Mammalian hybrids frequently form in nature between closely related species, and the majority are sterile due to the production of chromosomally unbalanced gametes. In Italy, the widespread wild boar has had negative consequences for free-range pig farming, which is considered the best practice for pig welfare and is a common method of farming most autochthonous pig breeds. This study aimed to analyze the sperm DNA integrity, sperm meiotic segregation and nuclear spatial organization in a boar–pig hybrid, for the first time, to evaluate its fertilizing capacity. The results show that the hybrid presented a high frequency (64%) of motile spermatozoa with a regular chromosome composition and a specific spatial distribution. This study underlines how fertile boar–pig hybrids represent a growing problem for conserving autochthonous pig breeds.Sus scrofa domesticus) and a wild boar (Sus scrofa ferus). Spermatozoa were also separated by a dual-layer (75–60%) discontinuous Percoll gradient, resulting in two fractions with a significantly better overall quality in the motile sperm fraction. These data were confirmed by FISH analysis also, where the frequencies of spermatozoa with a regular chromosome composition were 27% in total sperm fraction and 64% in motile sperm fraction. We also evaluated the nuclear architecture in all counted spermatozoa, showing a chromatin distribution changing when chromosome abnormalities occur. Our results demonstrate that the chromosome pairing has a minimal effect on the sperm segregation and semen quality of a boar–pig hybrid, making it fertile and harmful for the conservation of autochthonous pig breeds.A wide range of mammalian hybrids has recently been found by chance or through population-screening programs, but studies about their fertilizing capacity remain scarce and incomplete. Most of them are assumed to be sterile due to meiotic arrest caused by the failure of chromosome pairings. In this study, we evaluated both sperm meiotic segregation, by 2D fluorescent in situ hybridization (FISH) analysis, and sperm quality (Sperm Chromatin Structure Assay) by flow cytometer in a fertile boar–pig hybrid  originating from a Nero Siciliano pig breed ( Sus scrofa (Ssc) species [Sus scrofa domesticus, 2n = 38,XX/XY) and WB  seems to be very frequent, especially when they share the same area, grazing on high-altitude pastures and in forests [Over the last decade, several studies have reported a rising number of mammalian hybrids, originating by accident or design ,2,3 and  species . The hyb forests .With the introduction of assisted reproductive technology (ART), advanced techniques such as flow cytometry (FCM) have been applied in several studies to provide accurate and unbiased evaluation of sperm functions, such as sperm DNA integrity ,9,10. SpWB is characterized by both high reproductive potential (due to the untimely puberty)  and a hihttp://www.fao.org/dad-is/en/, accessed on 28 February 2021) and the herdbook of National Swine Breeders’ Association (ANAS). NS shows phenotypic resemblance to WB but has a smaller size , a darker coat and earlier sexual maturity. Moreover, it is a long-lived breed, disease-resistant and able to convert natural graze or poor food (roughage) into high-quality products (meat) [The NS is farmed in the Nebrodi Mountains (in the northeast of Sicily), and its population is increasing (6629 registered in 2020 vs. 900 recorded in 2001), according to Domestic Animal Diversity Information System (DAD-IS) data (s (meat) . Since ts (meat) ,19.In this study, we have analyzed the sperm DNA integrity, sperm segregation, aneuploidy and nuclear organization of one fertile boar–pig hybrid in both total (TSF) and motile (MSF) sperm fractions for the first time.During cytogenetic screening of 81 endangered local pig breeds, two NS boars (from the same farm) were found to have resulted in a boar–pig hybrid 2n = 37,XY rob . The twoThe evaluation of the quality and fertilizing ability of semen is an essential aspect in guaranteeing its “conservation value” and storing it in animal cryobanks. In this analysis, we included both a boar–pig hybrid 2n = 37,XY rob and a control DP 2n = 38,XY.v/v). Sperm cells collected were equilibrated for 90 min at 4 °C. After equilibration, sperm cells were diluted in the freezing medium Boarciphos B supplemented with 10% glycerol, 20% egg yolk and 2% Equex STM paste  (v/v), to achieve the final concentration of 500 × 106 sperm/mL that was then packed into 0.5 mL CBS High Security Straws  and sealed using polyvinyl alcohol powder. Semen straws were frozen in nitrogen vapor by placing on a rack at 7 cm above the nitrogen level for 15 min. Subsequently, straws were plunged into liquid nitrogen and stored at −196 °C in a liquid nitrogen tank.The epididymal semen samples were diluted using a two-step procedure: the first 50% was added ), followed by the second half ). In detail, sperm cells were flushed in a retrograde direction from the ductus deferens through the cauda epididymidis with a syringe loaded with approximately 4 mL of warmed (37 °C) Boarciphos A extender , supplemented with 20% of egg yolk  was split into two aliquots of 500 μL. One aliquot was evaluated as TSF. The second was subjected to a Percoll gradient to obtain an MSF, considered the highest-quality semen fraction. Then, 500 µL of the thawed semen (for each boar) was washed by centrifugation at 600× g for 8 min in 5 mL of a saline solution for washing spermatozoa (WS-PVA), consisting of 140 mM NaCl, 20 mM HEPES and 1 mg/mL polyvinyl alcohol (PVA), adjusted to pH 7.4.Frozen–thawed spermatozoa were separated through a dual-layer (75–60%) discontinuous Percoll gradient, following the protocol of Suzuki et al. with some modifications . For eacg for 5 min. After the centrifugation, the MSF obtained was resuspended in up to 3 mL of WS-PVA for washing centrifugation (900× g for 15 min). Supernatants were discarded and pelleted spermatozoa were resuspended in up to 3 mL of WS-PVA and washed twice by centrifugation (600× g for 8 min).The supernatant was discarded, and pelleted spermatozoa were resuspended in 1 mL of WS-PVA. It was overlaid on a two-step Percoll with the upper part of 60% and lower part of 75% concentrations in two 15 mL conical tubes from each boar. The tubes were centrifuged at 200× 5 spermatozoa were diluted in 200 μL of TNE buffer , pH 7.4) and added to 400 μL of an acidic solution . After 30 s, cells were stained with 1.2 mL of acridine orange solution . After 2.5 min, two replicates per sample were read on a flow cytometer. We quantified the type of DNA damage with the following parameters: (1) the extent of DNA denaturation, expressed as DNA fragmentation index (%DFI), which indicates the percentage of sperm cells outside the main sperm population with fragmented DNA; (2) the percentage of immature cells with reduced nuclear condensation (incomplete histone–protamine exchange), expressed as a percentage of sperm with high green (%HG) fluorescence.Sperm DNA fragmentation and chromatin condensation were assessed using the SCSA test by Evenson et al.  and furtData were acquired and analyzed using cytoSoft and IMV easySoft software , respectively. The latter created a %DFI and %HG histogram profile of the entire sperm population. Computer gates of the %DFI and %HG histogram were used to determine the %DFI and %HG.Preparation and decondensation of spermatozoa followed the protocol of Di Dio et al.  with somSsc lymphocyte R-banded metaphases to check their physical localization. The schematic representation of the translocation and localization of the BACs used in this study is illustrated in Each probe used in the study consisted of a pool of three bacterial artificial chromosomes (BACs) selected in a contig  to increase the size of the sequence covered by the probe (around 1 Mb) and thus the intensity of the FISH signals . We obtap-value of less than 0.001 was considered significant.The chi-square test  was usedp < 0.05.Data obtained from SCSA assessment were analyzed using the SAS package v 9.4 . The general linear model procedure (PROC GLM) was used to evaluate the Percoll separation’s efficiency on the SCSA sperm quality parameters. The model included fixed effects of the fraction and the animal. Results are given as adjusted least squares means ± standard error means (LSM ± SEM). We set the level of significance at Results relative to the sperm chromatin structure are shown in Sperm cells were successfully separated through the dual-layer (75–60%) discontinuous Percoll gradient, showing a higher subjective motility and lower %DFI in the motile sperm fraction. Considering the percentage of %DFI, a reduction in the DNA damage was noticed in the MSF fraction in both animals, and this reduction was significant in the hybrid sample. Hybrid boar showed higher values of %HG, representative of the percentage of immature spermatozoa with reduced nuclear condensation.Ssc chromosomes 15, 17 and rob. Segregation analysis was achieved on 10,000 spermatozoa  for TSF and MSF of the hybrid and 2000 for TSF and MSF of the control samples  and 17 , as shown in Ssc chromosomes) from rob spermatozoa, we considered all those that presented two detached signals as normal  detached signal was detected in the apical part of the sperm head (85%), the red (Ssc 17) detached signal was detected in the medial part of the sperm head (85%) and the green and red (attacked) or yellow signals were detected in the basal part of the sperm head (88%). We evaluated them in the control, finding the same localization for Ssc 15 and 17 chromosomes (96%) as shown in Furthermore, we evaluated the nuclear architecture of these chromosomes in all counted spermatozoa in specific regions: the green  have been broadly used for in vitro fertilization (IVF) and have been reported to have a positive correlation with sperm quality  ,34. In pMoreover, we found a positive correlation with alternate sperm segregations, proving that this method effectively reduced the percentage of spermatozoa carrying unbalanced chromosomal aberrations . Of the Ssc 15, 17 and rob in all sperm nuclei, not only to evaluate their nonrandom position but also to study sperm intranuclear localization chromosomes involved in a rob. These data are comparable with the results shown in pigs by Ducos et al. [We also analyzed the chromosome positioning of s et al.  and Rubes et al. .Ssc 1–18) and the sexual chromosome (X–Y) in a normal segregation. They demonstrated that all chromosomes are organized in specific chromosome territories, tracing Ssc 15 in the apical-medial and Ssc 17 in the medial-basal part of spermatozoa. We found the same medial localization of the Ssc 17 chromosome, but we localized Ssc 15 in the apical region . One probable reason could be the type of probe used for the analysis. Foster et al. used probes that painted the whole chromosome, producing larger signals due to the detection of entire chromosomes (20–30 Mb), while we used a pool of Bac probes with a smaller signal (1 Mb).The spermatozoa are highly differentiated cells resulting from the several-step process of spermatogenesis. During the stages following meiosis, chromatin undergoes structural reorganization, and DNA becomes very condensed and genetically inactive . The spaSsc 17 and Ssc 13, confirming our result for Ssc 17 in normal spermatozoa. Furthermore, they revealed a different localization of spermatozoa carrying the rob, probably induced by the fusion between the involved chromosomes. In our case also, we found a different localization  of spermatozoa carrying both the rob and any other abnormal segregation detected  studying the sperm segregation in a fertile hybrid, reporting also the percentage of each sperm fraction; (b) showing the correlation between the high-quality sperm fraction (selected by Percoll gradient according to DNA integrity) and a lower sperm aneuploidy percentage; and (c) showing the modification of nonrandom chromosome spatial organization in sperms carrying a CA.Finally, thanks to the cytogenetic monitoring of local pig breeds, CA can be easily detected, preserving the reproducers’ genetic selection.Further studies are needed for a better understanding of the correlation among motility, chromatin integrity and spatial chromosome distribution when a CA occurs.


