Various redox compounds are known to influence the structure of the gluten network in bread dough, and hence its strength. The cereal thioredoxin system (NTS), composed of nicotinamide adenine dinucleotide phosphate (NADPH)-dependent thioredoxin reductase (NTR) and thioredoxin (Trx), is a major reducing enzymatic system that is involved in seed formation and germination. NTS is a particularly interesting tool for food processing due to its heat stability and its broad range of protein substrates. We show here that barley NTS is capable of remodeling the gluten network and weakening bread dough. Furthermore, functional wheat Trx that is present in the dough can be recruited by the addition of recombinant barley NTR, resulting in dough weakening. These results confirm the potential of NTS, especially NTR, as a useful tool in baking for weakening strong doughs, or in flat product baking. As the raw material shows natural differences in many qualitative aspects, such as protein content, it has been crucial to develop tools to manipulate the bread properties to compensate for natural variations between wheat cultivars, or to adapt flour for specific applications. As an example, the variation in high molecular weight (HMW) glutenin contents in different flours influences the elasticity of the dough . An accu species  -dependent thioredoxin reductase (NTR) and thioredoxin (Trx) constitute a versatile multi-enzymatic NTS that has been extensively studied in plants. NTS functions by transferring reducing power from NADPH to various target proteins via a Trx-mediated disulfide bridge reduction  Figure B. Plant Plant Trx are highly expressed in seeds, and these very stable proteins likely remain functional in raw dough. In contrast, upstream reducing enzymes, e.g., the flavoprotein NTR, are rather unstable enzymes, and are likely to be lost during industrial flour processing. We therefore hypothesize that endogenous Trx that is present in wheat dough could be used as a natural reducing agent to modify dough properties, if supplemented with reducing equivalents provided by NADPH and barley NTR. We examined these hypotheses here in an experimental dough- and bread-making system.Saccharomyces cerevisiae) were provided by Danisco.Raw material. Wheat flour  and the yeast strain . Rabbit antibodies raised against wheat Trx h1 were a kind gift from Bob B. Buchanan .Enzymes. Recombinant barley NTR (HvNTR2) and Trx (HvTrxh1) were produced and purified as previously described ,18. The Dough preparation. Flour  was mixed with water to 55% water absorption using method AACC 54-21 and 2% (bakers’ percentage) NaCl on flour weigh. NADPH and either or both NTR and Trx were then added, and the dough was mixed for 6 min. Dough strength was monitored until a stable value of ca. 400 Brabender units (BU) was reached, as measured by farinograph. Yeast  and glucose (2% on flour) were included in the 300 g samples used in the baked products experiments. NADPH, NTR, Trx, and 10 mM Na-phosphate buffer (pH 6) were kept separately on ice, and mixed just before their addition to the flour in the mixing chamber, immediately after water addition.Kiffer rig measurements. After mixing, the dough was shaped into a bun, of which samples were cut out for Kiffer rig measurements. Eight dough strips were shaped in a specifically designed mold, and left to rest at 25 °C for 30 min in a tightly closed plastic bag (unless stated otherwise). Eight strips per sample were then assayed by measuring Kieffer uniaxial extension using a Stable Micro Systems model TA-XT2i texture analyzer. The resistance of the dough strip (in g) and the maximum elongation  were recorded using the Texture Expert Exceed version 2.64 software.Baked products. Minibreads and buns from yeast-containing samples were prepared by mixing 15 and 50 g of initial flour, respectively, to 450 BU. After fitting into molds, minibreads and buns were proofed at 34 °C, 85% relative humidity (RH) for 55 min, to allow for dough development, then minibread dough stability was tested by sliding half of them down a custom-made slide (“shock treatment”), and finally the products were baked at 220 °C for 8 min (MIWE aeromat CS oven). For enzyme testing, to the flour mix (300 g) was added either buffer (10 mM sodium phosphate pH 6) or a mix of 16 mg NADPH and 2 mg NTR in buffer. The weights and volumes of the minibreads and buns were measured after cooling down. The section and bottom surfaces of four minibreads and buns for each treatment were measured from scanned sections, using ImageJ software.2 after mixing and development. Samples were freeze-dried overnight and ground to powder