.  = 43.44, P < 0.001)  = 6.63, P = 0.003).The OPG value was significantly higher in groups treated with H3 formula than in the group treated with amprolium but not compared with the positive control group . Additionally, the chickens treated with 5 ml H3/l drinking water had fewer lesions than the positive control group, but with no statistical significance . However, the H3 formula at a dose of 10 ml/l water reduced the total lesion score by 50%.No mortality was registered in any experimental group. The chickens from all groups, with the exception of the ITA2 group, presented lesions in the duodenum, but all chickens had lesions in the caecum. Neither 5 nor 10 ml/l water of H3 formula significantly reduced the duodenal lesions compared with the positive control group. H3 formula at a dose of 10 ml/l water significantly reduced the caecal lesions  = 1.78, P = 0.182). However, compared with the positive control group, the chickens treated with H3 formula had higher weight gains and only slightly lower than the amprolium treated chickens.All the experimental groups presented lower weight gains than the negative control group . The synergistic effects of the combined herbs enhanced the anticoccidial activity of broiler chickens medicated with H3. Some of the herbs from the mixed extract already have proven anticoccidial activity such as Allium sativum powder, supplemented in broiler feed at 0.1% [Thymus serpyllum in ducks at a concentration of 2,500 mg/kg [Urtica dioica, Rosmarinus officinalis, Tanacetum vulgare, Coriandrum sativum or Glycyrrhiza glabra are known to have immunomodulatory effects [Eimeria infection, as stated above. The antioxidants can alleviate the damage to the intestinal tissue during parasite invasion by reducing the cytotoxic effects caused by the reactive oxygen species [As shown in the present study, the multi-herb product H3 highly reduced the coccidian multiplication rate and reduced the severity of intestinal lesions. The product had a lower anticoccidial effect on  at 0.1%  and Thym00 mg/kg . These t effects , 41–44.  effects . The ant effects . As show effects . Further effects , which n species , and thuH3 formula was effective in controlling experimental coccidiosis in chickens and can be used successfully as a natural anticoccidial. Field trials are, however, recommended in order to validate the data obtained in experimental studies.Additional file 1: Figure S1. Total ion chromatogram of the H3 formula.Additional file 2: Figure S2. Extracted ion chromatogram of the H3 formula.


Proteobacteria was the dominant phylum followed by Bacteroidetes and Firmicutes; whereas Clostridium was the dominant genus followed by Treponema, Propionibacterium, Syntrophus, and Desulfobulbus. The sludge samples could be clustered into six groups (C1 to C6) according their microbial structure similarities. Very high pH (≥11.9) was the main sludge attribute segregating C6, that presented very distinct microbial structure from the others. Its most dominant genera were Propionibacterium > > Comamonas > Brevundimonas > Methylobacterium ∼Stenotrophomonas ∼Cloacibacterium. The other clusters’ dominant genera were Clostridium > > Treponema > Desulfobulbus ∼Syntrophus. Moreover, high Fe and S were important modulators of microbial structure in certain sludges undertaking anaerobic treatment and having relatively low N-Kj, B, and P contents (C5). However, high N-Kj, B, P, and low Fe and Al contents were typical of domestic, unlimed, and aerobically treated sludges (C1). In general, heavy metals had little impact on microbial community structure of the sludges. However, our sludges shared a common core of 77 bacteria, mostly Clostridium, Treponema, Syntrophus, and Comamonas. They should dictate microbial functioning within WWTPs, except by SS12 and SS13.Sewage sludges generation and their disposal have become one of the greatest challenges of the 21st century. They have great microbial diversity that may impact wastewater treatment plant (WWTP) efficiency and soil quality whether used as fertilizers. Therefore, this research aimed to characterize microbial community diversity and structure of 19 sewage sludges from São Paulo, Brazil, as well as to draw their relations to sludge sources [domestic and mixed ], biological treatments (redox conditions and liming), and chemical attributes, using molecular biology as a tool. All sludges revealed high bacterial diversity, but their sources and redox operating conditions as well as liming did not consistently affect bacterial community structures.  