Comadia redtenbacheri. Those that did not amplify were also confirmed morphologically to be the larva of this species.Mezcals are distilled Mexican alcoholic beverages consumed by many people across the globe. One of the most popular mezcals is tequila, but there are other forms of mezcal whose production has been part of Mexican culture since the 17th century. It was not until the 1940–50s when the mezcal worm, also known as the “tequila worm”, was placed inside bottles of non-tequila mezcal before distribution. These bottled larvae increased public attention for mezcal, especially in Asia, Europe, and the United States. Despite these larvae gaining global interest, their identity has largely remained uncertain other than that they are larvae of one of three distantly related holometabolous insects. We sequenced the COI gene from larvae in different kinds of commercially available mezcals. All larval DNA that amplified was identified as the agave redworm moth,  Agave Linnaeus. While tequila is a specific, popular type of mezcal made from blue agave (A. tequilana F.A.C. Weber), mezcal can be distilled from 30 of the 159 species of Mexican agaves  , family Hesperiidae (Lepidoptera). The weevil is known as “picudo del agave,” a major pest of agave and yucca in Mexico   or the Tn Mexico . A gravin Mexico . The larC. Weber . Adult fthe leaf . Local ctat loss .Comadia redtenbacheri ) ) . The larsalmiana . A femalsalmiana . Larvae salmiana . Like mobranches . Becausebranches .Local worm collectors or “gusaneros” harvest wild larvae of red and white maguey worms by hand between May and September, when they are most abundant. Gusaneros identify infected agave plants and extract larvae using a metal hook or an agave spine, avoiding as much damage to plants as possible . Larvae 1, B2, B6, C, D, E, K), contain sodium, potassium, iron, zinc, calcium, copper, phosphorus, magnesium, and manganese (Red and white maguey worms are rich in protein (35–65% dry basis), fat (13–33%), vitamins  was recorded. All tissues are deposited in tubes containing 95% ethanol, stored in the −80 °C freezer collection at the MGCL.Specimens were obtained from mezcal bottles that were purchased between 2018 and 2022 . We atteSpecimens were removed from the −80 °C freezer and a small 0.5–1 mg piece of tissue from the cross-section of the thorax was dissected. Tissues were individually placed in wells of a 96-well plate and sent either to the Canadian Center for DNA Barcoding (CCDB) in Guelph, Canada, or to the Smithsonian Institution, Washington, D.C. for DNA extraction and sequencing. In both cases, specimens were sequenced with Sanger sequencing using the cytochrome c oxidase subunit 1 (COI) LCO-HCO primers . SamplesFor samples extracted using the OmniPrep DNA extraction kit, we used the following protocol, slightly modified from the manufacturer’s guidelines: A small piece of larval thoracic tissue was placed in a clean microcentrifuge tube, before 180 µL of OmniPrep genomic lysis buffer and 20 µL of OmniPrep Proteinase K were added to the same tube. After macerating the tissue in this solution, the tube was vortexed for 15 s before being incubated at 56 °C overnight. Afterwords, 100 µL of chloroform was added and the tube contents vortexed for 15 s before being spun on a centrifuge for 10 min at 14,000 RCF. The supernatant was transferred to a new tube and 25 µL OmniPREP DNA stripping solution was added to the tube before the tube was vortexed for 15 s. Tubes and their contents were incubated at 56 °C for 10 min before being cooled to room temperature and 50 µL of OmniPrep precipitation solution and 3 µL OmniPrep mussel glycogen was added. Samples were vortexed for 15 s before being spun on a centrifuge for 20 min at 14,000 RCF. The supernatant was transferred to a new Eppendorf tube and 250 µL of cold isopropanol was added before being incubated at −20 °C for 30 min. The sample was centrifuged for 10 min at 14,000 RCF and 350 µL of cold 80% ethanol was added. Samples were centrifuged for another 10 min at 14,000 RCF and the ethanol discarded. The tube was kept open at room temperature to allow ethanol evaporation. Once sample pellets were dry, 50 µL of OmniPrep TE buffer and 0.5 µL of RNAse were added. We attempted to sequence other genes in addition to COI, but those did not amplify, likely due to sample degradation.http://www.geneious.com). Sequence ends were trimmed to exclude primer regions. Additional COI sequences of all available Comadia species and two cossid outgroups  were downloaded from the Barcode of Life Data System V4 (https://www.boldsystems.org/) and GenBank databases (http://www.ncbi.nlm.nih.gov/Genbank). Specifically, we downloaded fourteen sequences, and their associated data are listed in Forward and reverse COI sequences were aligned and assembled using Geneious 9.1.3 (Comadia (C. redtenbacheri and C. henrici Grote) have COI data available on GenBank, as of January 2023. We compared our sequences visually in Geneious and then calculated similarity scores  to published sequences with nucleotide BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi). We also calculated pairwise distances between every sample in our dataset to examine the percent sequence difference. Pairwise distances were calculated in PAUP* version 4.0a169  optimality criterion in the software IQ-TREE v. 2.1.0 , using dv. 2.1.0 . We usedv. 2.1.0  for treeFinally, the second and third authors of this study compared the morphology of larvae in bottles  to the kC. redtenbacheri.We first examined larval morphology. All larvae appeared superficially very similar, with a distinct head capsule and prolegs that are characteristic of lepidopteran larvae. Some specimens were white, others were pinkish red. For samples that had visible diagnostic morphological features, all had a small angular head capsule, reduced legs and prolegs, an upcurved prominent spine on A10, and lacked a pair of long appendages on A10 . AlthougC. redtenbacheri based on morphology). The 18 sequences had >99.39% hit  and varied by <2.5% similarity to publicly available COI sequences of C. redtenbacheri for the sequences with known locality information , but there was no notable clustering of specimens based on geography for the sequences with known locality information  which, given its name, implies that it is included in tequila and other mezcals , makes it unlikely that a mezcal distiller would include A. hesperiaris larvae in mezcal bottles , the plant used to make tequila  is driven by health benefits and by beliefs that these larvae contain aphrodisiac properties (Our finding that all larvae are a single moth species affirms the importance of n Mexico , and addn Mexico . Adding operties . This troperties . Opportuoperties . Unfortuoperties .C. redtenbacheri is to rear larvae on agaves in greenhouses in low larval densities and spaced irrigation conditions (In response to the declining number of mezcal larvae, researchers have begun to develop methods to cultivate these larvae in captivity . The optnditions . Howevere.g., Many studies have used molecular diagnostics to examine food content e.g., , as thes10.7717/peerj.14948/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.14948/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.14948/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.14948/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.14948/supp-5Supplemental Information 5Click here for additional data file.


Cnesterodon decemmaculatus, respectively, to 135, 309, and 70 μg/L during 96 h. Then the bioconcentration kinetic of SIL was comparatively assessed in C. decemmaculatus and Piaractus mesopotamicus exposed, respectively, to 44.1 and 16.2 μg/L during a one-week, followed by a four-day depuration phase. HPAIs concentrations in water and tissue were measured by HPLC-MS after 0.22 μm filtration and direct injection or solid-liquid extraction, respectively. Bioconcentration factors obtained empirically (BCFe) for C. decemmaculatus were CBZ = 1.5, SIL = 1.4, and ENA = 0.007. Parameters estimated by the SIL bioconcentration kinetic model for C. decemmaculatus were: uptake rate constant (k1) = 5.5 L/kg d, elimination rate constant during uptake phase (k2u) = 0.00175 d−1, maximum predicted tissue concentration (Ct(max)) = 138588 μg/kg, estimated bioconcentration factor (BCFm) = 3143, lag time between the exposure and the first detection in tissue (tlag) = 0 d, elimination rate constant in the depuration phase (k2d) = 0.49 d−1 and half-life in the tissue (t1/2) = 1.4 d. The model parameters for P. mesopotamicus were k1: 7.3 L/kg d, k2u: 0.0836 d−1, Ct(max): 1423 μg/kg, BCFm: 88, tlag: 3.8 d in the uptake phase and k2d: 0.31 d−1 and t1/2: 2.3 d in the depuration phase. The reached conclusions were: 1) the bioconcentration capacity of CBZ and SIL are similar but around 200 times higher than ENA, 2) the time to reach the bioconcentration equilibrium for SIL is longer than 1 week, then estimated BCFm are between 1 and 3 orders of magnitude higher than BCFe obtained after 96 h exposure, but actual values need to be verified, 3) substantial differences (≈30 fold) were observed in the estimated BCF of SIL among species, indicating the need for further studies toward understanding such diversity to improve HPAIs ecological risk assessment worldwide.Sewage effluents are the main source of entry of Human Pharmaceutical Active Ingredients (HPAIs) to surface water bodies. Carbamazepine (CBZ), psychiatric drug, enalapril (ENA) antihypertensive, and sildenafil (SIL), to treat erectile dysfunction, have been frequently detected in receiving wastewater and in wild fish species from Argentina. This study aimed to assess the bioconcentration of selected HPAIs in native fish species of the Del Plata Basin. In a first trial, the bioconcentration factors of CBZ, ENA, and SIL were obtained by exposing  Human pharmaceuticals active ingredients (HPAIs) are considered emerging pollutants, and manufacturing, hospitals, and domestic wastewater discharges are their relevant sources for aquatic ecosystems . In pollPimelodus maculatus, Prochilodus lineatus, and Megaleporinus obtusidens, with maximum concentrations of 71.6, 69.4, 56.9, and 45.8 μg/kg of SIL, ATE, ENA, and CBZ  and Piaractus mesopotamicus  are Neotropical fish species