Background: Data suggest that pediatric patients might react differently to influenza vaccination, both in terms of immunity and side effects. We have recently shown that using a whole virion vaccine with aluminum phosphate adjuvants, reduced dose vaccines containing 6 µg of viral hemagglutinin (HA) per strain are immunogenic, and well tolerated in adult and elderly patients. Here we show the results of a multicenter clinical trial of pediatric patients, using reduced doses of a new, whole virion, aluminum phosphate adjuvanted vaccine . Methods: A total of 120 healthy volunteers were included in two age groups . We used hemagglutination inhibition testing to assess immunogenicity, based on EMA and FDA licensing criteria, including post/pre-vaccination geometric mean titer ratios, seroconversion and seropositivity rates. Safety and tolerability were assessed using CHMP guidelines. Results: All subjects entered the study and were vaccinated (ITT population). All 120 subjects attended the control visit on Day 21 (PP population). All immunogenicity licensing criteria were met in both age groups for all three vaccine virus strains. No serious adverse events were detected and the vaccine was well tolerated by both age groups. Discussion: Using a whole virion vaccine and aluminum phosphate adjuvants, a reduction in the amount of the viral hemmaglutinin is possible while maintaining immunogenicity, safety and tolerability in pediatric and adolescent patients. Influenza, though often unrecognized, is a major cause of illness and hospitalization in children . In addiWe have previously shown that a monovalent, prepandemic influenza vaccine (A H5N1) is immunogenic, safe and well tolerated in children and adolescents, when formulated with inactivated whole virion, and adjuvanted by aluminum phosphate, even with a smaller than usual amount of active ingredient, 6 µg of viral HA/strain . FurtherIn the present trial, we studied a reduced dose, trivalent seasonal influenza vaccine, produced by the above principle , in pediatric populations, in terms of immunogenicity, safety and tolerability, based on criteria by the U.S. Food and Drug Administration , as well as the European Medicines Agency  ,12.This was a multicenter, stratified (for age: 3–11 years and 12–18 years old), prospective, uncontrolled study, conducted in the European Union  at 2 centers, between 18 October 2014 (enrollment of the first participant) and 9 December 2014 (last visit of the last subject participating). No changes were made to methods after trial commencement. The study protocol, the patient information sheet, the informed consent form and other appropriate study-related documents were reviewed by the Ethics Committee for Clinical Pharmacology of the Medical Research Council (ETT KFEB), and approved by the National Institute of Pharmacy of Hungary (OGYI) in accordance with domestic legal requirements. The study was conducted in accordance with the rules of good clinical practice. The conditions were in compliance with the Declaration of Helsinki . The stuParticipants were recruited by their primary care physicians. Subjects eligible for enrolment into this study were: male and female patients aged 3–18years, with power of attorneys mentally competent, able to understand and comply with all study requirements, able to give written informed consent prior to initiation of study procedures, in good health  and stable medical condition.Exclusion criteria included: pregnancy, hypersensitivity to eggs, chicken protein, thiomersal, formaldehyde, gentamycin, ciprofloxacin, neomycin or any other components of the vaccine, history of anaphylactic shock or neurological symptoms following administration of any vaccine; history of Guillain-Barre syndrome; malignant tumor, autoimmune disease, advanced arteriosclerotic disease, complicated diabetes mellitus, acute or progressive hepatic disease, acute or progressive renal disease, congestive heart failure; immunosuppressive therapy within 36 months prior to vaccination; concomitant corticosteroid therapy ; receiving immunostimulants; receipt of immunoglobulin, blood products and/or plasma derivates within 3 months prior to vaccination; HIV, HBV or HCV infection; fever of >37 °C within 3 days prior to vaccination; vaccine therapy within 4 weeks; influenza vaccination within 6 months prior to vaccination; experimental drug therapy within 4 weeks prior to vaccination; concomitant participation in another clinical study.The vaccine was a whole virus, aluminum phosphate adjuvanted, trivalent vaccine against seasonal influenza containing 6 µg of HA per virus strain. The influenza viruses included in the vaccine were grown in embryonated hen eggs, inactivated by formaldehyde, purified and concentrated, and absorbed on aluminum phosphate gel, as described in detail elsewhere . The virSubjects were assigned to one of the following age groups: participants aged 3–11 years received one 0.25 mL intramuscular injection of the investigational vaccine, containing 3 µg per virus strain, while patients 12–18 years of age were given one 0.5 mL injection of the vaccine, containing 6 µg per strain. Doses of the study vaccine administered were determined based on previous clinical trials by our group .All subjects were observed for 30 min after vaccination for any immediate reactions and were instructed to complete a diary card to record local  and systemic reactions , and axillary temperature starting on the day of vaccination and for each of the 7 days following the immunization. All adverse events were collected from the day of vaccination until day 120. All adverse events necessitating a physician’s visit or consultation and/or leading to premature study discontinuation and all serious adverse events were collected throughout the entire trial and data were reconciled at study termination visit.Baseline evaluations on Day 0 included obtaining demographic data, medical history, and performing a complete physical examination. Blood samples were drawn for baseline hemagglutination inhibition test (HI) for all three vaccine virus strains. Serum antibody titers against the vaccine virus strain were measured by HI, using chicken red blood cells (CRBC) and following standard procedures, as recommended by the WHO . BrieflyThe primary outcome measure was the immungenicity of the reduced dose vaccine containing 3 or 6 µg of HA per strain as measured by HI test 21 days after vaccination. We used HI testing to assess immunogenicity, based on EMA and FDA licensing criteria, including post/pre-vaccination geometric mean titer ratios, seroconversion and seropositivity rates ,12.The safety objective was to evaluate the tolerability and safety of the administration of the vaccine doses containing either 3 or 6 µg of HA of seasonal A/H1N1, A/H3N2 and B influenza antigens as determined by the licensing requirements of the European Union . No chanA sample size of at least 50 evaluable participants per group was selected based on CHMP licensing criteria  and baseIt was estimated that a number of 50 subjects would be necessary in each age group to have 80% power for detection of a minimum expected GMTR of 3.125-fold by Day 21–28 post-vaccination in case of all of the investigated influenza virus strains. This sample size was thus sufficient for detecting with 80% power a minimum expected seroconversion rate of 44.54% and a minimum expected seroprotection rate of 76.68%, for all of the investigated influenza virus strains. Considering an approximately 17% of drop-out (or one participant out of six), a total of 120 healthy volunteers were included, (sixty (60) subjects in each age group) in order to achieve at least a total of 100 evaluable subjects, meaning 50 subjects in each age group).Descriptive statistics  for age at enrolment were calculated overall and by age group. Distributions of subjects by gender and previous influenza vaccination status were summarized overall and by age group.HI antibody titers were determined at baseline, and at Day 21. HI titers were used to calculate seroconversion rates, seroprotection rates, and increase in GMTRs, according to EMEA and FDA guidelines ,12. DistIn order to confirm protective immunogenicity, at least one of the following three requirements had to be met in both aged groups: the rate of seroconversions or significant increase in antihaemagglutinin antibody titre is >40%; mean geometric increase >2.5; rate of subjects achieving an HI titre ≥40 >70% of subjects.All calculations are made with the R programming language for statistics version 2.15.0. No interim analyses were performed. No changes in the outcomes were made after the trial commenced. No interim analyses were performed.A total of 120 healthy subjects  were selected for inclusion in the study, and screened prior to vaccination. All subjects screened were found to be eligible, entered the study and were vaccinated (ITT population). All of the 120 subjects (60 subjects per age group) attended the control visit at Days 21–28, and their data were available and evaluated at Days 21–28 (PP population).The baseline characteristics, including age and gender distribution are summarized for the two vaccination groups in Both vaccine doses fulfilled all applicable immunogenicity licensing criteria by EMA and FDA guidelines for the age groups investigated for all three vaccine virus strains .Administration of the vaccine was well tolerated by the study subjects in both age groups. All possibly or probably related AEs occurred during the study were mild (37 cases) or moderate , and recovered without sequels. No serious and no severe possibly or probably related adverse event was observed. The vaccine proved to be safe at both doses, no vaccine-related clinically significant changes in the physical condition or vital signs of the volunteers were observed .Most currently available influenza vaccines are split virion or subunit vaccines, without adjuvants, containing 15 µg of HA per virus strain. However, a renewed interest in whole virion vaccines has recently developed, at least in cases when increased immunogenicity or cross protection was desired, such as with emerging new strains or during pandemics ,21,22,23We have previously shown that using inactivated whole virion vaccines and aluminum phosphate adjuvants, a reduction in the amount of the viral hemmaglutinin antigen is possible, while maintaining adequate immunogenicity, safety and tolerability profiles in adult and elderly patients in case of seasonal trivalent influenza vaccines . We have found similar efficacy with monovalent pandemic H1N1 and prepandemic H5N1 whole virion, aluminum adjuvanted influenza vaccines in adult, elderly, and pediatric patients ,17,27. AVaccine doses for children 3–18 years of age for seasonal influenza are currently recommended at 15–30 µg/strain  based on age and previous vaccination status . In the We believe that the acceptable reactogenity profile we have seen in the present and previous trials with whole virion influenza vaccines can be at least in part explained by the endotoxin content of the vaccine being well below the values permitted by the European Pharmacopia.We did not study children younger than 3 years of age, and thus, we do not know if our results can be generalized for them. Since we did not test the whole virion vaccine without adjuvants, we are unable to determine whether the immunogenicity results seen in the present trial at reduced doses were due to using a whole virion vaccine, using an adjuvant, or both. Our study recruited only white Caucasian subjects. Although there are currently no licensing requirements to indicate such, there is at least some published evidence that there might be race-related differences in the antibody response to influenza vaccination . Our goa