using a mortar and pestle. Sixty milligrams of powder was used for protein extraction  was added. Samples were incubated for 2 hr at 4 °C with shaking, centrifuged at 14,000 rpm for 20 min, and supernatants were collected (soluble protein fraction). Twenty milligrams of protein were loaded and run on NuPAGE Novex 12–15% Bis-Tris minigels , and stained with Coomassie blue.Dough protein extraction and thiol titration. Dough samples were frozen in liquid N412 was recorded, and the thiol concentration was calculated from εM = 13,600 M−1cm−1 for free TNB anion, and the results were normalized by setting a value of 100 for the control sample.Volumes of 20 and 200 µL of extract were used to measure free thiols with Ellman’s reagent. Briefly, the sample and 5 µL 20 mM DTNB in ethanol were mixed in 10 mM Tris pH 8 buffer . After 30 min in the dark, AWestern blot. A total of 30 mg soluble protein were loaded and run on NuPAGE Novex 12–15% Bis-Tris minigels (Invitrogen), and blotted onto Hybond-ECL membranes . The quality of the protein transfer was checked by Ponceau staining, and membranes were afterwards incubated overnight in blocking solution consisting of 2% bovine serum albumine (BSA) in 30 mM Tris pH 8, 200 mM NaCl, 0.05% Tween 20 buffer (TBST). Membranes were incubated with 1/2500 diluted anti-wheat Trx rabbit primary antibody in blocking solution for 2 hr, washed three times for 10 min in TBST, and incubated for 1 hr in 1/2500 diluted secondary goat anti-rabbit horseradish peroxidase-coupled antibody . After three washes with TBST, membranes were put in plastic film, and an ECL Plus kit  and a UVP-Bioimaging system  were used for detection.As a starting point, a concentration of thiols was chosen based on the 90 ppm cysteine FDA regulation, taking into account that our enzymatic system would probably be less active in the non-optimal dough environment . An amount equivalent to 300 ppm thiol was added in the form of 120 mg Trx, and 13 mg NTR with 140 mg NADPH, for a 50 g flour assay. The amounts of each component were then progressively decreased and mixed in various combinations to assess the effects of the different compounds on the dough system. NADPH and NTR were observed to exert strong effects on the rheological properties of the dough . Control2 bubbles. This effect was suppressed when a light shock treatment was applied, resulting in dramatic collapse of the dough, due to the weakened gluten network. In this case, the measured height dropped below those seen for the control dough. When no enzyme was added to the dough, the shock treatment had no significant effect on minibread volume  we only analyzed the effect of enzyme addition. This addition caused an increase in the bottom surface of the rolls, while the maximum height decreased B,C. Thisn = 2) of measurements done. In yeast-containing dough, the free thiol content was higher than in samples without yeast, and NTS seemed to induce a slight decrease in thiol content  and Western blot, and free thiols were also quantified. Quantification showed a higher content of free thiols that were extractible from the dough when it was treated with high amounts of NTS A. Unfort content B. Wester content C. This cWe showed in this work that NTS is a powerful tool for the remodeling of the gluten network and the weakening of the dough, likely through functional wheat Trx being recruited in the dough upon addition of recombinant NTR. The addition of NTS induced dough weakening, as expected from reducing agents. It was possible to extract protein from dough and detect recombinant Trx spiked in the dough, together with a background signal from other proteins including most probably wheat Trx. Soluble dough extracts were also used to estimate the contents of soluble free thiols in the dough, which likely increased when NTS was added. Furthermore, this softening both of dough with and without yeast showed that added NTR and Trx activity were not lost due to elevated yeast metabolism in the developing dough. It is suggested that the optimization of the systems can involve increasing amounts of NTR added to the mix. The synergistic effect of Trx will allow for the reduction of the levels of NADPH needed for efficacy, and lead to a cost-effective procedure to soften strong flour systems using endogenous cereal NTR and Trx, where dough weakening is required, e.g., in flat product baking or laminated dough. Relaxation of the gluten network by enzymatic reduction is also increasing the volume of developed dough, although the shock sensibility observed here is a severe issue for any application in this direction. Using the NTS system, the costly addition of NADPH could be circumvented via the development of yeast strains containing high endogenous NTS system activity and/or to strains designed for specific applications, such as pizza or laminated dough making. The development of yeast GSH biosynthetic pathway mutants has already shown increases in their contents of GSH and derivatives , opening