characteristics of the Del Plata Basin . It is also used in ecotoxicological studies. Unlike C. decemmaculatus, P. mesopotamicus is important for local aquaculture and human consumption  and formic acid (88%) were Sigma-Aldrich.Carbamazepine (CBZ) CAS No 298-46-4, log Kow 2.77, pKa 2.77 and 13.9 , enalaprC. decemmaculatus were caught at the La Plata city park lake (Lago del Bosque), La Plata City, Buenos Aires, Argentina. Juvenile P. mesopotamicus were acquired from Teko Company, Province of Chaco, Argentina. Fish were maintained 2 weeks before the test in the aquaria of the Centro de Investigaciones del Medioambiente (CIM-CONICET-UNLP). The 2,000 L aquaria system was filled with dechlorinated and activated charcoal-filtered tap water at 24°C ± 2°C, pH 7.5 ± 0.5, and oxygen 8 ± 1.2 mg/L with 16:8 h light/dark cycle, constant aeration, and fish were fed twice daily based on a feed for omnivorous fish from Mixes del Sur. The weight and standard length of the fish at the start of the experiment were 0.3 g and 23 mm for C. decemmaculatus and 48 g and 100 mm for P. mesopotamicus. Fish were handled according to the protocol approved by the Commission for the Care and Use of Laboratory Animals of the Faculty of Exact Sciences of the National University of La Plata (CICUAL # 006-26-17).Adult females of C. decemmaculatus adult females were randomly distributed (12 per tank in 3 tanks) and exposed to 200, 600, and 100 μg/L of CBZ, ENA, and SIL, respectively, for 96 h. The exposure concentrations were set based on the acute lethal toxicity (≈1/100 LC50) obtained for C. decemmaculatus in a preliminary test. Also, a control group was included using the same concentration of vehicle solvent [0.01% (v/v) methanol]. Exposure time was selected according to the bioconcentration kinetic found in the literature for most HPAIs and because it is a broadly used exposure time in fish ecotoxicity tests  and aerated for 48 h before the trial. The bioconcentration tests were performed at the same light and temperature conditions used for fish maintenance. Partial media renewal, 80% of the volume, was done at 48 h, just after fish feeding. Fish were sampled, killed in ice-cold water, and stored at −80 °C until processing. Composite samples were performed using the whole fish.C. decemmaculatus: an 11-d test was conducted, including a 7 d uptake phase exposing fish to 50 μg SIL/L, followed by a 4 d depuration phase in which the test solution was replaced by dechlorinated and filtered water. Also, a control group with dechlorinated water and vehicle solvent [0.01% (v/v) methanol] was included. The tested concentration was based on the BCF obtained in the 96-h trial (1.4), and the concentration measured in fish collected in the field (71.7 μg/kg) in a former study  methanol] was included. Each treatment was carried out in triplicate, placing three juveniles in each of the eighteen 20 L stainless steel aquaria filled with a volume of 10 L of test solution. Aquaria were randomly sampled at 0, 2, 4, 6, 8, and 10 d. Partial renewal of the test solution (80%) was done every 48 h, just after fish feeding. Fish were sampled, killed in ice-cold water, and dissected with stainless steel instruments. The dorsal muscles of both flanks were sampled, wrapped in aluminum foil, placed in food-grade bags, and stored at −80 °C. Muscle instead whole fish was used because it is an eatable fish. The test was performed under the same conditions used for fish maintenance.Bioconcentration kinetic trial with g for 10 min at 4°C. Supernatants were transferred to 15 mL conical glass tubes and taken to dryness under a gentle nitrogen stream. Finally, extracts were resuspended in 500 µL of methanol-acetonitrile (50:50), transferred to a chromatographic vial, and stored at −20 °C.Water samples from each trial were collected at the beginning and the end of the exposure period (96 h) and analyzed by direct injection after filtration through a 0.22 μm nylon filter. Extraction and analyses of samples were carried out following the analytical methodology by ® ultraperformance liquid chromatography system ACQUITY UPLC® equipped with an autosampler and coupled to a single mass spectrometer ACQUITY SQD®. LC-MS instrumental settings have been detailed in C. decemmaculatus, spiked at 100 μg/kg.Analysis of pharmaceuticals was done by LC-ESI-MS, using a Waterse) were obtained as the ratio between the average concentration of the pharmaceutical in the tissue after 96 h exposure (Ct(96h)) and the average concentration in the testing solution (Cts(av)) (Eq. ts(av)) were expressed as the average concentration in water during the test using the concentration in water at the start Cw(t0) and the end of the test Cw(96h) ) Eq. . Concent96h) Eq. .BCFe=Ctm) were calculated presuming first-order kinetics, as the ratio between the uptake rate constant (k1) and depuration rate constant of the uptake phase (k2u) (Eq. t (µg/kg): is the pharmaceutical concentration in the tissue at time t, Cts(av) (µg/L): is the pharmaceutical average concentration in the testing solution during the entire test, k1 (L/kg d): is the uptake rate constant, k2u (d−1): elimination rate constant in the uptake phase, k2d (d−1): elimination rate constant in the depuration phase, t (d): exposure time, tlag (d) latency time (time delay between the exposure and the detection in tissue).Bioconcentration factors estimated by means of the kinetic model (BCFk2u) Eq. . Both pak2u) Eq.   Eq. . Adjustet(max) (µg/kg), (∞), the term {1-exp [-k2u x (t-tlag)]} of Eq. The model applied describes the accumulation through time that eventually approaches steady-state equilibrium with the source. The concentration in the fish at steady-state equilibrium is represented by C(µg/kg),  Eq. 5)..t(max) (2d (d−1)] was obtained based on Eq. The elimination rate constant of the depuration phase [k1/2 (d)), the time required for the concentration of SIL to fall to 50% of its initial value, was calculated with Eq. Half-life  between renewals. Internal concentrations of SIL in the whole C. decemmaculatus are shown in e of 40. Quick drug clearance was observed during the 2 days depuration phase, showing only 13% of the maximum accumulated concentration on day 11.The average concentration of SIL in water during the 2u 280-fold smaller than k2d. The estimated values for the maximum concentration at the plateau and the BCFm were comparatively high due to the small k2u value. No lag time was predicted in the uptake phase by the model. Additionally, the depuration half-time was estimated in less than 2 days.The first-order bioconcentration kinetic model fitted to empirical internal concentrations during the uptake and depuration phases is also shown in P. mesopotamicus test was 16.2 μg/L, dropping by 99% between renewals. Internal concentrations of SIL measured in P. mesopotamicus during the uptake and depuration phase are shown in e at that point was 15. During the four-day depuration phase, 60% of the maximum concentration was eliminated.The average concentration of SIL in the water during the 2u 3.70-fold smaller than k2d. The estimated values for the maximum concentration at the plateau and the BCFm were higher than measured from empirical data at the end of the uptake phase. The depuration half-time was quick, estimated at around 2 days.The first-order bioconcentration kinetic model fitted to the empirical internal concentrations during the uptake and depuration phases is also plotted in w 0) and partially due to the absorption by the fish during the time between renewals. These results remark the need to measure the testing water concentrations and, if possible, use flow-through aquaria or reduce the time between renewals.Analytical methods performance was according to previous studies , with sim of pharmaceuticals studied in C. decemmaculatus after 96 h of exposure, showed the order CBZ ∼ SIL > ENA. The log Kow values of these drugs in decreasing order are 3.18 for SIL, 2.77 for CBZ, and 2.45 for ENA  has been reported between whole fish and muscle concentrations for hydrophobic non-ionized organic pollutants , which was more than 40 times lower than the one estimated for P. mesopotamicus. A lower k2 value would reflect both a reduced capacity of depuration and/or biotransformation of SIL in C. decemmaculatus during the exposure period. On the other hand, the uptake rate constants (k1) were similar, as well as the elimination rate constants (k2d) and half-live (t1/2) estimated in the depuration phase. Particularly, the low k1 and k2u values were consistent with the behavior of hydrophilic compounds, in which diffusion through membranes might be hindered by de pH of the media  previously found in the surface waters of Argentina , and the Stockholm Convention on Persistent Organic Pollutants consider “bioaccumulative” or “very bioaccumulative” substances with BCF values between 1,000 and 5,000. According to the BCF values obtained for CBZ, ENA, and SIL in The BCFs for CBZ, ENA, and SIL were measured for the first time in the neotropical fish species, being for CBZ and SIL 1.5 and 1.4, respectively, and for ENA two orders of magnitude lower (0.007), possibly because of its biotransformation in enalaprilat. Obtained BCF values were like those reported for frequently studied holarctic fish species and range among the ones for compounds classified in the category of low bioconcentration potential.C. decemmaculatus, the smaller fish than in the muscle of P. mesopotamicus, the bigger one. Compared with the other assessed pharmaceuticals, SIL bioconcentration kinetics showed a longer time to reach the plateau and at least one order of magnitude higher BCF. Further studies assessing longer exposure times than 1 week are encouraged for precisely estimating its BCF.The bioconcentration kinetics of SIL was evaluated for the first time in fish, comparing the process in two native species. Not only differences were observed among species, but also in comparison with the other tested drugs. Bioconcentration of SIL was faster and higher in the whole body of Finally, the study contributes new information about the bioconcentration of three frequently detected pharmaceuticals in the Del Plata Basin for poorly studied neotropical fish species, which results in helpful inputs for future ecological risk assessments.