Iridoviridae, Parvoviridae, Alloherpesviridae, Papillomaviridae). Interestingly, we also detected a ‘core virome’ that was shared by all the organs and was mainly composed of Caudovirales, Microviridae and Circoviridae. These results show that tuna host a mosaic of viral niches, whose establishment, role and circulation remain to be elucidated.While planktonic viruses have received much attention in recent decades, knowledge of the virome of marine organisms, especially fish, still remains rudimentary. This is notably the case with tuna, which are among the most consumed fish worldwide and represent considerable economic, social and nutritional value. Yet the composition of the tuna virome and its biological and environmental determinants remain unknown. To begin to address this gap, we investigated the taxonomic diversity of viral communities inhabiting the skin mucus, gut and liver of two major tropical tuna species (skipjack and yellowfin) in individuals fished in the Atlantic and Indian Oceans. While we found significant differences in the virome composition between the organs, this was totally independent of the tuna species or sex. The tuna virome was mainly dominated by eukaryotic viruses in the digestive organs (gut and liver), while bacteriophages were predominant in the mucus. We observed the presence of specific viral families in each organ, some previously identified as fish or human pathogens (e.g.,  Marine animals live in close and complex interactions with an abundant and diverse assemblage of viruses, typically made up of bacteriophages and eukaryotic viruses. Both are suspected to play an essential role in the health of the host, either by contributing to its immune system ,2 or, coTuna are an interesting model for investigating the fish virome since they are (i) a keystone species and top predators in pelagic ecosystems, (ii) one of the most consumed fish species worldwide , and  in individuals of similar sizes sampled in the Atlantic and Indian Oceans. We sought to identify shared and specific viral taxa in these three key organs and to characterize the influences of species, sex and sampling site on the composition of their virome.Tuna. All skipjack  and yellowfin  tuna used in this study were sampled around fish aggregating devices (FAD), in the Atlantic  and Indian Oceans , during campaigns in July (10–11) and September (26–29) 2018, respectively. In total, 21 tuna were sampled in the Gulf of Guinea (6 skipjack and 15 yellowfin) and 27 off Reunion Island (18 skipjack and 9 yellowfin). In both locations the sampling and euthanasia of the fish were performed by professional fishermen working for the French National Research Institute for Sustainable Development (IRD) Exploited Tropical Pelagic Ecosystems Observatory (certified ISO 9001:2015). A hook and line were used, and the tuna always handled by the mouth using a clamp; euthanasia was performed by cervical dislocation (following European directive 2010/63/UE) immediately after capture. All participants wore gloves to avoid contamination during sampling.Skin mucus layer. The superficial mucus layer was sampled immediately after the death by swabbing one side of the body surface, which remained untouched. Avoiding the head, which was manipulated during euthanasia, buccal swabs  were rubbed from the back of the operculum to the caudal peduncle.Gastrointestinal content. After the sampling of the skin mucus, fish were individually placed in plastic bags and immediately stored on ice before dissection (within 5 h after sampling). Back in the laboratory, each individual was dissected using sterile tools: the gastrointestinal tract was extracted and cut from below the stomach to the rectum. Placed on a sterile surface, the content of each gut was expelled by squeezing, and the collected contents (minimum volume of 5 mL) were homogenized before sampling.Liver. Using a sterile cutter and forceps, a longitudinal piece of about 1 × 0.2 × 0.2 cm was cut from the right lobe (the largest) of the liver in each individual tuna. To avoid contamination from any other internal organs or fluids, the collected liver samples were rinsed with distilled water (through 0.2-µm filters).Storage. All skin mucus, gut and liver samples were placed in 5 mL sterile cryovials, frozen in liquid nitrogen and stored at −80 °C in the laboratory until nucleic acid extraction.Samples preparation. Three individuals (triplicate) of each sex  of each species (skipjack and tuna) in each ocean (Atlantic and Indian) were selected . In each of the 24 tuna, skin mucus, gut and liver viral communities were sampled as described above .n = 72) were suspended in 0.02 μm filtered PBS. For gut (n = 24) and liver (n = 24), 1 g sub-samples were placed in 2 mL Eppendorf and completed with 0.02 μm filtered PBS. For skin mucus samples (n = 24), individual swabs were placed in 1.5 mL Eppendorf with 500 μL of 0.02 μm filtered PBS. Gut and skin mucus samples were vortexed at maximum speed, respectively, 10 and 5 min, while liver samples were crushed with a FatsPrep for 45 s at 6 ms/ms. All samples were then centrifugated 10 min at 1500× g at 4 °C and supernatants were collected. After a second centrifugation at 14,000× g at 4 °C for 15 min, supernatants were collected and put in clean Eppendorf  [®Invitrogen, Carlsbad, CA, USA), 20 μL of TurboDNase , 5 μL of RNase A , 5 μL of Exonuclease I  and 5 μL of Benzonase  for 1 mL of sample [To separate virus from bacteria and organic matter, all biological samples  . All resf sample .Nucleic acid extraction. Viral nucleic acids were extracted from each sample (n = 72) using the Roche High Pure Viral Nucleic acid extraction kit  following the manufacturer’s protocols. Following the extraction, 1 μL of RNase OUT  was added in all samples, then nucleic acid concentration was assessed using Qubit dsDNA high sensitivity (®Life Technologies), following the manufacturer’s instructions.After quantification, triplicate individuals were pooled prior to the sequencing. Subsamples of 100 ng of extracted nucleic acid were taken from each of the three individuals replicates and pooled. Thus, for each organ a single replicate per sex , per species (skipjack and yellowfin) and per sampling site (Atlantic or Indian ocean) was obtained, for a total of 24 samples to be sequenced (n = 3 organs × 2 species × 2 sex × 2 sampling site = 24 samples).DNA amplification and purification. DNA amplification was then performed in duplicate, using Genomiphi V3 kit  following the manufacturer’s protocols. Duplicate amplifications of each sample were pooled, diluted with 60 μL of biomolecular water and purified using NucleoFast plates (®Macherey Nagel). Plates were centrifugated 30 min at 4000× g, 100 μL of molecular water were added in each well before a second 30 min centrifugation at 4000× g. After the addition of 30 μL of molecular water in each well, plates were agitated during 10 min before the collection of the eluded sampled placed in clean Eppendorf. Samples were sequenced using the Illumina MiSeq Technology with Nextera XT library kit in 2 × 250 bp format.nr database using DIAMOND [ggplot package in RStudio (R version 3.5.3).A total of 70,552,236 raw paired reads were obtained and trimmed, filtered, mapped and assembled into contigs/scaffolds using CLCgenomics7. Trimming parameters allowed the selection of sequences with a minimum length of 50 nucleotides and a quality above 0.05, resulting in 70,124,886 trimmed paired-reads. After assembly, the 828,771 contigs, with an average length of 596 bp , were aligned (BLASTx) against the  DIAMOND . The tax DIAMOND . Contigsvegan package [p value < 0.05.The effect of tuna species, sex, sampling site and organs on the composition of viral communities was determined by a one-factor PERMANOVA with 999 permutations on Bray–Curtis matrix, using the “adonis” function of the  package . For allThe most striking finding was the discovery of a specific viral signature in the skin mucus, liver and gut, whatever the tuna species, sex or sampling site considered , Table 1Although the liver and gut are well-known bacterial niches in vertebrates , bacteriAlloherpesviridae, a well-known fish pathogen family responsible for skin lesions [Podoviridae and Siphoviridae phage families, and to the Phycodnaviridae family for eukaryotic viruses. The former are common in marine environments [Cyclovirus, Megalocytivirus and several genera of Parvoviridae, recognized as severe fish pathogens [Papillomaviridae, well-known as human and mammal pathogens, were detected in the tuna liver—they have also been found in gilthead seabream, where they co-occurred with Iridoviridae and Polyomaviridae [By analyzing the virome composition in the skin, gut and liver samples, we identified several organ-specific viral taxa . Alloher lesions  and able lesions , was onlronments  and haveronments ,25 and mronments ,8. The lronments  and haveathogens ,28. Papiaviridae . One shoMyo-, Podo-, Sipho- and Microviridae families, as well as genera of Circoviridae and Geminiviridae (Caudovirales, Microviridae, Hepadnaviridae and several RNA viruses has been previously reported in the gills and liver of horse mackerel and gilthead seabream [While the virome composition was highly variable between the three organs, the findings also revealed the presence of several common taxa shared across the three organs. This ‘core virome’ was mainly composed of viral genera belonging to the iviridae . The preseabream . Circulaseabream , thus thOur results demonstrated that whatever the organ considered, the composition of the virome did not differ between the two species, nor sexes . Adult sIn line with the strong convergences between male and female tuna mentioned above, skipjack and yellowfin do not show sexual dimorphism and are distinguished only by the gonads . We knowThe skin mucosal virome of both skipjack and yellowfin varied significantly across the two sampling sites . The varThese results allow a pioneering characterization of the tuna meta-virome and provided new knowledge on the microbiome of marine organisms. Demonstrating the remarkable diversity of viruses associated with tuna, these findings raise questions about the role and implication of these viruses in the health of the host. Public health questions are also emerging as to whether these viruses can affect the health of fish consumers.