Urban centers fast growth and industrial activities intensification generate high volumes of effluents daily , which aProteobacteria phylum (21–65%) was predominant in municipal WWTPs (domestic sewage), mostly belonging to Betaproteobacteria that represents a class of microorganisms related to organic matter degradation and nutrient cycling. Other less dominant taxa were Bacteroidetes, Acidobacteria, and Chloroflexi ], biological treatment (redox) conditions, liming, urbanization, and industrial activity; as well as to sludge chemical attributes. It would supply useful information about hygiene measures needed and/or potential contamination resulting from sludge application as soil amendment.Table 1). Sample collection was performed at the sludges dewatering points, as described by EPA SW-865.Sewage sludge samples were collected from 19 WWTPs of São Paulo State, Brazil. Five samples  were collected from metropolitan area of São Paulo City, the most urbanized and industrialized region within São Paulo State, whereas the others were collected from other municipalities  taken in 10-min intervals, mixed, and properly homogenized. They were conditioned in glass bottles and refrigerated until analysis according CONAMA Resolution 375/2006 .-1 KCl, 0.2 g of MgO, and 0.2 g of Devarda alloy, which were taken in 5 mL of 20 g L-1 H3BO3 and titrated with 0.0025 mol L-1 H2SO4  for 3 h, distilled with 20 mL of 10 mol L-1 NaOH, which were also taken in 20 mL of 20 g L-1 H3BO3 and then titrated with 0.0025 mol L-1H2SO4 , according to manufacturer’s instructions. Integrity of the extracted DNA was checked by electrophoresis (1% agarose gel), which was stained with ethidium bromide and visualized under ultraviolet light.4-Forward and 4-Reverse primers with pre-adapters (Supplementary Table 2 (50 mM), 2.0 μL of DNTPs (2.5 mM), 0.1 μL of each primer mix, 0.3 μL Taq DNA polymerase (0.05 U/μL), 16 μL mili-Q water and 1.0 μL template DNA were utilized. Amplification conditions involved initial denaturation at 95°C for 3 min, 30 cycles at 95°C for 45 s, 57°C for 1 min: 45 s; 72°C for 1 min; followed by a final extension at 72°C for 4 min , targeting the V4 region of the 16S rRNA gene. This was amplified using a mixture of or 4 min . PCR pro2 (50 mM), 2.0 μL of DNTPs (2.5 mM), 5 μL of each adapter (index), 0.3 μL of Taq DNA polymerase (0.05 U/μL), 17.2 μL of mili-Q water, and 15 μL of previous reaction product . Amplification conditions consisted of 95°C for 3 min, five cycles at 95°C for 45 s, 57°C for 1 min: 45 s; 72°C for 1 min; followed by a final extension at 72°C for 4 min. Sequencing was carried out at the University of São Paulo (USP/ESALQ), by the Animal Biotechnology Laboratory within the Animal Science Department.After DNA purification, another PCR reaction was performed to bind adapters (an index pair) to identify sequence origin. This consisted of 3.0 μL of PCR buffer, 2.5 μL of MgCl® software (v.3.2)  program was used for DNA sequencing analysis . Sequenc (v.3.2) . ANOSIM 4+, N-NO2-/NO3-, organic N (N-Kjeldahl = N-Kj), organic carbon (OC), K, Ca, Fe, P, S, Mg, Na, Cd, Cr, Cu, Hg, Mn, Mo, Ni, Pb, Se, Zn, Al, As, and Ba) were settled by redundancy analysis (RDA) on Canoco® software (v.4.5). Graphics were plotted on Origin® software (v.10.5), but heatmap graphical scales were built in R software, using “gplots” and “RColorBrewer” packages1.Relationship between bacterial community composition and sludges sources, treatments, and chemical attributes , OTUs matrixes showed that all sludges presented high diversity indexes ; whereas the most abundant classes were Saprospirae > Betaproteobacteria > Bacteroidia > Clostridia > Deltaproteobacteria . In addition, Betaproteobacteria was the most abundant class within the Proteobacteria phylum (∼37% of the sequences), followed by Deltaproteobacteria (∼26%), Alphaproteobacteria (∼16%), and Gammaproteobacteria (∼11%); whereas Saprospirae was the most abundant class within the Bacteroidetes (∼46%), followed by Bacteroidia (∼36) and Flavobacteria (∼3%) . Within the Firmicutes, the most abundant classes were Clostridia (∼87%) and Bacilli (∼9%) . Finally, the most abundant genera were Clostridium > Treponema > Propionibacterium > Syntrophus > Desulfobulbus > Brevundimonas > Paludibacter > Cloaci-bacterium > Methylobacterium . Despite distinctions in sewage sources and treatments, their bacterial community presented a common core of 77 genera, being Clostridium, Treponema, Syntrophus, and Comamonas the most abundant ones . This result was also validated by similarity analysis of their bacterial communities (Table 2).The sludge samples could be grouped in six clusters according to PCoA: C1 , C2 (SS9 and SS16), C3 (SS11 and SS18), C4 , C5 , C6 (SS12 and SS13). Its main two coordinates explained 36.7% of sludges’ bacterial community structures . The other clusters (C1-C5) generally presented great abundance of Clostridium, Treponema, Syntrophus, and Desulfobulbus, except that C1 showed low abundance of Syntrophus and high abundance of Dechloromonas; C2 showed relative high abundance of Sedimentibacter; C3 showed relative high abundance of Paludibacter; C4 showed relative high abundance of Sedimentibacter and also of Dok59 and Bacillus; and C5 showed relative high abundance of Paludibacter, PD-UASB-13, Desulfovibrio, and E6 .C6 showed bacterial community very distinct from the others, with relative dominance of Table 3), suggesting that clusters were formed due to other factors, likely related with sludges chemical attributes as suggested by RDA . In fact, pH , Fe ; B and Mg ; Na ; and P, Ba, organic N (N-Kj), and Ca  contents were the sludge attributes most related to microbial community structuring and clustering; whereas organic carbon (OC), inorganic N (in the different forms), Hg, Se, and As contents, C/N ratio, and moisture were not correlated with sludges bacterial community structures  (Supplementary Table Sewage sources (domestic or mixed) and biological treatments (redox conditions) did not affect consistently the bacterial community structuring . In all samples, independently of sewer operating condition, the most abundant phyla were Proteobacteria > Bacteroidetes > Firmicutes . Similar results were reported for sludges from China   even for distinct geographic regions and sludge treatments   . Their aia (NH3) . Firmicu systems  and were systems . They maSaprospirae > Betaproteo-bacteria > Bacteroidia > Clostridia > Deltaproteobacteria > Alphaproteobacteria > Gammaproteobacteria > Actinobacteria > Spirochaetes . Betaproteobacteria, which is often associated with organic matter degradation and S cycle , corroborating with the literature . A great diversity of bacterial genera were also described in the literature  showed higher bacterial community differentiation among WWTPs  and diversity  (Supplementary Table Sewage sources (domestic or mixed) and biological treatments (redox conditions) did not affect microbial community structure . High pH values (≥11.9) resulted from liming were responsible for segregating C6 (SS12 and SS13) and enhancing Ca contents . Its most abundant phyla were Actinobacteria, Proteobacteria, and Bacteroidetes; whereas the most abundant genera were Propionibacterium, Comamonas, Brevundimonas, Methylobacterium, Stenotrophomonas, and Cloacibacterium . Other limed samples  presented lower pH (Table 1) and; therefore, very distinct microbial structure from C6. Despite having similar operating conditions as SS12 and SS13, SS11 also showed slightly lower pH as well as lower Cu and Zn and higher Fe and Pb contents (Table 1). It has been demonstrated that 1 pH-unit may considerably affect bacterial community structure and composition  at lower pH values (∼8.0). Liming to high pH values usually decreases microbial community diversity . Several studies showed that pH affected microbial community diversity and composition in soils  relative abundance did not depend on sludge location, but on pH instead. Therefore, pH may modulate microbial community by controlling nutrients availability and