Helicobacter pylori (H. pylori) infection and various inflammatory factors on G17 secretion level.To analyze the difference of serum gastrin‐17 (G17) level in healthy people with different sex, age, and body mass index (BMI), to explore the correlation between G17 and pepsinogen, and to study the influences of H. pylori, serum amyloid A (SAA), C‐reactive protein (CRP) and erythrocyte sedimentation rate (ESR). The difference of G17 secretion in different subjects and its correlation with PG were analyzed to investigate H. pylori infection and expound the effects of inflammatory indicators on G17.A total of 531 subjects who received physical examination in our center from April 2019 to December 2019 were enrolled in the study. All subjects were tested for G17, pepsinogen I (PGI), pepsinogen II (PGII), PGI/PGII ratio (PGR), p > .05). G17 positively correlated with PGI and PGII, but negatively correlated with PGR. The G17 level of H. pylori‐positive subjects was 10.16 ± 12.84, and prominently higher than that of H. pylori‐negative subjects (3.27 ± 6.65). SAA and H. pylori infection were the greater risk factors for G17 abnormality among various indicators. CRP and ESR had no effect on G17 abnormality.There was no significant difference in G17 secretion level in people with different sex, age and BMI (H. pylori. Combined screening contributes to early screening of gastrointestinal diseases in normal people or groups at high risk for gastric cancer, but the influence of inflammatory indicators on G17 should be excluded to improve the reliability of the results.G17 secretion is closely related to PG and  These serological markers may be conducive to identifying people with precancerous lesions who should undergo gastroscopy.H. pylori IgG, but also other biomarkers such as G17 and PGII.Compared with the markers in tissues, serological markers are superior due to their less invasiveness, easy availability, lower cost and shorter time consumption. Currently, available serological markers associated with gastric diseases include Pepsinogen I (PGI), Pepsinogen II (PGII), PG I/PG II (PGR), gastrin‐17 (G17) and anti‐G17 is closely related to inflammation, immunity, invasion and metastasis of gastric cancer cells, and is lowly expressed in healthy people. When the pathological changes of gastritis, gastric cancer, etc. occur, G17 expression level will increase significantly.22.1H. pylori typing, C‐reactive protein (CRP), erythrocyte sedimentation rate (ESR) and serum amyloid A (SAA) were detected. None of these subjects was diagnosed with acute and chronic inflammatory disease, liver and gallbladder disease, diabetes, blood disease, kidney disease, rheumatic immune disease, tumor, etc. Groups were constructed according to age, body mass index (BMI), and H. pylori typing.A total of 531 healthy subjects  who received physical examination in the Healthcare Department Center of Wuhan Union Hospital from April 2019 to December 2019 were selected by continuous and effective sampling method, and approved by the Ethics Committee of the hospital (No. S426). In addition to routine physical examination, G17, PGI, PGII, PGR, 2.2About 3 mL fasting venous blood was collected and centrifuged to obtain serum. The serum PGI, PGII, PGR, and G17 were quantitatively detected by enzyme‐linked immunosorbent assay. The testing equipment was procured from BIOHIT Healthcare (Hefei) Co., Ltd and Wuhan Yolins Technology Trade Co., Ltd. As per the instructions of the kit, PGI <70 ng/mL concurrently with PGR <3 was defined as PG‐positive, and G17 <1 or >15 pmol/L was regarded as G17‐positive.2.313C breath test early in the morning on an empty stomach. The test was implemented using the kit produced by Shenzhen Zhonghe Headway Bio‐Sci & Tech Co., Ltd., and the HCBT‐02 13C breath test instrument. The positive value of H. pylori infection was 4.0 times higher than the reference value.Subjects underwent the 2.4CRP was measured by immunofluorescence quantitative detection using an I‐CHROMA Rester immunofluorescence analyzer (Boditech Med Inc.). ESR was calculated according to the Westergren method. SAA was detected with a SIEMENS BN prospect specific protein analyzer as well as matching reagents in strict accordance with the operation manual.2.5t test, while contrast among multiple groups was achieved through one‐way analysis of variance. The correlation analysis was performed by Spearman rank correlation test, with the calibration level of 0.01. The simple Logistic regression analysis was used to evaluate the influencing factors. A p < .05 was considered to be statistically significant.SPSS19.0 statistical software was used for analysis. Comparison between two groups was completed by 33.1p > .05)  than in males (5.32 ± 10.11). The G17 secretion level in the different age groups  was 5.01 ± 10.68, 4.17 ± 8.13, 4.81 ± 7.40, 7.66 ± 12.73, 6.58 ± 11.11, and 8.65 ± 6.15, respectively, with no significant difference among them. Moreover, G17 secretion level did not differ in the three BMI groups . Collectively, the G17 secretion level was not statistically different in sex, age, and BMI groups  and H. pylori infection ( p = .017) were the major risk factors for G17 abnormality. Other factors had no effect on the secretion of G17 (Table p > .05).The risk factors of G17 secretion abnormality were analyzed, including sex, age, 17 Table , p > .053.3H. pylori‐infected subjects (10.16 ± 12.84) was remarkably higher than that in uninfected subjects (3.27 ± 6.65) (Table p < .05).The secretion of G17 in 3.4p = .000). G17 positively correlated with PGI and PGII (r = .322 and .452), but negatively correlated with PGR (r = −.367)  should be performed.Further, the combination of serum PG and G17 levels can help diagnose gastric mucosa, reflect the degree of gastric aging, and distinguish pathological state from healthy state.H. pylori detection as reference to further improve the diagnosis rate of AG and early gastric cancer, and also to directly screen high‐risk groups. On the basis of G17, PG, and H. pylori detection results, endoscopy and histological examination were scheduled to enhance the compliance of screening and treatment of gastric diseases, and avoid unnecessary radiation and discomfort caused by endoscopy. Besides, former findings have unveiled that G17 combined with serum tumor markers  can significantly improve the sensitivity of the diagnosis of gastric cancer.Therefore, G17 is not the major diagnostic indicator, but can be combined with PG and H. pylori, based on the overseas screening mode of gastric cancer and combined with the situation of China and the current epidemiological investigation. Through this test, it is expected to find and optimize the early gastric cancer screening method suitable for China's national conditions, and timely provide evaluation and intervention recommendations for high‐risk groups. Also, it is hopeful to reduce mortality, prolong life, and protect people's health by early screening, early diagnosis and early treatment. Nevertheless, in fact, some limitations exist in our study. Reportedly, several separate and combinatorial transcripts of H. pylori virulence genes are associated with clinical outcomes .H. pylori genotype and G17 level needs further study. Besides, the grading and staging of gastric cancer also need to be examined to confirm the relationship between G17 level and gastric cancer.In recent years, Chinese scholars have successively promoted the “four items for gastric function” test combined with G17, PG, and 5Since China is challenged by high incidence rate and mortality rate of gastric cancer, and precancerous lesions are the key stage in the development of gastric cancer, it is necessary to find effective means to screen gastrointestinal diseases. Combined screening is conducive to early screening gastrointestinal diseases in normal people or groups at high risk of gastric cancer, but the influence of relevant factors on G17 should be excluded to enhance the reliability of the results.Junchao Zeng: conceptualization; writing—original draft. Yan Shen: project administration; writing—review & editing. Sanping Xu: formal analysis; investigation; resources; software. Rui Yang: investigation; validation; visualization.The authors declare no conflicts of interest.A preprint has previously been published.


Enterococcus durans strain K11 and Lactiplantibacillus plantarum strain St3 using 16S rRNA gene sequencing. The antioxidant activity of HA produced by E. durans strain K11 and L. plantarum strain St3 was (65.4 0.2%) and (66.6 0.1%), respectively. The viability of UVB-irradiated keratinocytes pre-treated with HA produced by E. durans strain K11 and L. plantarum strain St3 was 91.3 and 91.4%, respectively, compared with the control. While the viability of UVB-irradiated keratinocytes post-treated with HA produced by E. durans strain K11 and L. plantarum strain St3 was 86 and 88.5%, respectively. To the best of our knowledge, this is the first recordation of HA production by Enterococcus durans and Lactiplantibacillus plantarum which revealed a significant radioprotection of the human keratinocytes against UVB radiation.Exposure to ultraviolet (UV) radiation is one of the major factors that causes skin aging, erythema, sunburns, and skin cancer. This study aimed to select probiotic bacterial isolates able to produce high yield of hyaluronic acid (HA) to be employed for skin photoprotection and other possible biological applications. The selected isolates K11 and St3 were able to produce the highest yields of HA 4.8 and 4.4 mg/ml, respectively. Both isolates were identified as The online version contains supplementary material available at 10.1007/s00284-023-03377-y. Major health problems linked to overexposure to UV radiation, such as skin cancer (melanoma and non-melanoma), premature aging of the skin, actinic keratoses, sunburns, cataracts, pterygium, degeneration of the macula, and suppression of body’s immune system and the skin’s natural defenses .Oxidative stress, brought on by prolonged exposure to UV radiation from sunlight, usually affects human skin, can lead to cellular damage , apoptosis, and cell death. Skin overexposed to UV is linked to a variety of abnormal responses, such as inflammatory responses, epidermal hyperplasia, the breakdown of collagen, and the formation of melanin . AccordiL. plantarum extracts in skin care, even though bacteria are assumed to play a significant influence in the prevalence of acne, skin hydration, and nutrient metabolism [Bifidobacterium, Saccharomyces, Enterococcus, Bacillus, and Lactobacillus [L. plantarum protects hairless mice and human skin fibroblasts from UVB-induced photoaging [Oral and topical probiotics have the potential to prevent and treat skin photoaging by altering the skin microbiome and gut–skin microbial interactions. These mechanisms include lowering oxidative stress, preventing extracellular matrix remodeling, reducing inflammatory cascade reactions, and maintaining immune homeostasis . A few rtabolism . There hbacillus . Also, rotoaging .Hyaluronic acid, also known as hyaluronan, is a linear polysaccharide which is one of the main components of connective tissue, forming a gelatinous environment that surrounds cells. HA is made up of repeated units of -D-glucuronic acid (GlcUA) and -N-acetylglucosamine (GlcNAc), which are connected alternately by glycosidic linkages. In humans, HA is found in all body tissues and fluids, but it is most prevalent in the skin , the vitreous of the eye, the umbilical cord and synovial fluid. It is also found in skeletal tissues, heart valves, the lung, the aorta, the prostate, tunica albuginea, corpora cavernosa, and the corpus spongiosum of the penis. Mesenchymal cells produce the majority of HA; however, other cell types can as well .Streptococcus species have been employed to produce HA at industrial scale. S. zooepidemicus is the most broadly applied strain in the production of HA. Many studies have revealed that certain strains of lactobacilli and bifidobacteria are able to produce hyaluronic acid, which could improve the barrier, immunological, and homeostatic functions of the skin [Commercial hyaluronan is mainly produced by biotechnology . HA generated from microorganisms is compatible with the human body . Conventthe skin –13.The antioxidant activity of hyaluronic acid in the body as an effective free radical scavenger is one of its most important functions . HyaluroEnterococcus durans and Lactiplantibacillus plantarum were reported as HA producers for the first time. The extracted HA showed an antioxidant activity and good protective effect to the human dermal keratinocytes against UVB damage.In the current study, Three different samples, kefir (K), breast milk 1 (H), and breast milk 2 (Z), were collected from different sources. Kefir sample was purchased from Healthy Corner, Heliopolis, Cairo (30°05′24.8″ N 31°19′06.4″ E). Two breast milk samples were collected from two different newly delivered women. From each sample, 1 ml was serially diluted in phosphate-buffered saline solution (PBS) (pH ~ 7.4) then, separately inoculated into three different agar media: deMan Rogosa and Sharpe (MRS) , KanamycLactiplantibacillus plantarum strain M and Lactiplantibacillus plantarum strain St3) were isolated by Shaheen [Lactobacillus casei NRRL-1922 and Lactobacillus acidophilus NRRL-23431) were kindly supplied from Agricultural Research Services Culture Collection, Peoria, United States.In addition to the isolated cultures four probiotic strains  then incubation for 1 h at 4 °C, followed by centrifugation for 20 min at 10,000 g at 4 °C . One volAccording to the method reported by Hor et al.  