Solanum lycopersicum L.). Among the different abiotic stresses, drought is considered to be the most critical one, since limited water availability negatively impacts plant growth and development, especially in arid and semi-arid areas. The aim of this study was to understand how biostimulants may interact with critical physiological response mechanisms in tomato under limited water availability and to define strategies to improve tomato performances under drought stress. We investigated the physiological responses of the tomato genotype ‘E42’ grown in open fields under optimal conditions (100% irrigation) and limited water availability (50% irrigation) treated or not with a novel protein hydrolysate-based biostimulant . Plants treated with the protein hydrolysate showed a better water status and pollen viability, which also resulted in higher yield under drought stress compared to untreated plants. The treatment with the biostimulant had also an effect on antioxidant contents and activity in leaves and fruits depending on the level of irrigation provided. Altogether, these results indicate that the application of protein hydrolysates on tomato improved plant performances under limited water availability and in different experimental fields.Abiotic stresses adversely affect crop production causing yield reductions in important crops, including tomato ( Tomato is one of the most important crops, with more than five millions hectares cultivated, worldwide . Being aMoringa oleifera Lam.) increased yield and growth in tomato, basil, cabbage and pepper, as well as the quality of tomato, lettuce, radish, spinach, rocket and pepper [Medicago sativa L.) and red grape (Vitis vinifera L.) extracts improved growth and yield in Capsicum chinensis L., and triggered the accumulation of secondary metabolites in leaves. On rocket, the use of two vegetal-based biostimulants enhanced productivity under both optimal and sub-optimal nitrogen fertilization rates [One of the most promising strategies that can be used to improve plant response to drought stress is the use of biostimulants. These are substances or micro-organisms whose application is beneficial for plant growth and productivity . The appd pepper . These ed pepper , salinitd pepper , heat [1d pepper  and heavd pepper . Ertani d pepper  found thon rates .Despite the high number of scientific papers in which protein hydrolysates and plant-based biostimulants have been proven to increase crop productivity and abiotic stress tolerance, the functional cause–effect relationship and physiological basis that determine the growth stimulant and/or protective action of these products is still unclear .The aim of this study was to link the physiological responses and agronomic performances of tomato plants treated with a plant-derived protein hydrolysate. This biostimulant  was previously found to be effective in heat stress protection on different tomato varieties grown in open field. In this study, we tested whether the application of this biostimulant could be beneficial for tomato productivity and fruit quality under two different water regimes  and in different environmental fields. The physiological bases of these responses are discussed.We investigated the performances of one tomato genotype (‘E42’) grown in the year 2019 in an open field located in Battipaglia in the Campania Region  under optimal conditions (100% irrigation) and limited water availability (50% irrigation), and treated or not with a protein hydrolysate-based biostimulant. Pollen viability decreased by 27% under water deficit in non-treated plants. On the contrary, plants treated with the protein hydrolysate and subjected to water deficit showed an increase of 51% in pollen viability compared to non-treated plants a. Water In order to further confirm the effect of the protein hydrolysate on plant growth and on final yield and yield components, an additional experiment was carried out in the year 2020 in another experimental field located in Benevento  on plants grown in optimal conditions . These aIn the plants grown in the open field located in Battipaglia under optimal conditions (100% irrigation) and limited water availability (50% irrigation), treatment with the biostimulant had a significant effect on stomatal conductance, which, under full irrigation, increased by 84% after treatment a. The 50The content of total and reduced ascorbic acid (AsA) in the leaves of the plants grown in the open field located in Battipaglia was significantly reduced by biostimulant treatment only under 100% irrigation  a,b. UndeIn fruits of the plants grown in the open field located in Battipaglia carotenoid and lycopene contents were significantly affected by the interaction between biostimulant treatments and water regimen . In partThe aggregated data heat-map analysis , summariIn this study, we investigated the performances of one tomato genotype (‘E42’) grown in open fields located in Battipaglia and in Benevento in the Campania Region , treated with a protein hydrolysate rich in glutamic acid, glycine betaine and micronutrients as boron, manganese and zinc. Biostimulants have demonstrated beneficial effects in promoting growth and alleviating the effects of abiotic stresses in horticultural crops . Herein,In this study, we found a higher number of fruits in treated plants compared to non-treated ones under drought in 2019, and also under optimal conditions in 2020 . This caThe biostimulant had a positive effect on the total antioxidant activity (FRAP) in leaves of plant grown under limited water availability c. The hiRegarding fruit quality, the content of carotenoids and lycopene was higher in the fruit of treated plants compared to the non-treated ones under well-watered conditions . These r3 ha−1 and mean daily air temperature was between 17 and 27 °C (−1 (one emitter per plant). Water deficit was induced at 22 Days After Transplant (DAT) and continued until the end of the experiment. The biostimulant was applied by fertigation at a concentration of 3 g/l of water (400 mL per plant) at transplanting and, thereafter, every 15 days until the end of the cultivation cycle for a total of four applications. The biostimulant tested was CycoFlow, a protein hydrolysate produced by Agriges  by mixing sugar cane molasses with yeast extract obtained by autolysis of previously grown Saccharomyces cerevisiae yeasts. Its composition was previously reported [One experiment was carried out at the agronomy farm of the University of Naples “Torre Lama” located in Battipaglia, Salerno, Italy  on a clay-loam soil. Four weeks after seeding, at the third true leaf fully expanded, tomato plants  were trnd 27 °C . The expreported , which d3 ha−1 and mean daily air temperature was between 14 and 29 °C  in both experimental years. The number of fruits, the average single fruit weight and their total biomass were recorded in each experiment. During the cultivation cycle, the confirmation of plant stress was obtained measuring stomatal conductance and leaf water potential after 45 DAT (25 day after stress induction). Following procedures reported in other works ,47,48,49Pollen viability was analyzed using five flowers per plant sampled from three different plants per replicate in both years. In the laboratory, pollen grains were spread on microscope slides. Then, one droplet of DAB solution  was added to each pollen sample; slides were gently warmed with a gas lighter and mounted with a cover slip . Scoringv/v). The mixture was centrifuged at 15,000 rpm for 5 min at 4 °C. Supernatants were collected and stored at −20 °C until analyses. To determine the level of carotenoids and chlorophylls a and b, absorbance of lipophilic extracts was read at 470, 663, and 645 nm, respectively. For lycopene and β-carotene levels’ absorbance was read at 505 and 453 nm, respectively. Three separated biological replicates for each sample and three technical assays for each biological repetition were measured.Samples of freshly harvested, fully ripened tomato fruits and leaves were collected from each plot to determine pigments content by a colorimetric assay on freeze-dried and finely ground samples. The evaluation of total carotenoids, chlorophylls, lycopene and β-carotene was carried out according to the method reported by Wellburn et al.  and by Z2O and 80 µL of color reagent solution. This solution was prepared by mixing solution A (31% (w/v) H3PO4, 4.6% (w/v) TCA and 0.6% (w/v) FeCl3) with solution B (4% (w/v) 2,2′ -Dipyridyl). For total AsA, to 20 µL of sample, 20 µL of 5 mM dithiotreitol in 0.4 M phosphate buffer (pH 7.4) was added and the mixture was incubated for 20 min at 37 °C. Ten microliters of N-ethyl maleimide  in water) were added and left for 1 min at room temperature. Eighty microliters of color reagent were added as previously described for reduced AsA. Both the final mixtures were incubated at 37 °C for 40 min and measured at 525 nm using a Nano Photometer TM . The concentration was expressed in mg/100 g of fresh weight (FW). Three separated biological replicates for each sample and three technical assays for each biological repetition were measured.Measurements of reduced ascorbic acid (AsA) and total ascorbic acid (AsA + dehydroascorbate—DHA) contents were carried out by using a colorimetric method , with mo3·6H2O solution (12 mM), and 150 µL of methanolic extract obtained by adding 5 mL of 60% methanol base solution to 250 mg of frozen powder. The mixture was incubated for 30 min in the dark, and then readings of the colored products (ferrous tripyridyltriazine complex) were taken at 593 nm using a spectrophotometer. Results were expressed as micromoles of Trolox equivalents (TE) per 100 g FW. Three separated biological replicates for each sample, and three technical assays for each biological repetition, were measured.The antioxidant capacity was analyzed by FRAP assay carried out by using the ferric reducing/antioxidant power method  with slip < 0.05 were considered significant. ANOVA was performed by using SPSS  Package 6, version 23. A heat map, generated by using the http://biit.cs.ut.ee/clustvis (accessed on 1 April 2021) program package with Euclidean distance as the similarity measure and hierarchical clustering with complete linkage heatmap, summarized all the plant responses to both water-deficit and plant-based biostimulants.Data were subjected to analysis of variance using a two-way ANOVA. To separate means within each parameter, Duncan’s test was performed. Differences of In this study, a novel protein hydrolysate-based biostimulant was effective at enhancing tomato growth and productivity in different experimental fields, and also under limited water availability. This was possible thanks to the enhancement of the water status of the treated plants coupled with higher antioxidant activity, which are part of a common tolerance strategy generally employed by plants to overcome different abiotic stresses. Even if additional research is needed to fully understand its mechanisms of action, these results can be valuable to functionalize the use of this class of biostimulants in real agricultural contexts, which is necessary to increase their efficacy. Finally, this biostimulant increased fruit quality thanks to the accumulation of antioxidant molecules, including carotenoid and lycopene, which is an added value in regard to the increasing interest in the nutraceutical properties of food.