enzymatic processes that are essential to microbial metabolism . These samples underwent anaerobic treatment, except for SS10 (Table 1). Under anaerobiosis, both Fe and S have important roles in redox reactions (3+ → Fe2+) . In parallel, S reduction generates energy in anaerobic environments . Desulfovibrio, Desulforhabdus, and Smithella were very efficient in removing S from anaerobically treated sludges (Table 1). C5 samples had lower N-Kj likely due to denitrification (Table 1). Systems operated initially under anaerobic followed by aerobic conditions usually contribute most to N loss since they warranty anoxic denitrification and aerobic nitrification, thus converting ammonia (NH4+) to gaseous N  (Table 1).C5  segregation was related mostly to Fe but also to S, B, P, and N-kj, and contents   and redu → Fe2+) . Severalronments ; and it  sludges . SS10 wafication , resultiand N2O) . These sTable 1). They presented higher B, P, and N-Kj as would be expected from their higher organic matter pool, thus generating sludges with lower C/N ratios (Table 1). They also presented high Na and low Fe and Al contents (Table 1), which would be expected by their source nature (domestic). The most abundant genera were Clostridium > Dechloromonas >> Treponema > Desulfobulbus > Dok59 . Likewise, Clostridium, Treponema, and Desulfobulbus were also abundant in C2 and C3 . High abundance of Clostridium in domestic sludges was expected since it represents 10–40 % of human intestinal microbiota  removal from WWTPs  derived from domestic sewers, aerobically treated and without liming . These element contents were below those set by the Brazilian legislation for sludge use in agriculture (CONAMA 375/2006). Only one sample (SS7) exceeded threshold concentration for Ni and three  for Zn, but both are plant micronutrients. On the other side, Cd, Cr, and Ag inhibited important microorganisms for biological treatment, thus impacting sludge bacterial community , did not impact microbial community structure , Saprospirae (∼46%), and Clostridia (∼87%), respectively. Clostridium was the dominant genera, followed by Treponema, Propionibacterium, Syntrophus, and Desulfobulbus. Moreover, the samples were clustered into six groups according similarity of microbial community structures, which were related to their chemical attributes. High pH values (≥11.9) resulted from liming impacted mostly bacterial community structures and segregated C6, in which predominated Propionibacterium, Comamonas, Brevundimonas, Methylobacterium, and Cloacibacterium that are extremotolerant organisms. However, Clostridium, Treponema, Desulfobulbus, and Syntrophus were usually the most abundant ones in the other clusters, except that C1 presented relatively high abundance of Dechloromonas; C2 and C4 presented relatively high abundance of Sedimentibacter, and C3 and C5 presented relatively high abundance of Paludibacter. High Fe and S contents were important modulators of microbial structure for certain sludges undertaking anaerobic treatments and having relatively low N-kj, B, and P contents (C5); whereas high N-Kj, B, and P contents were important modulator for domestic, aerobically treated, and unlimed sludges having low Fe and Al contents (C1). Toxic inorganic elements, such as heavy metals (excluding micronutrients), had little impact on microbial community structure of the sludges. Nevertheless, the sludges shared a common core of 77 bacteria, being Clostridium, Treponema, Syntrophus, and Comamonas the most abundant ones.All sewage sludges presented high bacterial diversity. Their sources and biological treatment (redox) conditions did not consistently affect bacterial community structures. Overall, AN contributed to sample collection and processing as well as chemical and microbiological analyses. AS contributed to overall data analyses and manuscript writing. PA performed bioinformatic analyses. FA contributed to research idealization and manuscript revision. AC performed chemical attributes analyses. FO contributed to sample collection and research idealization. JR idealized, wrote, and revised the manuscript and coordinated the research.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.