HA samplThe concentration of the extracted HA from different cultures was measured by reversed phase (RP) HPLC according to Vigliano et al.  with som−1.The extracted HA from different cultures of the selected bacterial isolates was compared with standard HA using FTIR spectroscopy . FTIR anTaq  according to instruction manual. A pair of universal bacterial primers was used to partially amplify target 16S rRNA gene from the bacterial isolates 27F (5′-AGA GTT TGATCC TGG CTC AG-3′) and 1492R (5′-GGT TAC CTT GTT ACG ACT T- 3′). A portion of 2 μl of bacterial DNA was transferred into a 50-μl final volume as template for PCR. PCR was performed using Maxima Hot Start PCR Master Mix (Thermo K1051) according to manufacturer’s instructions. PCR was performed in genius model FGENO2TD thermal cycler . The PCR conditions were adjusted to 5 min for initial denaturation at 94 °C, then 35 cycles of 1 min at 94 °C, 1 min at 54 °C, and 1 min at 72 °C and then finally 10 min at 72 °C. The amplified genes were run on 1% agarose gel and visualized with standard marker of known size  to determine the size and purity of products and then amplified bands were cleaned using GeneJET™ PCR Purification Kit (Thermo K0701).The PCR was carried out at Microbiology Department, National Center of Radiation Research and Technology (NCRRT), Atomic Energy Authority (AEA). Total DNA of the selected bacterial isolate showed the highest production of HA was extracted using DNA extraction kits  according to manufacturer’s instructions. PCR was performed using Premix Sequencing of forward and reverse directions of PCR product of 16S rRNA gene was carried out by Macrogen, Korea. The obtained sequences were identified using BLAST search program, National Center for Biotechnology Information (NCBI), National Library of Medicine, USA  and the The scavenging effect of the produced HA on 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals was evaluated. Two milliliters from each HA sample of concentration 100 µg/ml (the produced and the standard) were added to 0.1-mM DPPH dissolved in 95% ethanol. The mixture was shaken and left for 30 min at room temperature and the absorbance of the resulting solution was measured at 517 nm , 27. A lStock solutions (1 mg/ml) of the produced and commercial HA from L'Oréal (1.5%) were prepared in cell culture media Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum (FBS), glutamine (0.29 mg/ml), and gentamycin (50 µg/ml). The tested concentration of HA (1 mg/ml) was selected according to Hašová et al. .2 of tissue, by each foreskin, was harvested for double-enzymatic digestion. Subcutaneous fat was removed with scissors and the remaining tissue was cut into roughly 0.5 cm2 pieces and incubated in 20-ml Dispase for 12 h at 4 °C. After incubation the epidermis was lifted off the dermis with pliers and transferred into 5-ml trypsin. The tissue was incubated at 37 °C in 95% humidity and 5% CO2 for 15 min and during this time it was repeatedly removed from the incubator and triturated. After incubator and triturated, the suspension was centrifuged at 1400 rpm for 10 min then the supernatant was removed, and the pellet of human keratinocyte cells was resuspended in culture medium [2 at 37 °C [The study was applied on keratinocytes from foreskin in children. After circumcision, pediatric foreskin was transferred to the laboratory in DMEM at 4 °C. The skin was rinsed in phosphate-buffered saline with antibiotics and antimycotics. As much as 4 cme medium . Human kat 37 °C .2) used in this experiment was selected according to Hašová et al. [2). The cells and cell culture media were incubated and harvested 24 h after UVB irradiation. In case of post-treatment, immediately after the UVB irradiation (single dose 10 mJ/cm2), culture media was replaced by cell culture media with or without HA and then were incubated and harvested after 24 h.UVB irradiation was carried out by means of UVB broadband TL Philips lamp emits radiation in the B bandwidth of UV spectrum (290–315 nm) and the UV intensity was measured using Waldmann Variocontrol UV meter, Germany and then the dose was calculated. The irradiation dose -2,5-diphenyltetrazolium bromide  assay . The optAll tests are performed in triplicates, and data were expressed as mean ± standard error (SE). Statistical significance was assessed using one-way ANOVA  test and the means were compared with Duncan’s test at 0.05 level.L. plantarum strain M, L. plantarum strain St3, L. casei 1922 and L. acidophilus 23,431 were examined to produce hyaluronic acid by turbidimetric method. Fifteen out of twenty-two bacterial isolates were able to produce hyaluronic acid  in the concentration of HA produced by the isolate K11 and isolates St3 and K33. The highest HA production was recorded by the isolates K11, St3, and K33 compared with the other isolates.Eighteen bacterial isolates were selected from different sources grown on MRS agar, Kanamycin aesculin azide agar, and KF streptococcus agar media. In addition to the selected isolates, P≤0.05) about 4.8 ± 0.3 and 4.4 ± 0.2 mg/ml, respectively  showed high HA productivity ranged from 3 to 4.8 mg/ml. The production of HA by K11 and St3 was significantly high . The produced HA by the selected isolates showed characteristic peaks at 3322, 2173, 1638, 605, and 587 cmLactiplantibacillus plantarum strain St3 and recorded at the GenBank under the accession number (KY508301). Bacterial isolate K11 was identified using 16S rRNA gene as Enterococcus durans. The nucleotide sequences were submitted to GenBank under accession number (ON359827). Neighbor-joining phylogenetic tree with alignment of 16S rRNA gene revealed that L. plantarum strain St3 was placed in the phylogenetic cluster with L. plantarum strain CIP 103151 type strain, while E. durans strain K11 in the cluster with E. durans strain DSM 20633 type strain. The topology of the phylogenetic tree clearly confirmed the NCBI-BLAST results and the belonging of St3 and K11 strains to genus Lactobacillus and Enterococcus, respectively and thus identification of strain St3 as L. plantarum and strain K11 as E. durans  were selected as the most promising HA producing isolates. The isolate St3 was isolated and identified using 16S rRNA gene by Shaheen  as LactiE. durans strain K11 and L. plantarum strain St3, respectively  compared with that of the produced HA. Also, there is no significant difference of antioxidant activity between the produced HA by E. durans strain K11 and L. plantarum strain St3. The produced HA by E. durans strain K11 and L. plantarum strain St3 showed good antioxidant activity which is very close to that of ascorbic acid and standard HA. The variation in significance may be attributed to the difference in the parity between the highly purified standard HA and the extracted HA samples.The antioxidant activity of the produced HA was 65.4 ± 0.2%) and (66.6 ± 0.1%) by  ± 0.2% a2) revealed that the viability of irradiated untreated cells significantly decreased (at P≤0.05). There is a significant difference (at P≤0.05) in the viability of UVB-irradiated cells pre-treated with HA extracted from K11 and St3 compared with that pre-treated with commercial HA. The results presented that HA extracted from K11 and St3 significantly maintained the viability of the cells by 91.3% and 91.4% in comparison to the absence of HA by 76%, respectively. Also, there is a significant difference (at P≤0.05) in the viability of UVB-irradiated cells post-treated with HA extracted from K11 and St3 compared with that post-treated with commercial HA. The results showed that HA extracted from K11 and St3 significantly maintained the viability of the cells by 86% and 88.5% in comparison to the absence of HA by 76%, respectively  is frequently used as a biomaterial for both wound healing stimulation and bioengineering applications. HA is frequently employed in ophthalmology, rheumatology, pharmacology, and drug delivery in addition to dermatological and cosmetic products .L. plantarum strain M, L. plantarum strain St3, L. casei 1922, and L. acidophilus 23,431.In the present study, 18 probiotic isolates were isolated on MRS, kanamycin aesculin azide, and KF streptococcus agar media which are proper media used to isolate probiotic strains, from different three sources , 33. In S. thermophilus on skimmed milk medium. Also, Lew et al. [All studied isolates were screened for their ability to produce hyaluronic acid on skimmed milk medium. This is parallel to Izawa et al.  and Kanaw et al.  reportedThe concentration of the produced hyaluronic acid using CTAB turbidimetric method was assessed. This method is a quick, safe, and accurate method to determine HA concentration in fermentation culture compared with colorimetric carbazole method . The amoS. thermophilus TH-4 and S. thermophilus BLM 58, respectively. Also, Izawa et al. [S. thermophilus using HPLC technique.The hyaluronic acid produced by the selected isolates was quantitatively analyzed using HPLC analysis. The results of HPLC analysis revealed that the HA standard and all the produced HA had the same retention time and the HA concentrations produced by the selected isolates ranged from 0.2 to 4.8 mg/ml. Kakehi et al.  reporteda et al.  and Mohaa et al.  confirmeFrom the previous studies, turbidimetric assay is less accurate in determining the hyaluronic acid concentration compared with that obtained by HPLC. Also, the HA-CTAB complex formation depends significantly on the ionic composition and strength. Therefore, the CTAB precipitation is applicable only for rough estimations of HA in certain samples, such as a microbial fermentation medium, whereas HPLC method is sensitive and specific . This isThe FTIR spectroscopy is an important technique to identify the functional groups and organic compounds by assessing the transitions between vibrational states of bonds contained within the molecule . FTIR an, as L. plantarum strain St3 (KY508301), while the isolate K11 was identified using 16S rRNA gene sequence as E. durans strain K11 (ON359827). This is the first record of HA production by E. durans and L. plantarum. The production of hyaluronic acid using novel probiotic bacteria was a main objective of this study and our results revealed that it has been successfully attained. Previous studies reported that HA producing bacteria was achieved by pathogenic strains S. zooepidemicus. It was reported for the first time in 2009 that HA is produced in milk broth through fermentation by a putative probiotic strain Streptococcus thermophilus YIT2084 [S. thermophiles, L. casei, L. fermentum, L. gasseri, L. acidophilus, L. bulgaricus, Bifidobacterium bifidum, and B. longum which recorded as HA- producing bacteria [L. acidophilus PTCC1643, L. rhamnosus PTCC1637, L. casei PTCC1608 and S. thermophilus PTCC1738 were able to produce HA.Data obtained from CTAB turbidimetric assay and confirmed with HPLC revealed that the highest HA production was achieved by K11 and St3 isolates which were selected as the most potent HA producing probiotic isolates. The probiotic isolate St3 was identified using 16S rRNA gene sequence by Shaheen , as L. pin K11 ON9827. Thibacteria . Also, Fbacteria  reportedEnterococcus durans strain K11 and L. plantarum strain St3 were (65.4 ± 0.2%) and (66.6 ± 0.1%), respectively. This is in accordance with Pan et al. [Out of all the in vitro methods, DPPH scavenging method is the most easy, simple, and reasonably costly method for estimating the free radical-scavenging activities of antioxidants. The antioxidant activity of the HA produced by n et al.  who recon et al. .Enterococcus durans strain K11 and L. plantarum strain St3, respectively. Also, the viability of irradiated post-treated cells was 86 and 88.5% for HA produced by Enterococcus durans strain K11 and L. plantarum strain St3, respectively. This is in accordance with Hašová et al. [Streptococcus equi had significant protective effects for HaCaT keratinocytes against UVB irradiation.In the present study, the response of human dermal keratinocytes to UVB irradiation was in vitro evaluated. The viability of the irradiated untreated cells was 76% while the viability of pre-treated irradiated cells was 91.3 and 91.4% for HA produced by á et al.  who recoEnterococcus durans strain K11 and L. plantarum strain St3, which belongs to probiotic bacteria, can produce exopolysaccharides, such as HA. Second, in an in vitro antioxidant assessment, the produced HA showed much higher antioxidant activity. Third, the application of HA could protect the human dermal keratinocytes before and after exposure to UVB radiation. HA could, therefore, be act as a protective agent that prevents the harmful effect of solar irradiation on skin.Data obtained from all experiments suggest three significant conclusions. First, Supplementary file1 (TIFF 665 KB) Figure (S1): HPLC analysis of the produced hyaluronic acid by the selected isolates A: standard HA, B: K2, C: K4, D: K11, E: K13, F: K33, G: K44, H: H3, I: H4, J: H5, K: Z3, L: Z4, M: M, N: St3, O: 1922, and P: 23431.Supplementary file2 (TIFF 450 KB)Supplementary file3 (TIFF 733 KB)Supplementary file4 (TIFF 610 KB) Figure (S2): FTIR analysis of the produced hyaluronic acid by the selected isolates A: standard HA, B: K4, C: K11, D: K33, E: H3, F: H4, G: H5, H: St3, and I: 1922).Below is the link to the electronic supplementary material.