The choice of a green space metric may affect what relationship is found with health outcomes. In this research, we investigated the relationship between percent green space area, a novel metric developed by us , in three different types of buffers and type 2 diabetes (T2D). We obtained information about diagnosed T2D and relevant covariates at the individual level from the large and representative 45 and Up Study. Average contiguous green space and the percentage of green space within 500 m, 1 km, and 2 km of circular buffer, line-based road network (LBRN) buffers, and polygon-based road network (PBRN) buffers around participants’ residences were used as proxies for geographic access to green space. Generalized estimating equation regression models were used to determine associations between access to green space and T2D status of individuals. It was found that 30%–40% green space within 500 m LBRN or PBRN buffers, and 2 km PBRN buffers, but not within circular buffers, significantly reduced the risk of T2D. The novel average green space area metric did not appear to be particularly effective at measuring reductions in T2D. This study complements an existing research body on optimal buffers for green space measurement. Residential environments have a significant influence on health outcomes ,2,3. TheWhile green spaces may indicate a myriad of areas with vegetation ranging from nature strips and backyard gardens to public parks, in this paper we confine ourselves to green spaces that can be utilised to engage in meaningful physical activity or that can affect mental health. Thus, green spaces can be defined as outdoor sports fields, school playgrounds, any vegetated land adjoining an urban area including bushland, nature reserves, national parks, parks, and rural or semi-rural areas immediately adjoining an urban area .To understand how access to green space impacts on health outcomes such as T2D, it is important to define a spatial unit that best represents an individual’s accessible neighbourhood green space. The choice of a spatial metric of access to green space may influence its relationship with health outcomes. For instance, in a landmark study, Oliver et al. found that using line-based road network buffers resulted in stronger associations between built environment attributes and walking than circular buffers . CommonlThe choice of an appropriate geographic container or spatial unit is important as it can influence the exposure-outcome associations . Some exAppropriately quantifying the amount of green space within a container is also important. Thus for instance, to quantify the amount of green space within a buffer, studies count the number of discrete green spaces within each buffer ,20, whilThe goal of this study thus, is to explore the consistency in direction and magnitude of associations using different buffer types , buffer sizes , and green space metrics (percent green space or ACGSA) as a proxy of access to green space . A seconThe study area was the Sydney Statistical Division (SSD), in New South Wales (NSW), Australia, which had a population of approximately 4.42 million in 2016 and covers an area of 12,137 square kms . We obtaThe 45 and Up Study was approved by the University of New South Wales Human Research Ethics Committee and the University of Sydney Human Research Ethics Committee.Green space data were obtained from Pitney Bowes Australia StreetPro 2016. As mentioned earlier, in this dataset, green space includes national parks, nature reserves, historic sites, state forests, state recreation areas, wildlife refuges, conservation parks, protected areas, wildlife reserves, urban recreation parks and other urban green spaces.The proportion of green space within 500 m, 1 km, and 2 km circular buffers, LBRN buffers and PBRN buffers around participants’ residences were used as proxies for geographic access to green space. Circular buffers of the specified radii were created around the centroid of each participant’s residence location. LBRN buffers were created using the Pitney Bowes StreetPro 2016 data, which also included all roads in the study area excluding highways. We used ArcGIS network analyst  to calcuThe PBRN buffers were also created using the StreetPro road network data and ArcGIS network analyst . We calcTwo proxy measures of green space were then created using the specified buffers: proportion of green space and ACGSA. The proportion of green space within the specified buffers were categorised into 0%–10%, 10%–20%, 20%–30%, 30%–40%, and >40%. Note that we use standard mathematical interval notation, where for instance, 10%–20% implies that the category ranges from 10% (inclusive) to less than 20%.To evaluate ACGSA we first calculated the number of green space parcels that overlapped the specified buffers. Green space polygons which were completely or partially situated inside the buffers were included. The total area of these green space parcels including those partially located outside the buffers were calculated. The average area within these green space polygons was then calculated.2 followed by 1000 m2 (green space 2), 2364 m2 (green space 3), 4267 m2 (green space 4), 5542 m2 (green space 5), 9011 m2 (green space 6) and the largest green space polygon (green space 7) is 54,275 m2. The ACGSA within the 1 km circular buffer is thus 0.011 km2, 0.022 km2 for the 1 km PBRN buffer, and 0.006 km2 for 1 km LBRN buffer.An example is provided in 2 ranged from 1.1% to 1.8% for 500 m buffers, 1.6% to 2.6% for 1 km buffers, and 2.9% to 4.2% for 2 km buffers, we categorized this variable into the following categories:Average green space area was grouped into distinct categories. Since the percentage of average green space area over 50 km2],  and specificity (97.7%) compared to administrative hospitalisation data .Participant reported socio-demographic characteristics, including age, gender, body mass index (BMI), marital status, educational attainment , high school and less than 10 years of schooling), ancestry  and employment status were included as covariates in the regression models. We also included physical functioning  Physical Functioning Scale; it ranges from 0 to 100 and was categorised into no limitation (100), minor limitation (95–99), moderate limitation (85–94), or severe limitation (0–84)) , psycholArea-level deprivation was measured by the 2006 Index of Relative Socio-Economic Disadvantage (IRSED) quintiles at the postcode level. The IRSED was created by the Australian Bureau of Statistics to compare social and economic disadvantage across geographical areas in Australia. The index is derived from the 2006 Census variables such as low income and educational attainment, high unemployment, and people working in unskilled occupations ,33.Moderate to vigorous physical activity  and sitting (hours per week) were also included in the model as covariates. Physical activity was assessed in the 45 and Up study using questions from the Active Australia Survey  which haWe used generalised additive models (GAMs) to explore the relationships between the green space metrics and the prevalence of T2D, and to categorise green space metrics based on points of inflexion in the nonlinear relationships (not shown). We expect individuals in the same geography  to behave similarly. Thus, we used generalized estimating equations (GEE) which include an additional variance component to accommodate for correlated data and to allow for differences among local government areas. GEE logistic regression models were used to determine associations between access to green space and T2D prevalence. We assumed a compound-symmetric (exchangeable) correlation structure for the GEE model. Since GEE is a non-likelihood-based method, QIC (quasi-likelihood under independence model criterion) was used for variable selection and selecting the working correlation matrix. A lower QIC value indicates better model fit. As T2D may lead to physical disability, participants with severe physical functional limitation were excluded when fitting the GEE models.Of the 94,075 participants, 7192 (7.65%) participants reported T2D. Participants who were male, older, reported non-English ancestry, had lower education qualification, lived in more disadvantaged areas, were unemployed, obese, reported psychological distress, were engaged in less amount of time in MVPA, and spent more time in sitting were more likely to report T2D .Descriptive statistics for the various green space metrics are presented in the 2 were used as reference groups. There were significant reductions in odds of T2D when 30%–40% of LBRN or PBRM buffers of 500 m radius, or PBRN buffers of 2 km radius were covered with green space. A U-shaped pattern in odds ratios is also apparent, across all buffer sizes, with a dip in the odds ratios at 30%–40% and peaks on either side at 20%–30% and greater than 40%. The models with the 500 m and 2 km LBRN buffers also had the lowest QIC in their respective buffer size groups.Results from GEE models for the risk of T2D by the percentage of green space within the various buffer types and sizes are presented in 2 of ACGSA were associated with a significant reduction in T2D odds when buffer sizes are less than 2 km. The models with ACGSA in circular buffers less than 2 km in diameter also have the lowest (500 m buffer) and second lowest (1 km buffer) QICs. A general U-shaped trend was also visible with the odds of diabetes dipping when the average green space area in the buffer ranged from 3–20 km2.Circular buffers with 5–10 km2 to 20 km2 with 5–10 km2 of average green space within circular buffers showing the greatest reduction in odds.Our paper is the first to investigate the relationship between three different buffer types and two different ways of measuring green space on T2D, including a novel metric of average contiguous green space area. We report three significant findings. First, we showed that while using percent green space in LBRN and PRBRN buffers for analyses showed similar reduction in diabetes odds, circular buffers tended to show lower reduction in odds compared to LBRN and PBRN buffers. Second, the optimal amount of green space in buffers appears to be between 30% and 40% with increased odds of diabetes with either more or less green space. Third, the optimal amount of average contiguous green space area for reducing the odds of T2D was found to be between 3 kmThis study has a number of strengths. First, it introduces novel comparisons and an entirely new metric. Second, it leverages a large survey dataset from a diverse metropolitan area. Finally, it accounts for a number of lifestyle factors and demographic factors at individual level. We adjusted for socio economic status which has been previously found to confound the relationship between diabetes  and greeThirty to forty percent green space within 500 m LBRN or PBRN buffers, and 2 km PBRN buffers, but not within circular buffers significantly reduces the risk of diabetes. This underscores the importance of accessible green space within close proximity to residence in reducing the odds of diabetes, and the importance of LBRN and PBRN in measuring this. This is consistent with a study by Maas et al.,  which fo2 of average green space within circular buffers. These are very large green space areas, and since corresponding LBRN and PBRN buffers do not show a relationship, this may imply that these areas are not necessarily accessible to the resident. Thus, the reduced odds of diabetes are likely through the mental health, reduced stress and better sleep related pathways, that are manifested from living close to large green space areas [Circular buffers capture a larger area than LBRN or PRBN buffers, and may be able to capture multiple entrance points to extended regions of green space such as national parks located outside the buffer area, and thus be more relevant to the average green space area metric . We founce areas . It doesce areas . Most ofA U-shaped trend was visible for analyses using both average contiguous green space area and with percent green space. This is expected, given that small patches of green space may be of limited utility, and very large green spaces may be perceived to be unsafe. This may be mediated through the relative paucity of park features associated with safety, such as good lighting, visibility of houses, and high trafficked roads, all of which are rarely found in very large green spaces such as national parks and large undeveloped areas with vegetation .While, as expected, the associations between T2D and green space varied across the different buffer types, this study indicates that network buffers are optimal in analyses of T2D and green space relationships. Second, this study found that 30%–40% green space within walkable distance from home is optimal for the reduction of T2D odds. Third, large contiguous green spaces that extend beyond buffer boundaries may have limited influence on T2D odds. Future studies on the effect of different buffers in green space and health evaluation could focus on newer green space metrics such as google street view that provide a more realistic estimate of the individual’s green space experience.