The aim of this study was to assess the association of IL1A SNPs with the risk of colorectal cancer in a Chinese Han population.Colorectal cancer (CRC) is one of the most common cancers worldwide, and genetic variations exert distinct roles in its pathogenesis. Single nucleotide polymorphisms (SNPs) in IL1A polymorphisms and CRC risk, Agena MassARRAY platform was used for genotype determination among 248 CRC patients and 463 controls. The relationships between IL1A variants and CRC susceptibility were examined by logistic regression analysis. Stratified analysis was conducted for the association detection in males and females. Haplotype construction and analysis were applied to evaluate the potential relationship between the genetic block and the risk of CRC. SNP functional exploration was performed with available bioinformatics datasets.To evaluate the correlation between p = 0.036). With stratified analysis, the recessive models of rs3783550 , rs2856838 , rs1609682 , and rs3783521  revealed significant relationships between these variants and an increased CRC risk only in females. Bioinformatics analysis also revealed the putative functions of the selected SNPs.After adjusting for age and gender, the “AA” genotype of rs2856838 exhibited a risk association with colorectal cancer in the recessive model  contains supplementary material, which is available to authorized users. Colorectal cancer (CRC) is one of the most common cancers worldwide, and it is the third leading cause of cancer-related deaths in both genders. In the past two decades, an increasing incidence of CRC was found in China as lifestyles and eating habits have become progressively changed , 33. TheIL1A , is defined as an “alarm cytokine” that belongs to IL-1 cluster and plays dual roles in malignant tumour progression [Accumulating evidence has demonstrated that most cancers develop from long-time chronic inflammation, including CRC, which could be attributed to two common inflammatory bowel diseases, namely, Crohn’s disease and ulcerative colitis , 18. Infgression . Membrangression .IL-1A is associated with diverse diseases. Gao et al. has reported an insertion/deletion (ins/del) polymorphism  in IL1A that may contribute to hepatocellular cancer susceptibility, and revealed the regulatory function of the variation on IL1α expression by disrupting the binding sites for miR-122 and miR-378 [IL1A polymorphisms and the risk of papillary thyroid cancer, nasopharyngeal cancer and epithelial ovarian cancer have been reported as well [Furthermore, it is widely recognized that the polymorphisms of  miR-378 , 20, 31. as well , 29, 32.IL1A in colorectal adenocarcinoma, which indicated the potential role of IL1A in the etiology of CRC. However, few studies have examined the associations of IL1A polymorphisms with the risk of CRC. In our study, we investigated the effects of five IL1A variants  on the susceptibility to CRC, which is supposed to provide more evidence for IL1A in CRC pathogenesis and contribute to early CRC risk estimation among the individuals of Chinese Han ancestry.A recent work has elucidated a decreased tumour expression of The current research involved a total of 248 CRC patients  and 463 controls  with unrelated Chinese Han ancestries. All CRC cases were diagnosed and confirmed by two independent pathological examinations. As for the eligible CRC cases selection, the individuals without other types of cancer, familial adenomatous polyposis, hereditary nonpolyposis colorectal cancer or undergone previous radiotherapy and chemotherapy were included. With regard to healthy controls, the subjects who had suffered from chronic or severe endocrine and metabolic diseases, digestive system disorders and malignant diseases were excluded from this study. The controls were polyp free individuals at recruitment. Moreover, the individuals with family colorectal disease and cancer history were excluded from control group as well.IL1A polymorphisms, we selected SNPs which could impact cancer risk, and searched the genetic data of them in dbSNP database (https://www.ncbi.nlm.nih.gov/snp/) and 1000 Genomes database . Only the SNPs whose minor allele frequency (MAF) beyond 5% in Asian populations were included in this study in order to achieve adequate statistical power. Finally, five