In recent years, the hypothalamus has emerged as a new neurogenic area, capable of generating new neurons after development. Neurogenesis-dependent neuroplasticity seems to be critical to continuously adapt to internal and environmental changes. Stress is a potent environmental factor that can produce potent and enduring effects on brain structure and function. Acute and chronic stress is known to cause alterations in neurogenesis and microglia in classical adult neurogenic regions such as the hippocampus. The hypothalamus is one of the major brain regions implicated in homeostatic stress and emotional stress systems, but little is known about the effect of stress on the hypothalamus. Here, we studied the impact of acute and intense stress , which may be considered as an inducer of an animal model of posttraumatic stress disorder, on neurogenesis and neuroinflammation in the hypothalamus of adult male mice, focusing on three nuclei: PVN, VMN and ARC, and also in the periventricular area. Our data revealed that a unique stressor was sufficient to provoke a significant impact on hypothalamic neurogenesis by inducing a reduction in the proliferation and number of immature neurons identified as DCX+ cells. These differences were accompanied by marked microglial activation in the VMN and ARC, together with a concomitant increase in IL-6 levels, indicating that WIRS induced an inflammatory response. To investigate the possible molecular mechanisms responsible for neuroplastic and inflammatory changes, we tried to identify proteomic changes. The data revealed that WIRS induced changes in the hypothalamic proteome, modifying the abundance of three and four proteins after 1 h or 24 h of stress application, respectively. These changes were also accompanied by slight changes in the weight and food intake of the animals. These results are the first to show that even a short-term environmental stimulus such as acute and intense stress can have neuroplastic, inflammatory, functional and metabolic consequences on the adult hypothalamus. The hypIn recent decades, the hypothalamus has emerged as a new neurogenic area, containing new neurons generated after early development . The preThe effects of stress on hippocampal neurogenesis are widely known, and it has been described that stress strongly suppresses adult hippocampal neurogenesis , as thesOn the other hand, in the hippocampus, although not entirely conclusive, it appears that changes in neuroinflammation mediated primarily via microglial cells may play an essential role in regulating neurogenesis . HoweverSince the study of acute stress-induced effects can provide valuable information on the mechanisms involved in the stress response and pathological responses to stress, we set out to study the neurogenic changes and neuroinflammatory response in the hypothalamus as a consequence of exposure to acute and intense stress, which can act as a model of posttraumatic stress disorder (PTSD), an anxiety disorder associated with biological disturbances, with the hypothalamus being a particularly affected structure . We have2.2.1.All animal procedures were conducted following the European regulations for animal research  No. 1946/2003) and Spanish National Guidelines for Animal Experimenting , approved by the University of Malaga Ethical Committee (CEUMA 51-2018-A) and corroborated by the Agriculture, Farming, Fishing and Sustainable Development Council (10/06/2019/114).2.1.1.ad libitum. Animals were caged individually. The experiments were carried out in the morning from 9:00 to 13:00.For this experiment, a total of 16 male C57BL/6 J mice that were 3 m.o.  were used following standard housing conditions  with food and water administered 2.1.1.1.n = 6) suffered a unique stress episode for 2 h, whereas the control animals  remained home-caged  paradigm. The stress animals  (50 mg/kg in NaCl 0.9%) was injected on Day 1 into all groups after the stress procedure. This was done for the purpose of identifying cells newly generated after stress.2.1.1.4.Animals were anaesthetized using 200 mg/kg sodium pentobarbital (ip. injected), and the circulatory system was washed using PBS, pH 7.4. Later, animals were perfused using a 4% PFA solution for fixation. Brains were extracted from the skull and left for postfixation for an additional 24 h at 4°C. They were changed to PBS + 0.02% sodium azide at 4°C.2.1.2.2.1.2.1.Brains were embedded in 4% agar prior to sectioning using a Leica VT1000S vibratome. Whole brains were sliced in coronal 40 μm thick slices and stored in six series. Thus, each section was 240 μm apart from the next section within each series for the histological analysis. Sections were stored in cryoprotectant solution  at −18°C until further use.2.1.2.2.Several histological markers were analysed using a rabbit anti-Iba-1 IgG antibody for microglia , a rat anti-BrdU IgG antibody  for new cells and a goat anti-doublecortin IgG (DCX) antibody  for immature neuron identification. The following biotinylated secondary antibodies were used at a 1:1,000 dilution: goat anti-rabbit IgG , rabbit anti-rat IgG , and donkey anti-goat IgG .2O2, 10% methanol in PBS) by agitation for 45 min away from light at room temperature. After three 10 min washes in PBS, tissue saturation was followed by a 1 h incubation in PST . Tissue was later incubated in primary antibody solution in PST o/n at 4°C with agitation. The next day, the primary antibody solution was removed, and after three washes in PBS, the secondary antibody was added and incubated for 90 min with agitation at room temperature. After three washes in PBS, an ExtrAvidin-Peroxidase solution  was incubated for 1 h at room temperature with agitation. Finally, after washing the tissue three times, the brain slices were visualized using 2.5% 3,3-diaminobenzidine (DAB) and 0.1% H2O2 in PBS for 6.5 min. Later, slices underwent three quick washes and three 10 min washes. For DCX, DAB was supplemented with 0.4% NiCl2 to intensify staining. Slices were mounted on a gelatine-coated slide following the rostrocaudal axis. For Iba-1 slides, a contrast staining was performed by staining for 5 s in 0.3% toluidine blue. Slides were dehydrated and mounted using Eukitt mounting media. For the negative control, the primary antibody was omitted.First, slices were washed in PBS three times for 10 min with agitation at room temperature. For the BrdU protocols, an additional step of antigen retrieval was necessary. It consisted of a 15 min incubation in 2 N HCl at 37°C followed by three quick PBS washes and three additional 10 min washes. Then, endogenous peroxidase was inactivated  and Alexa Fluor 647 donkey anti-rabbit IgG  were used at a 1:1,000 dilution. After secondary antibody incubation and during the second wash, sections were incubated with DAPI   for 20 min followed by a regular PBS wash for 10 min. Sections were imaged using confocal microscopy .2.1.2.3.For each animal, up to seven slices were studied to account for three hypothalamic regions: paraventricular nucleus (PVN), ventromedial nucleus (VMN) and arcuate nucleus (ARC) following Paxino’s Atlas reference . Section2.1.3.n = 8). After administration of the thymidine analog BrdU after stress, proliferation was measured by manually counting cells on a bright field microscope ). The average number of BrdU+ cells per section was determined. DCX+ cells were manually identified and counted using the same microscope setup.For this study, four animals per group were randomly selected . For each slice, a 10 μm range was scanned, and that stack was combined into one single image.Slides were scanned at ×20 magnification using an Olympus VS120-5S and 2.1.4.2.N = 12). Using ImageJ Fiji , density and regularity index. The regularity index was calculated by dividing the distance’s standard error by the total number of microglia in that area  in the three hypothalamic nuclei, a total number of 10 animals was used. For Control-Stress comparison, a number of 6 animals for group was included . As a quantitative approximation of microglial processes, a threshold analysis was performed , following a stress procedure similar to the histological study but with different times of perfusion (1 h or 24 h after stress)  . Animals2.2.2.™ Multiplex Immunoassay  was conducted following the manufacturer’s instructions. Reads were performed using a Luminex™ instrument . Lower limits of quantification were 0.470 pg./mL for IL-6, 0.313 pg./mL for TNF-α and 18.768 pg./mL for IFN-γ. Statistical analysis was performed using the software StatSoft Statistica 8 and one-way ANOVA (p < 0.05). A post hoc LSD Fisher analysis was performed to determine differences among groups.Inflammatory cytokines  were measured in the left hypothalamus. Brain tissue was homogenized in lysis buffer  and then centrifuged at 14,000 × g for 15 min at 4°C for supernatant recovery. The lysate was brought to a concentration of 7 μg/mL total protein. Next, a ProcartaPlex2.2.3.Right hypothalami from four animals per group were processed for protein extraction using TEAB-SDS buffer and centrifuged at 16,000 × g for 10 min at 4°C to recover the supernatant. The protein concentration was calculated by the BCA method and adjusted to 1 μg/mL. Protein extracts were readied using an isobaric tandem mass tag procedure . Samples were fractioned using a reversed-phase column in a high pH and a Thermo Scientific Pierce High pH Reversed-Phase Peptide Fractionation Kit, obtaining 8 fractions per sample for better resolution.™ (Thermo Fisher Scientific) ionization source and Easy-nLC 1200 UHPLC system (Thermo Fisher Scientific) was used for mass identification and relative quantification of proteins with a shotgun procedure. The SwissProt database for Mus musculus (ver 2017.10.25) was used to identify the 4,529 proteins found. Identification criteria also included those matched to a “Master” protein, an FDR < 0.01 and at least 2 unique peptides found for the protein, which reduced the total number of proteins to 1,850. For data analysis, the level of each protein was normalized to the total peptide concentration and later normalized to all the rest of the proteins. Ratios for experimental and control samples were calculated using the protein abundance-based method. Selection criteria for proteins included p < 0.05 and FC (fold change) > 1.200 for overexpression and FC < 0.833 for repression after stress  coupled with an EASY-Sprayr stress .2.2.4.p = 0.001 and FDR q-value < 0.05 for a restrictive interpretation of data) to obtain functional networks for protein clusters.Proteins meeting the criteria outlined above were analysed by STRING 11.0 , and a f2.3.Data compilation and mean/SEM calculation were performed using Excel 2016. T-student tests were used for two-group comparisons (control vs. stress), whereas one-way ANOVA tests were used to compare the three regions in the control group. Due to inter-individual variability in feed intake and body weight in animals, for food intake and body weight, ANCOVA was performed to determine whether weight differences were due to the treatment and not animal variability prior to the stressor. Data collected prior to stress were treated as a covariate, and differences between the control and stress groups after stress were analysed. These analyses were performed using StatSoft STATISTICA 8. For all tests, a 95% confidence interval was set. Outlier values were removed from the analysis using IBM SPSS Statistics 20.n = 12) was used for the analysis and tested for sampling adequacy by Bartlett sphericity and the statistical adequation of the samples. The Kaiser–Meyer–Olkin sample adequation index (KMO) was studied and considered for values >0.5  with varimax rotation was performed to make associations with all different datasets to group them into factors and study their relationship . Input dues >0.5 . The resp ≤ 0.05.Data were considered statistically significant at 3.3.1.F = 10.993, p = 0.009, N = 12], with no significant effect observed on the prestress food intake   = 14.516, p = 0.004, N = 12], and there was a significant difference between pre- and post-stress measurements when controlling for experimental groups   = 0.430, p = 0.682, N = 8] or in the VMN   = 3.239, p = 0.017, N = 8]. The periventricular region of the hypothalamus (PE) was of interest to