Vigna unguiculata (L.) Walp.) plants respond to three common agricultural abiotic stresses: hypoxia (applied with the increasing time of exposure to nitrogen gas), salinity , and water deficit . We hypothesized that the cowpea plants would increase in tolerance to these three abiotic stresses when their intensities rose in a stepwise manner. Following two weeks of treatments, leaf and whole-plant fresh weights declined, soluble sugar levels in leaves decreased, and lipid peroxidation of leaves and roots and the levels of leaf electrolyte leakage increased. Polyphenol oxidase activity in both roots and leaves exhibited a marked increase as compared to catalase and peroxidase. Leaf flavonoid content decreased considerably after hypoxia, while it increased under water deficit treatment. NO emission rates after 3 h in the hypoxically treated plants were similar to the controls, while the other two treatments resulted in lower values of NO production, and these levels further decreased with time. The degree of these changes was dependent on the type of treatment, and the observed effects were more substantial in leaves than in roots. In summary, the responses of cowpea plants to abiotic stress depend on the type and the degree of stress applied and the plant organs.Exposing plants to gradually increasing stress and to abiotic shock represents two different phenomena. The knowledge on plants’ responses following gradually increasing stress is limited, as many of the studies are focused on abiotic shock responses. We aimed to investigate how cowpea ( Vigna unguiculata (L.) Walp.) is an important legume and a key crop in the context of global climate change and food security [Cowpea  increased drastically when the plants were treated with increasing salinity or decreasing water levels for two weeks . Salt stRoot and shoot length, along with fresh and dry weight of leaves and of the whole plants, decreased significantly, while the plant water-use efficiency increased following gradually increased hypoxia and subsequent reaeration.Following the salt stress treatment, shoot and root lengths, the number of compound leaves, the fresh weight of the leaves and of the whole plants, the dry weight of leaves, and the plant water-use efficiency decreased significantly, whereas stomatal density increased markedly .Water deficit decreased the number of compound leaves, the fresh weight of leaves and of the whole plants, the relative water content of leaves, and the plant water-use efficiency significantly, whereas root lengths, stomatal density, and stomatal index increased . Other ma, chlorophyll b, and carotenoids—did not show significant differences following hypoxia; the levels of carotenoids in salt-stressed leaves and of chlorophyll a and total chlorophyll in water-deficient leaves showed a small but significant increase as compared to the controls  in both hypoxic and salt-stressed leaves and roots, as well as water-deficient roots, increased significantly with respect to the controls B. FurtheWhile the total antioxidant capacity (DPPH) A, catalaTotal antioxidant capacity (DPPH) A, polyphWhen the plants were exposed to water deficit treatment, the leaves increased the levels of all antioxidant parameters measured during the study . FurtherUnderstanding different avenues of plant metabolism and adaptive responses to identify the crops or their traits resistant to abiotic stresses, or to improve their physiological adaptability, is important for global food security. There is limited knowledge of how cowpea plants respond to gradually increasing abiotic stresses when the intensity is changed in a stepwise manner on the same plant; this is because many of the studies are focused on abiotic shocks, where the treatments are carried out separately on different plants, which seems to be an unrealistic phenomenon except in the case of a sudden environmental change ,12. Our According to our results, it is clear that soil parameters changing significantly following each treatment can pose an effect on the morphological, physiological, and metabolic processes of cowpea plants. Soil oxygen deficiency changes the composition of dissolved gases in the rhizosphere, and affects soil pH and, thus, the nutrient availability and ultimate plant growth . During Some legumes are particularly sensitive to environmental stresses, as they are dependent on nitrogen fixation via symbiotic associations . Floodin+  reduces shoot and root growth in cowpea during the vegetative and reproductive growth of plants [Reduction in the rate of leaf surface expansion is the immediate response to increasing salinity, which accounts for ~80% of plant growth reduction, while the remaining 20% of the growth reduction is due to a decrease in stomatal conductance . Howeverf plants . Salinitf plants . Increasf plants . Abeer ef plants  reportedV. unguiculata (L.) Walp. was found to be one of the most drought-resistant legumes, grown widely in the semiarid regions [3 plants to avoid water deficit effects causing highly negative water potential [2 assimilation rate and thus changes leaf photochemical and biochemical responses and reduces productivity [Although drought, high temperature and longer day-length alone or in combination cause physiological damage and substantial yield reduction in many cowpea cultivars, in general  regions . As seenotential . The latuctivity ,37,38. Auctivity . In our a and total chlorophyll under water deficit stress, and in carotenoid levels under salt stress, the contents of pigments generally remained largely unchanged after all three treatments, indicating that cowpea plants are relatively resistant to all three stresses when applied gradually over time  and non-enzymatic constituents  to withstand oxidative damage . Activitjor role . AccordiVigna unguiculata (L.) Walp.) cultivar ‘Waruni’ were surface sterilized with 10% NaOCl and washed three times with autoclaved distilled water. Seeds were germinated in Petri dishes containing sterilized filter paper for five days in growth chambers. The fully grown, healthy seedlings of the same sizes were transplanted into 2 L pots filled with equal volumes of a pot mixture (soil: sand: peat = 2:1:1 (v/v/v)). All pots were watered up to field capacity (FC) one day before sowing to allow for draining of excess water; they were arranged in a completely randomized design in growth chambers  with a 14 h photoperiod, temperature of 26 °C/20 °C (light/dark), relative humidity of 50%, and photosynthetic photon flux density of 150 μmol m−2 s−1. Plants were irrigated regularly, and after two weeks the poorly developed plants were removed from each pot and seedlings (five in one pot) were grown for another week. The initial dry weight of seedlings before the treatments was determined to calculate plant water-use efficiency later. Other plants were subjected to three treatments for two more weeks.Healthy seeds of the cowpea (Pots (containing three-week-old plants) were divided into three experimental groups  and one control group; they were subjected to gradually increasing stress conditions for two weeks. To reach 75% field capacity of pot media, 110 mL of water  needed to be added to each individual pot in each treatment. For the water deficit treatment, 110 mL of water was added to each pot except for the water deficit one until the end of the experiment. The amount of watering was reduced every two days for two weeks as the water deficit treatment . For salt stress conditions, plants were treated with different concentrations of sodium chloride (NaCl) solutions, and the concentration was increased every two days for two weeks .−1 . Each pot was watered with the same amount of water (110 mL) regularly, as in the control. Unidirectional air valves faced outwards in the openings to maintain ambient pressure within the chamber while preventing ambient air from entering [To test the plants under hypoxia and re-oxygenation conditions (post-hypoxia), they were placed in a custom-built, sealed, dark chamber (5 L) under a steady inflow of nitrogen gas at 120 mL minentering ,53. The For physiological evaluation, the pots in each treatment were separated and measurements were conducted in the morning, always starting at 08:00 each day. To perform the biochemical analysis, the rest of the pots were used, and the fully developed fourth or fifth leaves and the roots were harvested immediately after the treatment, frozen in liquid nitrogen, and stored at −80 °C. The tests were repeated with at least three biological replicates and three technical replicates.Soil pH, electrical conductivity (EC), and total dissolved solids (TDS) in the soil suspensions under each treatment and control were determined at the end of the experiments using a pH meter and a handheld portable EC and TDS meter , respectively, as described by Bang-Andreasen et al. .Fresh weight (FW) and dry weight (DW) of leaves, stems, and roots of 10 plants from each treatment and the control group were determined after measuring the length–width ratio (L/W), and leaf area was roughly estimated by the product of the length and width (L*W = LA) of leaves. Dry weight was determined after oven-drying at 80 °C for 48 h . Plant sa (Chl a), chlorophyll b (Chl b), and carotenoid contents were estimated spectrophotometrically at wavelengths of 646.8, 663.2, and 470 nm, respectively. The content of each pigment was calculated as follows:Plant pigments were extracted into 80% acetone . ChloropThe impression approach was followed to determine leaf stomatal and epidermal cell densities, which were expressed as the number of stomata per unit of leaf area . Twelve The stomatal density (SD) (number of stomata per leaf area) and stomatal index (SI) were estimated as described by Xu and Zhou :StomatalThe water-use efficiency (WUE) and relative water content (RWC) of the individual plant were determined under each stress condition. RWC was determined by using the following standard formula: RWC = (fresh weight − dry weight)/(weight at full turgor − dry weight) × 100. Water content was determined by oven-drying of leaves at 80 °C for 48 h, and full turgor was estimated by keeping the samples overnight in water, as described by Farrant . WUE was2SO4 method [v/v), and supernatants were collected after centrifuging at 1480× g to determine total soluble sugars using H2SO4. Total soluble protein content was determined as described by Bradford [The total soluble sugar content was determined using the H4 method . Dry masBradford .