find that the majority of BrdU+ cells were arranged just below the ependymal layer and located along the entire wall of the third ventricle  = 17.087, p < 0.001, N = 8]  = 4.550, p = 0.019, N = 10]  = 4.144, p = 0.027, n = 10].The cell morphology study showed differences in cell shape and distribution among the PVN, VMN and ARC in control animals . More sp N = 10]  and a le N = 10]    = 8.850, p = 0.001, N = 10]  = 8.670, p = 0.001, N = 10]  = 4.159, p = 0.027, N = 10]  = 5.452, p = 0.010, n = 10] , p > 0.05, N = 12] , p < 0.05, N = 12] , p < 0.05, N = 12], and only two of the morphological parameters were altered, namely, solidity  and circularity   = −5.220, p < 0.001, N = 12] , and therefore factors could not be extracted . However3.6.According to the high sensitivity molecular analysis performed in brain protein extracts, basal hypothalamic values for the studied chemokines were 5.40 ± 0.36 pg./mL for IL-6, 11.85 ± 0.85 pg./mL for IFN-γ and 5.32 ± 0.36 pg./mL for TNF-α. Thus, the most abundant chemokine under basal conditions was IFN-γ, which showed double the levels of the other inflammatory proteins.F = 10.449, p = 0.001, N = 20], namely, in the 1 h stress group, which was higher than that of the rest of the groups . This indicated there was a significant peak in IL-6 1 h after acute stress, which returned to basal levels 24 h after stress  = 12.203, p = 0.132, N = 20] nor TNF-α  showed significant effects in the interactions of Group or Time or in the individual effects of each variable (p > 0.05) (Neither IFN-γ [ > 0.05) ,D.3.7.Shotgun proteomics revealed that there was already a change in some protein levels after the acute stressor. After 1 h had passed, the animals showed increased expression of three proteins encoded by the genes Anks1b , Cpne6 and Camkv . No reprAfter network enrichment, nodes were added to the network by STRING. Topological analysis for these nodes revealed highly connected regions that qualified as clusters, leaving some of the enriched nodes provided by STRING behind. Seeds for each cluster were also identified. Each cluster was given a score based on topology, and the proteins (nodes) were classified into each cluster .Based on these results, a functional analysis was obtained for the protein level changes 1 h and 24 h after stress as a more comprehensive and integrative result for the obtained proteins. Proteins that changed 1 h after stress seemed to be only distantly related to each other, as three of the five functional clusters found  were deeFor the 24 h poststress groups, only overexpression Cluster 4 was found to be insignificant for the enrichment analysis. Functional clusters originating from overexpressed and repressed proteins interacted with each other to form a functional network . Alterat4.The hypothalamus is a core structure in regulating the neuroendocrine system and controls key physiological processes such as homeostasis and metabolism . In receOur data revealed that acute stress reduced proliferation in the PE and ARC. Moreover, acute stress reduced the number of DCX+ cells in the parenchyma of all studied areas, indicating a negative effect on hypothalamic neurogenesis. The hypothalamus is a core structure in the regulation of the neuroendocrine system and controls key physiological processes such as homeostasis and metabolism . ConsequHowever, how stress impacts hypothalamic neurogenesis remains unknown. Recent studies, which focus on other neurogenic regions, indicate that changes in inflammation, mediated primarily via microglial cells, may play an essential role in this process . MicroglMoreover, cytokine determination revealed a significant increase in the level of IL-6 1 h after stress. In this sense, although IL-6 may act as both proinflammatory and anti-inflammatory molecule, acute stress is an “injury-like stimuli,” under which IL-6 can act as a pro-inflammatory cytokine . Marked Microglia may act as injury-responsive mediators of hypothalamic dysfunction. Environmental factors modulate microglia, which can regulate brain function, namely, neurogenesis, which participates in different stages and is necessary for proper adjustment of the process . AlthougOn the other hand, we tried to determine whether proteomic changes take place at 1 h and 24 h after the application of WIRS. The data revealed that WIRS induced changes in the hypothalamic proteome, modifying the abundance of three and four proteins after 1 h or 24 h of stress application, respectively.The modified protein data revealed that 1 h after stress, the levels of proteins were increased, including Cpne6, Camkv and AIDA-1 (Anks1b). These proteins have been directly or indirectly linked to calcium regulation, neuroplasticity and transcriptional activity . More spOn the other hand, among the proteins under- or overexpressed 24 h after the application of the stressor, the Hba protein, a subunit of hemoglobin, is noteworthy. Expression of this protein in various brain regions  has been linked to the regulation of oxidative stress and has been identified in various neurodegenerative diseases . The funIn conclusion, our data revealed that following an acute stressor, particularly an intense stressor that may be considered to establish an animal model of PTSD, many changes that provide valuable insights into the mechanisms involved in the impact of the stress response on the physiologically relevant hypothalamus were initiated . Thus, ohttps://dx.doi.org/10.24310/riuma.26229.The data presented in this study are deposited and made publicly available in: Dataset Orbitrap Raw-Data Stress Hypothalamus(1h-24h): The animal study was reviewed and approved by University of Malaga Ethical Committe (CEUMA 51-2018-A) and corroborated by the Agriculture, Farming, Fishing and Sustainable Development Council (10/06/2019/114).MII-L, AN-Q, and EZ-I: procedures with animals. MII-L, AN-Q, and PC-P: immuno assays and cell counting. AN-Q and MII-L: hippocampal molecules analysis. MC, JM, CP, and MP-M: design and supervision of the experiments, and draft of the article. MII-L, AN-Q, CP, and MP-M: writing of the article. CP and MP-M: funding for the study. All authors contributed to the article and approved the submitted version.This study was supported by Consejería de Conocimiento, Investigación y Universidades, Junta de Andalucía P20_00460-Co-financing by the European Regional Development Fund (ERDF/FEDER) to CP; Ministerio de Ciencia e Innovación—Plan Nacional I+D+I from Spain Grant PID2020-117464RB-I00 funded by MCIN/AEI/10.13039/501100011033 to CP and MP-M; FEDER/Junta de Andalucía—Proyectos I+D+I en el marco del Programa Operativo FEDER Andalucía 2014-2020 (UMA20-FEDERJA-112) to CP and MP-M.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Tritrichomonas foetus and Trichomonas vaginalis are extracellular flagellated parasites that inhabit animals and humans, respectively. Cell division is a crucial process in most living organisms that leads to the formation of 2 daughter cells from a single mother cell. It has been assumed that T. vaginalis and T. foetus modes of reproduction are exclusively by binary fission. However, here, we showed that multinuclearity is a phenomenon regularly observed in different T. foetus and T. vaginalis strains in standard culture conditions. Additionally, we revealed that nutritional depletion or nutritional deprivation led to different dormant phenotypes. Although multinucleated T. foetus are mostly observed during nutritional depletion, numerous cells with 1 larger nucleus have been observed under nutritional deprivation conditions. In both cases, when the standard culture media conditions are restored, the cytoplasm of these multinucleated cells separates, and numerous parasites are generated in a short period of time by the fission multiple. We also revealed that DNA endoreplication occurs both in large and multiple nuclei of parasites under nutritional deprivation and depletion conditions, suggesting an important function in stress nutritional situations. These results provide valuable data about the cell division process of these extracellular parasites.IMPORTANCE Nowadays, it's known that T. foetus and T. vaginalis generate daughter cells by binary fission. Here, we report that both parasites are also capable of dividing by multiple fission under stress conditions. We also demonstrated, for the first time, that T. foetus can increase its DNA content per parasite without concluding the cytokinesis process (endoreplication) under stress conditions, which represents an efficient strategy for subsequent fast multiplication when the context becomes favorable. Additionally, we revealed the existence of novel dormant forms of resistance (multinucleated or mononucleated polyploid parasites), different than the previously described pseudocysts, that are formed under stress conditions. Thus, it is necessary to evaluate the role of these structures in the parasites' transmission in the future. Tritrichomonas foetus (from class Tritrichomonadea and order Tritrichomonadida) has a worldwide distribution, and is the causative agent of bovine tritrichomonosis, a venereal disease that causes early embryonic death, abortion, and infertility  causes trichomoniasis, a common human sexually transmitted infection linked to pelvic inflammatory disease, adverse pregnancy outcomes, and an increased risk of HIV, papillomavirus infection, and cervical or prostate cancer . Historically, the Parabasalia have been treated as 2 groups: the “trichomonads” up to 6 flagella), and the “hypermastigotes” (multiflagellate) , 2. Tricertility , 4. T. f animals , 6. On t flagella animals . Trichome cancer –11.T. foetus and T. vaginalis claimed that the mode of reproduction is exclusively by binary fission , and despite being an integral part of their life cycle, there is a lack of evidence concerning its potentially associated functions  cell-cell fusion (syncytium); or (ii) multiple rounds of nuclear division within a common cytoplasm (coenocyte) , 17. It unctions –23. Furtost cell . Specifirowazeki , 26. Spe to date , 28. Pse to date .Trichomonas augusta, and other trichomonad flagellates, as a normal phase of the life cycle .Here, we show that multinuclearity is a phenomenon regularly observed in different T. foetus were mostly observed during nutritional depletion, numerous cells with 1 larger nucleus has been observed under nutritional deprivation conditions. We also revealed that DNA endoreplication occurs in both large and multiple nuclei of parasites under nutritional deprivation and depletion conditions, suggesting an important function in stress nutritional situations. Taken together, our findings contribute to our understanding of T. vaginalis and T. foetus cell division, and provide the basis for further research in this area.Additionally, we demonstrated that nutritional depletion or nutritional deprivation led to different dormant phenotypes. Although multinucleated T. vaginalis and T. foetus divide by longitudinal binary fission, where a parent cell splits into 2 identical mononucleate daughter cells (T. foetus (K strain) contained multiple nuclei in some parasites under standard growth conditions  and 16.56 μm2 (RU384 strain). Based on the observed nuclei size differences, we subsequently evaluated whether T. foetus and T. vaginalis strains exhibited variation in their DNA content. To this end, the parasites were fixed, the DNA was stained with propidium iodide (PI), and the DNA content was measured by flow cytometry. We observed that T. foetus DNA content was heterogeneous: OLI strain showed 90.19%, 9.55%, and 0.25% cells with 1C, 2C, and > 2C nuclear DNA content, respectively. In T. foetus K parasites, we observed that 63%, 26.13%, and 10.95% of the cells showed 1C, 2C, and > 2C nuclear DNA content, respectively. V47 strain presented a 42% of cells with 2C DNA content. Likely, the difference between the cell count of 1 or 2 nuclei, and cells with twice the DNA content could be to the presence of parasites with nuclei with more than 1C DNA content. While T. vaginalis showed greater homogeneity in DNA content, in B7268 and G3 strains, most of the parasites (90%) presented 1C nuclear DNA content , a nonspecific fluorochrome that binds to the pseudocysts/cysts surface (T. foetus.The trophozoites (a polar and flagellated