−1 inflow of nitrogen gas through one opening, while the other opening was attached to a chemiluminescent detector  by a vacuum pump that pulled in air at 120 mL min−1 and was connected to an ozone destroyer [Nitric oxide (NO) emission of plants was measured as described by Cochrane et al. , by immeestroyer . A nitriestroyer . The prog for 5 min at 4 °C. To a 1 mL aliquot of supernatant, 4 mL of 20% TCA containing 0.5% TBA was added. The mixture was incubated at 95 °C for 30 min, quickly cooled in crushed ice, and centrifuged at 10,000× g for 10 min, and then the absorbance was measured spectrophotometrically at 532 nm and 600 nm. The nonspecific absorbance at 600 nm was subtracted from the absorbance at 532 nm, and the concentration of MDA was calculated using an extinction coefficient of 156 mM−1 cm−1.Lipid peroxidation was measured in terms of malondialdehyde (MDA) content, as described by Heath and Parker , using tLeaf electrolyte leakage was determined as described by Dionisio-Sese and Tobita . Leaf pig for 30 min at 4 °C. Supernatants were stored at −70 °C and used for enzyme activity assays.Leaf or root samples (0.5 g) were homogenized on ice in 4 mL of ice-cold buffer containing 1 M Tris–HCl (pH 6.8) and 0.3 M sucrose. The homogenates were centrifuged at 13,000× 2O2 (w/w). The reaction was initiated by adding 0.03 mL of plant extract to the reaction buffer solution. The absorbance was measured at 240 nm, and the activity was expressed in units per mg of protein. The unit of activity was defined as 1 μmol of H2O2 decomposed per min. The molar extinction coefficient for H2O2 (39.4 M−1 cm−1) was used to calculate the equivalent catalase activity.Catalase  activity was measured as described by Guilbault et al. . The reaPolyphenol oxidase  activity was determined as described Raymond et al.  at 40 °C2O2, 0.05 mL of 20 mM guaiacol, and 0.01 mL of enzyme extract. The increase in absorbance was recorded at 435 nm for a duration of 3 min. POD activity was expressed as units of enzyme activity (U). Units were calculated using a molar absorptivity of 2.66 × 104 M−1 cm−1 for tetraguaiacol .Peroxidase  activity was measured as described by Malik and Singh , with a v/v) acetone with 0.2 % (m/v) formic acid at a ratio of 1:10, with 8 h of shaking at 4 °C followed by centrifuging at 20,000× g for 20 min at 4 °C, as described by Vyas et al. [DPPH  radical scavenging activity, total soluble phenolics, and flavonoids were estimated after extracting them into 80 % , using Minitab version 17 software . The t and p values is provided to assist interpretation of the results.Data are presented as the mean of three independent experiments with the respective SD bars. Statistical significance between means was analyzed with the Student’s Progressive stress application alters soil properties, inducing changes in the morphological, physiological, and metabolic parameters of cowpea plants. Cowpea plants showed varying growth, morphological, and physiological responses under these stresses. Under post-hypoxia, photosynthetic pigments and plant NO production remained initially unchanged, while sugar production and flavonoids in leaves decreased, but leaf and root protein production and cell damage increased. When the salt stress was applied with the increased intensity, sugar production in leaves and NO emission rates decreased, while protein levels remained unchanged. The increased salt application resulted in the induction of cell damage mainly in leaves, despite their higher antioxidant capacity. Water deficit in soil stimulated protein production, induced cell damage, and increased the antioxidant capacity of cowpea plants, while NO production and leaf sugar production declined. All three stresses resulted in stronger responses in leaves than in roots. We conclude that the responses of cowpea plants to the gradually increased abiotic stress factors depend on the type and the degree of stress applied, and on the plant organ. These responses indicate a high plasticity of cowpea plants in the course of their adaptation to environmental changes—a feature that is overlooked in the conventional stress–shock experiments but can be clearly seen when the intensities of the treatments simulate naturally occurring phenomena. Overall, this study provides evidence that plants are able to increase their tolerance to abiotic stress when its intensity changes in a stepwise manner.


Prenatal cocaine exposure (PCE) is associated with behavioral, cognitive, and social consequences in children that might persist into later development. However, there are still few data concerning epigenetic mechanisms associated with the effects of gestational cocaine exposure, particularly in human newborns.OXTR) gene in the umbilical cord blood (UCB). The relationship between UCB DNA methylation levels and the severity of the mother’s cocaine use during pregnancy was also evaluated.We investigated the effects of PCE on DNA methylation patterns of the Oxytocin Receptor (in utero and 30 UCB samples of non-exposed newborns (NEC) were compared for DNA methylation levels at two genomic loci located in exon III of the OXTR gene (OXTR1 and OXTR2) through pyrosequencing. Maternal psychopathology was investigated using the Mini International Neuropsychiatric Interview, and substance use characteristics and addiction severity were assessed using the Smoking and Substance Involvement Screening Test (ASSIST).In this cross-sectional study, 28 UCB samples of newborns with a history of crack cocaine exposure No differences between newborns with a history of PCE and NEC were observed in OXTR1 or OXTR2 DNA methylation levels. However, regression analyses showed that maternal addiction severity for crack cocaine use predicted OXTR1 DNA methylation in newborns.OXTR methylation levels in the UCB of children are affected by the severity of maternal crack cocaine usage. Larger studies are likely to detect specific changes in DNA methylation relevant to the consequences of PCE.These data suggest that  Crack cocaine, a smoked form of cocaine, is a highly addictive drug . Crack-cPrenatal cocaine exposure stimulates the fetal central nervous system, acting mainly on the dopaminergic system . OxytociOXTR gene (Chr:3p25.3), a polypeptide of 389 amino acids with seven transmembrane domains. The OXTR is a class I G coupled receptor. The OXTR gene spans 17 kb and contains three introns and four exons. Exons I and II correspond to the 5′-non-coding region, followed by exons III and IV, encoding the amino acids of the receptor  of prenatal crack cocaine-exposed newborns and compare these to those in non-exposed newborns. The relationship between UCB DNA methylation levels and the severity of the mother’s cocaine use during pregnancy was also evaluated. This approach makes it possible to identify a potential cocaine-induced epigenetic effect during early development, prior to exposure to the extrauterine environment.Given the relationship between OT, addiction, and social behavior, the present study aimed to evaluate The biological, clinical, and demographic data of the study participants were obtained from a previous cross-sectional study conducted from January 2012 to September 2013 in Southern Brazil, assessing 2,228 births in a public hospital . This stn = 2,228). Among those reporting crack cocaine use during pregnancy 105 mothers (4.7%) had their use confirmed through positive urine drug tests  and clinical interviews. All of them were invited to participate in this study and donate UCB from their children. However, only 30 were considered eligible due low DNA quality obtained from UCB, presence of Human Immunodeficiency Virus (HIV) or Hepatitis C Virus infections in mothers, or low quality of clinical data.In this cross-sectional design study, the factor studied was prenatal crack cocaine exposure, and the outcome was OXTR DNA methylation levels in the UCB of newborns. To find mothers who used crack cocaine during pregnancy, we consecutively interviewed women about their use of illicit and licit substances  upon admission to a public hospital maternity unit in Southern Brazil (n = 30) consisted of newborns not exposed to prenatal crack cocaine, and their UCB samples were obtained from the hospital biobank. Mothers who were invited to donate UCB samples for the hospital biobank had an extensive investigation on exposure to toxic, infectious agents and health records, establishing the absence of any mother/baby pathology. In addition, the control group only included mothers who denied any substance use during pregnancy and showed negative findings in urinary analysis for drugs . The selection of control samples also accounted for matching procedures based on age of mothers who used crack cocaine and were included in the study.The control group (It is important to note that this study focused on the effects of crack (smoked cocaine), instead of snorted/injected cocaine, during gestation. Crack produces all the effects of cocaine. However, its smoked route of administration reaches the lung and brain in a few minutes. Thus, its effects happen much faster than those of snorted cocaine. Crack effect lasts for about 5 min, while the effect of injected/snorted cocaine lasts for about 20–45 min. The short duration of the effect of crack is associated with a pattern of consumption of greater quantities. This behavior causes a much faster pattern of addiction compared to snorted or injected cocaine. Finally, many substances in addition to cocaine might be added to crack rocks, to increase the profit of drug dealers. Crack contains a mixture of cocaine with sodium bicarbonate or ammonia, powdered milk, and lime, in addition to other substances that enhance its neurotoxic effects, such as lidocaine, phenacetin, levamisole, benzocaine, procaine, and hydroxyzine. Based on that, we decided to maintain this distinction.OXTR DNA methylation analyses, interviews, and reviews of medical records. The independent variable was a history of crack cocaine use during gestation. Maternal information, including age, self-reported ethnicity, prenatal care, education, and the socioeconomic level, was collected at the time of enrollment using a standardized questionnaire. The socioeconomic level was dichotomized into “low socioeconomic status”  and “high socioeconomic status” (classes A and B). This was established based on the Criterion of Economic Classification of Brazil (CCEB), which is a standard classification method of socioeconomic status in Brazil. This indicator has five different classes  based on parameters that include the physical structure of the residence, the existence and quantity of certain goods , access to public services , the education level of the head of the family , and family income. Participants classified within the “high socioeconomic status” have an estimated monthly income higher than 10390,00 R$ (1853 US$). Ethnicity was dichotomized as caucasians and others. Educational level was dichotomized as “incomplete or completed primary education, or incomplete secondary education” and “completed secondary education, or incomplete or completed higher education.” The following newborn variables were assessed by reviewing medical records: weight, sex, and Apgar scores at 1 and 5 min of life.The study variables were obtained by Maternal psychopathology was investigated using the Mini International Neuropsychiatric Interview, Brazilian version 5.0.0/DSM-IV/Current . Also, d® tubes with ethylenediaminetetraaceticacid (EDTA) and centrifuged at 4.0 rotations per minute for 10 min at 4°C. Mononuclear cells were lyzed, and DNA was isolated and purified using the Gentra Puregene® Blood Kit , in accordance with the manufacturer’s standard protocol. Genomic DNA was quantified by ultraviolet absorption using NanoDrop® Lite . DNA purity was estimated . Samples containing approximately 1,000 ng DNA were stored at −20°C for subsequent DNA methylation analysis.All umbilical cord blood samples were collected in BD Vacutainer® Bisulfite Kit (Qiagen) according to the manufacturer’s standard protocol. After bisulfite treatment and purification, the concentration of DNA was reevaluated using NanoDrop® Lite (Thermo Scientific). Bisulfite-converted DNA was used as a template in a polymerase chain reaction (PCR) for the amplification of two target sequences located in exon III of the OXTR gene. For this, we used two PyroMark CpG assays  according to the manufacturer’s instructions.Sodium bisulfite modification was performed using 1,000 ng of genomic DNA using the EpiTectOXTR containing four CpG sites . The PCR primer sequences for OXTR1 were as follows: forward, 5′-TTTAGGG ATATGAGTAGTAGTAGGTAGG-3′ and reverse, 5′-ACCCCTCTTCTTCTTCTTCATAAAACACC-3′, which generated a 193-bp amplicon. Similarly, the OXTR2 assay allows analysis of the sequence TCGTAGTAGGTAG CGAGTACGATGATCGGTACGA . The OXTR2 PCR primers generate a 248-bp amplicon . Before the experiment, the PCR primer sets were tested thoroughly to determine reaction efficiency and specificity, and the absence of primer-dimer products.The PyroMark CpG assay OXTR1 allows analysis of the sequence AGGCGGTATAGTAGGTCGGGTTCGTAG AAGCGGA , and a fully unmethylated negative control (EpiTect® Control Unmethylated DNA) to standardize and test the quality of the reactions. Successful PCR amplification of a single fragment was confirmed using agarose gel electrophoresis for each sample. Finally, we prepared single-stranded biotinylated PCR products for sequencing using the Pyrosequencing Vacuum Prep Tool (Qiagen) in accordance with the manufacturer’s instructions.A 50-ng sample of bisulfite-converted DNA was used as the starting template in a PCR volume of 25 μL using the PyroMark PCR Kit (Qiagen). The PCR program cycle was as follows: 95°C for 15 min; 42 cycles at 95°C for 30 s, 56°C for 30 s, and 72°C for 30 s; and a final extension at 72°C for 5 min. In each plate, we added 0.3 μM of the primer in 25 μL of annealing buffer per well. Further, for each run, we added a blank control (distilled water), a fully methylated positive control  followed by hybridization of the pyrosequencing primer in a PyroMark® Q24 Vacuum Workstation (Qiagen). Pyrosequencing was performed using PyroMark® Gold Q24 Reagents (Qiagen) in accordance with the manufacturer’s instructions. We analyzed the data using PyroMark® CpG software version 2.0.6 (Qiagen), which identifies cytosine methylation (%) at each CpG unit within a given sequence. The PyroMark® CpG software generates a quality score for each CpG site throughout the sequencing run, which allowed us to exclude low-quality samples from the pyrosequencing analysis.Polymerase chain reaction products were purified using Streptavidin SepharoseIn summary, the DNA is first incubated with sodium bisulfite. As a result, unmethylated cytosine residues are converted into uracil, while methylated cytosines remain unchanged, giving rise to two different sequences that can be distinguished. Cytosines that are not methylated are converted to thymine by bisulfite treatment and PCR. Then, in the resulting report, the ratio of cytosine to thymine at each analyzed CpG site is presented, which reflects the proportion of methylated DNA.Following pyrosequencing analysis, two patients were excluded because of low-quality scores in the methylation analyses. Thus, the final sample consisted of 28 prenatal crack cocaine-exposed newborns and 30 non-exposed newborns.t-test or by using the Chi-squared test for categorical variables. DNA methylation data in the region denominated as OXTR1 were analyzed by calculating the mean values for four CpG units (OXTR1 methylation). A similar calculation was performed for the region denominated as OXTR2, which consisted of the mean values for five CpG units (OXTR2 methylation). Zero-order correlation was applied to explore the relationship between OXTR1 methylation and OXTR2 methylation and clinical and sociodemographic variables . For DNA methylation prediction analysis, a stepwise linear regression analysis was conducted with OXTR1 methylation and OXTR2 methylation as independent variables and the ASSIST crack cocaine score as the dependent variable. For p-value corrections, we used the Bonferroni test. Statistical analysis was performed using SPSS 20.0 . The level of significance was set at p ≤ 0.05.All variables were tested for normality of distribution by the Shapiro–Wilk test. Sociodemographic and clinical data were compared between crack cocaine user mothers and non-user mothers, or between crack cocaine-exposed and non-exposed children using the Student’s Sociodemographic and clinical characteristics of PCE newborns and non-exposed to cocaine (NEC) newborns, as well as their respective mothers, are shown in Mothers of NEC newborns showed an absence of substance abuse  throughout their lives. Therefore, we analyzed the ASSIST total scores for alcohol (8.10 ± 7.89), nicotine (15.17 ± 8.97), cannabis (6.4 ± 8.71), and crack cocaine (25.53 ± 8.52) usage within CCUMs. Further, we examined the frequency of drug use during the 3 months prior to delivery .The OXTR1, OXTR2, and overall OXTR DNA methylation data of PCE and NEC newborns are shown in OXTR methylation and maternal and neonatal sociodemographic and clinical variables. OXTR1 and OXTR2 methylation levels of the PCE newborns were not associated with Apgar scores at 1 and 5 min; newborn weight; maternal age; ASSIST scores for tobacco, alcohol, or cannabis (p > 0.05). Additionally, OXTR methylation was compared between groups of socioeconomic status , ethnicity , and education , showing that methylation levels also were not associated with these variables. However, OXTR1 methylation showed a moderate correlation with crack cocaine ASSIST scores  = 0.160, p = 0.003] (p = 0.03).To examine the influence of crack cocaine exposure on UCB = 0.003] . Only OXOXTR in the UCB of PCE children. We found that maternal ASSIST crack cocaine scores predicted DNA methylation levels of the CpG island within exon III of the OXTR gene among PCE children, even after controlling for potential confounders. Although our analysis revealed this positive association, we found no differences between groups (newborns not exposed to crack). These data reveal that the magnitude of the effects of prenatal crack cocaine exposure at the epigenetic level might be dependent on the severity of maternal consumption patterns  are known to be related to OXTR CpG hypermethylation (OXTR in the brain (OXTR gene (OXTR gene, it is possible that group differences that were not evident in our analyses could be observed in future studies through a broader appreciation of the epigenome.Also, PCE may alter the expression of the hylation . Furtherhe brain . TherefoXTR gene . Due to OXTR methylation in UCB of PCE children.The ASSIST scale allowed us to measure the severity of psychoactive substance use of the mothers, making it possible to verify the degree of drug addiction for distinct substances . In our OXTR methylation levels, contrary to what has been observed in other studies. For instance, OXTR exon III CpG island.We have shown ethnic differences between mothers using and not using crack cocaine and between exposed and non-exposed newborn children; however, we found no association between ethnicity and It must be discussed, however, that there are significant limitations in this study. Although crack cocaine was required to be the drug of choice as an inclusion criterion in the study, the presence of other psychoactive substances was not an exclusion criterion. Previous research indicates that mothers who use cocaine during pregnancy are likely to also use other substances . WithoutWe provide preliminary evidence that the mother’s severity of crack use is associated with an increase in OXTR DNA methylation. These results provide additional evidence to the potential role of DNA methylation in the relationship between maternal substance use and later behavioral and developmental sequelae in offspring . ParticuThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Pontifícia Universidade Católica do Rio Grande do Sul. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.TB and RG-O conceived and planned the experiments. TB and LA carried out the experiments. MN, AP, and FM carried out pyrosequencing procedures. VM and CS contributed to sample recruitment and clinical assessment. TV, SC, and JS contributed to the interpretation of the results. TB took the lead in writing the manuscript. RG-O was in charge of the overall direction and plan, and supervised the findings of this work. All authors provided critical feedback and helped shape the research, analysis, and manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.