cell) can adopt a spherical shape with internalized flagella, known as “pseudocystic” under unfavorable environmental conditions . This psflagella . To conf surface . In conc surface . Our resT. foetus parasites (K and 62H strains) were synchronized by 12 h incubation in a media without serum , we found 18.7% and 6% of parasites with 4 nuclei (4N) and more than 4 nuclei (4N), respectively. In synchronized parasites (SP), we observed a 5.7% (4N) and a 0.3% (>4N); suggesting that the lack of nutrients enriched a population of parasites with 1 nucleus. At 24 h, 18% of T. foetus K parasites had 4N, and 2.4% had > 4N. At 36 h, the results were: 27.3% (4N) and 5.6% (>4N). T. foetus 62H strain showed 5.8% and 0.9% of parasites with 4N and > 4N, respectively at 24 h, and 0.3% of parasites with 4N at 36 h. Based on these results, we concluded that nutrient depletion induces the formation of multinucleate parasites, mainly in the T. foetus K strain.As shown in T. foetus K produced 34.15% and 50.2% of parasites with more than twice the DNA content (>2C), respectively, in parasites grown for 36 h without changing the media culture, and parasites grown in the absence of serum for 36 h. These results were consistent with previous results where, under unfavorable conditions, T. foetus K formed multinucleate structures. T. foetus 62H strain had 25.54% and 47.57% of parasites with more than twice the DNA content (>2C) in parasites grown for 36 h without changing the media culture, and parasites grown in the absence of serum for 36 h, respectively  for (HS+), and parasites grown in the absence of serum for 36 h (HS−), respectively. In the T. foetus 62H strain, the average nuclei size was 42.65 μm2 and 70.35 μm2 for parasites grown for 36 h without changing the media culture (HS+), and parasites grown in the absence of serum for 36 h (HS−), respectively. Interestingly, we concluded that nutrient depletion does not induce the formation of multinucleated parasites but induces an increase in the size of the nuclei in the T. foetus 62H strain . ectively . These fH strain .T. foetus K parasites were stained with CFW  (Trophozoite and pseudocyst forms of 2 phase) , 27. Hen2 phase) .Some organisms  regularly divide by multiple fission , 39. In T. foetus multinucleate parasites increased under unfavorable nutritional conditions. To evaluate if these multinucleate cells could produce several individual parasites when the media is restored, we transferred the T. foetus K parasites grown in the absence of nutrients (complete culture medium for 36 h or serum-free medium for 36 h) into fresh media and estimated growth rates. The parasites were counted at 36 h and 4 h after the changes in media, and we calculated the growth rate ; an anti-HA antibody, anti-alpha tubulin antibody, and DAPI for TvTSP6-HA parasites .To examine the cytoskeletal structures of multinucleated parasites during the egress, parasites flagella , were grT. foetus  was measured. As can be seen in T. foetus K parasites incubated in the water for 24 h were transferred to Diamond’s medium and incubated for 24, 48, and 72 h  was analyzed using propidium iodide (PI) staining by flow cytometry. As control, the DNA content of and 72 h . After 2and 72 h . We alsoand 72 h  divides and 72 h . These rE. histolytica, and in this organism, the formation of multinucleate cells is favored by the absence of nutrients  to determine the active DNA replication in parasites under stress conditions , different than the previously described pseudocysts, that are formed under stress conditions. In this context, we also consider that the change in shape of the parasite, from pyriform to spherical, could be due to osmotic shock, which should be considered in future analyses.Multinuclearity has been described as a highly relevant process in different organisms. The emergence of multinucleated cells in plants has been linked to an increase in cell size and, as a result, the organism's overall growth . In amebutrients . In Acanferation . Plasmodd phases . Additiod phases –66. HereTrichomonas augusta and other trichomonads  that these parasites use for fast and efficient multiplication when the optimal conditions are reestablished. In conclusion, multiple genome contents present in a single nucleus or distributed over multiple nuclei in a single cell could represent a survival and multiplication mechanism in these parasites in the face of unfavorable environmental conditions.A mechanism that could account for multinucleated cell formation is DNA endoreplication. During the endoreplication, the cells undergo DNA replication in the absence of subsequent cell division . This pr program . It has  program . Endorep program . Tumor p program . Endorepte cells . Here, wFinally, we consider that trichomonads could, therefore, represent an interesting model system to study endoreplication and polyploidy, their consequences, and potential advantages in stress response, development, and disease.T. foetus strains: K  , B7268 and CDC1132 or MSA1132 (high)  medium supplemented with 10% horse serum and 10 U/mL penicillin/10 μg/mL streptomycin (Invitrogen). Parasites were grown at 37°C and passaged daily. The TvTSP6-HA construct was generated and transfected into the T. vaginalis CDC1132 strain as previously described ( Brazil) , OLI, 972 (high) . T. foetescribed .T. foetus strains, as well as G3 and B7268 T. vaginalis strains. For these experiments, 1 × 105 trophozoites were inoculated in 8 mL of TYM medium, and incubated at 37°C for 72 h. After inoculation, cell counts were recorded at the following times: 20 h, 25 h, 43 h, 48 h, and 67 h using a Neubauer hemocytometer. Growth rates were determined as the natural logarithm of the change in the density of parasites per milliliter at time t compared with that at time zero  by the following equation: growth rate = [lnCC (t) – lnCC (0)]/(t − 0), where CC (t) and CC (0) are the parasites counts per milliliter at time t and time zero, respectively, and t is the time of incubation  labeling. Parasites in the absence of host cells were incubated at 37°C on glass coverslips for 4 h, then were fixed and permeabilized in cold methanol for 10 min. The cells were washed three times in PBS, and incubated with a 300 nM DAPI stain solution for 5 min, protected from light. After 3 washes with PBS, the coverslips were mounted onto microscope slides using fluoromount mounting media. The parasites' nuclei were stained with DAPI for the nuclear size analysis. The nuclear area in μmParasites were pipetted onto a glass slide, air dried, and fixed in methanol for 10 min at room temperature. Slides were washed three times in PBS for 5 min each time, and incubated with 0.01% Calcofluor White (Sigma) in PBS, pH 7.2, for 30 min at 26°C. The slides were then washed three times in PBS, mounted on fluoromount, and observed under a phase-contrast and fluorescence microscope (Zeiss Axio Observer 7 inverted fluorescence microscope).Parasites, grown in different conditions, were incubated at 37°C on glass coverslips for 4 h, and then fixed and permeabilized in cold methanol for 10 min. The cells were washed and blocked with 5% fetal bovine serum (FBS) in PBS for 30 min, and incubated overnight with the primary antibody diluted in PBS plus 2% FBS. Primary antibodies used in the different immunolocalization experiments were anti-HA (1:500) (Covance), anti-tubulin (1:500), anti-BrdU (1:300), and anti-PCNA monoclonal antibody (Abcam) (1:400). Coverslips were washed with PBS, and then incubated for 1 h at room temperature (RT) with a 1:5000 dilution of Alexa Fluor conjugated secondary antibody (Molecular Probes). The coverslips were mounted onto microscope slips using ProLong Gold antifade reagent with DAPI or 4′, 6′-diamidino-2-phenylindole (Invitrogen). All observations were performed on a Zeiss Axio Observer 7 (Zeiss) inverted fluorescence microscope. For image processing, Adobe Photoshop (Adobe Systems) was used.6) were harvested and fixed in 5 mL of ice-cold 100% EtOH at 4°C overnight. Following that, each sample was washed in 1 mL of PBS containing 2% vol/vol horse serum (HS), resuspended in 1 mL of PBS,180 μg/mL RNase A to digest RNA and 2% vol/vol HS, and incubated for 30 min at 37°C. Then, samples were stained with a 25 μg/mL propidium iodide (PI) solution, and incubated for 30 min at 37°C prior to flow cytometer analysis. Flow cytometry analysis was carried out using a FACS Calibur flow cytometer  equipped with a dual laser system (15 mW 488 nm argon ion laser and a 635 nm red diode laser). For measurement of DNA content, cells were excited with 480 nm light, and emission was measured through 585/42 . Data from 20,000 cells was recorded, and these sets were analyzed using the FlowJo 7.6 software. Correlation between the light scattering properties of cells with their DNA content was performed by setting electronic gates on the forward scatter (FSC) and side scatter (SSC) profiles, and checking for DNA content of every gate.To determine DNA content and cell size, parasites (5x10T. foetus parasites (K and 62H strains) were synchronized by serum starvation for 12 h to increase the abundance of uninucleate parasites in culture media. Then, the parasites were grown in the presence of serum (without media change), and samples were taken every 12 h until their death phase. Samples were fixed, and the nuclei were stained with DAPI and quantified by fluorescence microscopy. To evaluate the DNA content in low amounts or depletion of nutrients, we analyzed the T. foetus K and 62H strains in the following culture conditions: UP as a control, SP for 12 h, parasites grown for 36 h without change media (HS+), and parasites grown in the absence of serum for 36 h (HS−). The DNA content was determined by flow cytometry.For depletion of nutrients, For the deprivation of nutrients assay, the parasites were incubated in water for 24 and 48 h at 37°C. Afterwards, these samples were fixed, and the nuclei were stained with DAPI and quantified by fluorescence microscopy. The DNA content was determined by flow cytometry.To evaluate reversibility of multinucleated parasites induced by nutrient depletion, parasites were grown in complete culture medium, as well as in serum-free medium for 36 h, and then recovered in fresh medium in the presence of serum. Then, the parasites were counted in a Neubauer hemocytometer at 4 h after medium change, and the growth rates were calculated.To analyze whether the formation of spherical structures formed during nutrient deprivation is a reversible process, parasites cultured for 24 h in water were centrifuged and grown in standard culture medium for 24, 48, and 72 h at 37°C. The parasites were counted in a Neubauer hemocytometer, and the percentages of spherical parasites and trophozoites were calculated.T. foetus serum-starved parasites (12 h) were incubated with BrdU for 2 h. The parasites were fixed for 15 min in 4% paraformaldehyde at RT. The cells were washed with PBS, and incubated with 2 M HCl for 20 min at RT. Then, we add 0.1 M sodium borate pH 8.5 for 2 min at RT. The parasites were washed with PBS, and permeabilized with 0.2% Triton X-100 and 3% BSA in PBS for 5 min, washed three times with PBS/BSA for 10 min each, and incubated with anti-BrdU antibody diluted in PBS/2% FBS. Then, the parasites were washed three times with PBS, and incubated for 1 h with the secondary antibody. Finally, we washed the parasites three times, for 10 min each, with PBS, and added DAPI to stain the DNA.The parasites were incubated in TYM medium with colchicine (1.5 mM) dissolved in dimethyl sulfoxide (DMSO). The cells were maintained under drug pressure for 18 h at 37°C. All reagents were purchased from Sigma. Control experiments were performed using trophozoites grown with 1% DMSO. Afterwards, we determined the relative cellular DNA content. Parasites were fixed in 5 mL of ice-cold 100% EtOH, and incubated at 4°C overnight. Following that, each sample was washed in 1 mL of PBS containing 2% vol/vol HS, resuspended in 1 mL of PBS,180 g/mL RNase A, and 2% vol/vol HS, and incubated for 30 min at 37°C. Then, samples were stained with a 25 μg/mL propidium iodide (PI) solution, and incubated for 30 min at 37°C prior to flow cytometer analysis. Also, the nuclei were stained with DAPI and observed by a fluorescence microscope.


The Carpentier‐Edwards PERIMOUNT Magna Ease valve is a third‐generation bioprosthesis for aortic valve replacement (AVR). This is a postapproval study reporting on its 8‐year outcomes.Adults undergoing AVR with the Magna Ease valve between October 2007 and December 2012 were enrolled for this prospective, nonrandomized, single‐arm, and multicenter study. Assessments occurred preoperatively, at hospital discharge, 6 months, 1 year, and annually thereafter for up to 8 years. Outcomes included safety endpoints, hemodynamic performance, and New York Heart Association (NYHA) functional class.2, the mean gradient was 14.8 ± 8.3 mmHg, and 88.6% of patients had no or trivial aortic regurgitation.Of the 258 study patients, 67.5% were in NYHA Class I or II, and 32.5% were in NYHA Class III or IV at baseline. Concomitant procedures were performed in 44.2%. Total follow‐up was 1597.6 patient‐years, and median follow‐up was 7 years (interquartile range: 5.5–8.0 years). Eight years following AVR, the functional class remained improved from baseline with 93.9% in NYHA Class I/II and 6.1% in NYHA Class III; 38 deaths had occurred, 8 of which were valve related; freedom from all‐cause mortality was 80.7% ; freedom from valve‐related mortality was 95.8% ; freedom from reintervention, explant, major bleeding events, and structural valve deterioration was 89.8% , 94.8% , 85.1% , and 90.1% , respectively; effective orifice area was 1.5 ± 0.5 cmThis study demonstrated satisfactory safety and sustained hemodynamic and functional improvements at 8 years following AVR with the Magna Ease valve. AVRaortic valve replacementCECClinical Events CommitteeCIconfidence intervalNSVDnonstructural valve dysfunctionNYHANew York Heart AssociationSVDstructural valve deterioration1Stenosis and regurgitation are common conditions affecting the aortic valve.The effectiveness and durability of the Carpentier‐Edwards PERIMOUNT valve (Edwards Lifesciences), a stented bovine pericardial bioprosthesis, have been well described.22.1ClinicalTrials.gov NCT01171625). The FDA required data from at least 101 patients over 8 years. The study protocol projected that at least 225 patients would need to be enrolled at the beginning to achieve the required number at an 8‐year follow‐up and the study was terminated when this number was reached. The study protocol complied with ISO 14155:2011; European Medical Device Directive 2007/47/EC; and MedDev 2.12‐1, 2.7.4, and 2.12.2. The ICH E6 GCP Good Clinical Practices was also used for guidance.This study was a prospective, nonrandomized, single‐arm, postapproval, multicenter, 8‐year study of the Magna Ease valve , nonstructural valve dysfunction (NSVD), reintervention, and valve explant. All safety endpoints and serious adverse events were defined as per Akins et al.Effectiveness endpoints included the proportion of patients in New York Heart Association (NYHA) Functional Class I or II at 8 years, and hemodynamic performance as assessed by echocardiographic parameters: effective orifice area, mean gradient, and aortic regurgitation  severity. Hemodynamic data were collected and scored by the individual sites. Collection of these data in Years 3 and 7 was not mandated and therefore not reported.2.4The investigational sites were responsible for the accurate collection and recording of the clinical data. Edwards Lifesciences, the study sponsor, monitored and aggregated the clinical data, then analyzed them per the study protocol and statistical analysis plan. Summary statistics include absolute and proportional data for categorical variables and mean ± standard deviation for continuous variables. Early safety events were defined as those occurring within 30 days of the index procedure and were reported as the number of patients with an event divided by the number of implanted patients. Late events represented those occurring beyond 30 days postoperatively and through 8 years (postoperative days 2922). Actuarial Kaplan–Meier analyses were undertaken on each of the safety endpoints and reported with 95% confidence intervals (CIs). SAS version 9.4 was used for all statistical analyses.33.1A total of 283 patients across 14 investigational sites consented to participate. Of these, 258 patients met the eligibility criteria and received the Magna Ease valve, the outcomes of whom are reported here. Table 3.2Overall, 258 patients underwent a total follow‐up of 1597.6 patient‐years: median 7.0 years (5.5–8.0 years). At study closure, 103 patients (39.9%) had completed the 8‐year follow‐up and a further 67 patients (26.0%) were on protocol with the study valve in place.3.3One death (0.4%) occurred within the early postoperative period, and it was not valve related. Thirty‐seven deaths occurred late, eight of which were adjudicated to be valve related: two endocarditis, one thromboembolism/stroke, and five other unknown causes. Figure 3.4Safety endpoint events were experienced by 109 (42.2%) patients and are summarized in Table n = 4), SVD (n = 9), NSVD (n = 1), and major paravalvular leak (n = 1). Freedom from study valve reintervention was 89.8%  at 8 years. Freedom from explant was 94.8%  at 8 years.Study valve reinterventions were carried out in 18 patients, including 11 explants, 6 valve‐in‐valve insertions, and 1 repair procedure without explant. There were three early reinterventions, all explants that occurred during the index procedure due to complications during surgery not related to the study valve. The reasons for the 15 late reinterventions were endocarditis  at 8 years; freedom from major bleeding events was 85.1%  at 8 years.3.5Figure 3.6Of the 14 patients with SVD, 3 underwent explant, 7 had valve‐in‐valve procedures, and 4 cases were being monitored at study closure. Freedom from reintervention due to SVD was 93.6%  at 8 years.3.7Figure 4This is the first prospective evaluation of the Magna Ease bioprosthesis for surgical AVR. These results demonstrate good safety and effectiveness, with functional and hemodynamic outcomes that remain consistent over the 8‐year follow‐up.4.1p < .01).Kaplan–Meier analyses showed freedom from all‐cause and valve‐related mortality at 8 years were 80.7%  and 95.8% , respectively. A recent retrospective study of AVR with the Magna Ease valve, with a median follow‐up of 4.5 years, revealed overall survival at 12 years of 54% , although with limited numbers of patients.4.2Early and late complications in this study were consistent with others.4.3p < .0001),The mean gradient remained acceptable at 8 years: 14.8 ± 8.3 mmHg compared with 12.2 ± 5.0 mmHg at 1 year and was consistent with findings of a recent study.The hemodynamic improvement seen in this study resulted in a sustained improvement in NYHA functional class for nearly two‐thirds of patients.4.4Freedom from SVD was 99.1%  at 5 years and 90.1%  at 8 years. The 8‐year rate is slightly lower than the 10‐year freedom from SVD rates with the Magna Ease valve reported by Bourguignon et al.p = .009).p < .001).Freedom from reintervention due to SVD was 99.1%  at 5 years and 93.6%  at 8 years. Rates reported for the Trifecta valve were 97.3%  at 6 years.In an effort to reduce SVD and improve the durability of bioprosthetic valves, Edwards Lifesciences has developed RESILIA tissue with advanced anticalcification technology, but durability data beyond 5 years with this tissue are still awaited.5his study has some limitations. While it includes outcomes from 14 centers and used an independent CEC to adjudicate safety events, an echo core lab was not used, potentially introducing variability, as echocardiographic data were collected and evaluated by individual centers. Second, this study did not include a comparator study arm to compare these Magna Ease valve outcomes with those of other contemporary valves.6This is the first multicenter, prospective cohort study of the Magna Ease valve, with CEC adjudication of all safety events. The Magna Ease valve demonstrated satisfactory freedom from mortality and valve‐related complications requiring reintervention and sustained improvement in hemodynamics at 8 years. These data support the continued use of this valve, adding to the growing body of evidence that the Magna Ease valve represents a standard of performance against which other surgical valves may be compared.Financial support from British Standards Institute and 3R Life Sciences: Steven Tsui. Shareholder in Edwards Lifesciences: Michael Rosenbloom. Honoraria for meetings from Edwards Lifesciences; owns stock in Kardium Inc.: James Abel. Consulting fees from Edwards Lifesciences: Axel Haverich. Financial support from Edwards Lifesciences, Medtronic, CryoLife Inc., Ethicon, Cambridge Medical Robotics, and LSI Solutions®: Joseph Zacharias. Consulting fees from Edwards Lifesciences: Gideon Cohen. Financial support from Medtronic and owns stock in Highlife: Rüdiger Lange. Gilbert Schorlemmer, Jeffrey Swanson, and Michael Moulton declare no conflict of interest.All study sites obtained Institutional Review Board (IRB) approvals: UBC‐Providence Health Care Research Institute, Vancouver, BC, Canada, #H11‐00994, 06/24/11; Providence Health and Services IRB, Portland, OR, USA, #10‐063B, 07/28/10; Sunnybrook Health Sciences Centre REB, Toronto, ON, Canada, #382‐2007, 02/21/08; Dignity Health IRB, Sacramento, CA, USA, #033588, 11/17/11; Atlantic Health IRB, Morristown, NJ, USA, #R09‐12‐004 (455058), 01/07/10; The Cooper Health System IRB, Camden, NJ, #09‐190EX, 02/05/10; Mountain Star St. Mark's Hospital IRB, Salt Lake City, UT, USA, #0221, 01/21/10; MHH Ethics Committee 30623 Hannover, Germany, #5083, 08/22/08; NHS National Research Ethics Committee, Papworth, Cambridge, UK, #08/H0304/34, 05/30/08; NHS National Research Ethics Committee, Blackpool, Lancashire, UK, #08/H0304/34, 07/23/08; Ethics Committee of the University Hospital Puerta de Hierro, Madrid, Spain, #892, 02/26/08; Ethics Committee of the Faculty of Medicine of the Technical University of Munich, Munich, Germany, #1913/07, 12/04/07; Ethics Committee of the Medical University Innsbruck A‐6020, Innsbruck, Austria, #EK 1 25.09.07, 01/29/08; and Western IRB, Olympia, WA, USA , #20110574. All study participants provided informed written consent.