candidate variations rs3783550, rs3783546, rs2856838, rs1609682, and rs3783521 in IL1A gene were selected for genotyping. Five millilitres venous blood was collected from the subjects when they were recruited in this study. Genomic DNA was extracted from the blood with the Whole Blood Genomic DNA Purification Kit . The PCR primers used in multiplexed PCR assay were designed by Agena Bioscience Assay Design Suite V2.0 software and are showed in Additional file By reading the previous publications of 2 test. Logistic regression analysis with adjustment for age and gender was applied to the odds ratio (OR) and 95% confidence interval (CI) assessment [p ≤ 0.05 was regarded as statistical difference. The associations of the five candidate variants with the CRC risk were examined in codominant, dominant and recessive genetic models using SNPstats online software (https://www.snpstats.net/start.htm). Additionally, we used the Haploview software package, version 4.2  for linkage disequilibrium (LD) construction and haplotype evaluation [2 and D’. And D’ = 1 is defined as complete linkage disequilibrium. The allele combination of the variants in one LD block was considered as haplotype. We selected the haplotypes with frequency greater than 5% to evaluate whether the haplotypes were associated with CRC risk. These associations were detected using SHEsis software with available OR (95% CI) and p value.SPSS 18.0 software  was employed for primary statistical analysis. The heterozygosity for the five SNPs in control cohort was assessed by the Fisher’s exact test to detect their compliance with Hardy-Weinberg equilibrium (HWE). Allele and genotype frequencies of each SNP in CRC cases and healthy controls were calculated and evaluated for the difference with Pearson’s χsessment . Adjustmaluation . Using thttps://pubs.broadinstitute.org/mammals/haploreg/haploreg.php) was applied for SNP functional annotation using bioinformatics methods. Expression quantitative trait loci (eQTL) analysis were carried out with the data available on the Genotype-Tissue Expression (GTEx) project . The impact of each candidate SNP on IL1A expression was determined in several human tissues and p-values were provided for statistical significance assessment.HaploReg v4.1 database . An association between allele “A/A” of rs2856838 and an increased risk of CRC was observed under the recessive model  after adjustments for age and gender.Basic information and allele frequencies of the IL1A variants rs3783550, rs2856838, rs1609682 and rs3783521 did not show any significant association with CRC risk in males, whereas the statistical significance was detected in females , respectively. Moreover, rs3783550 and rs1609682 were associated with an increased risk of CRC based on the results of the recessive model . The frequency of homozygous “A/A” genotype of rs2856838 differed significantly between patients and controls in females  as well. The recessive genetic model also provided the evidence for the role of rs2856838 in CRC susceptibility among Chinese Han women . Additionally, rs3783521 homozygous genotype “G/G” distributed differently between the case and control group . Further genetic model analysis revealed that SNP rs3783521 was linked to an increased risk of CRC in females according to the recessive model . However, no statistically significant association was detected between SNP rs3783546 and CRC risk in Chinese Han women.In accordance with the stratified analysis by gender, we found that the IL1A polymorphisms  were mapped to a 10-kb LD block and displayed three haplotypes with frequencies of more than 5% in our subjects (Table IL1A LD block represented significant linkage between the five SNPs. The LD degree is displayed by standard color schemes with bright red for very strong LD (D’ = 1). According to the haplotype analysis, there were no statistically significant differences appearing between patients and controls among any of the IL1A haplotype frequencies.Finally, five IL1A expression in skin, testis or pituitary, which indicated the effects of different genotypes on gene expression  were associated with CRC susceptibility in Chinese Han females.In this study, we genotyped five polymorphisms of IL1A polymorphisms may play causal roles in the development of cancer.Interleukin-1 alpha (IL-1α) is a major member of the interleukin-1 (IL-1) family, which has been well established as pro-inflammatory cytokine cluster involved in tumorigenesis. Several studies have showed that IL-1α contributes to tumour invasion, tumour proliferation, tumour metastases and interaction between the host immune system and malignant tumour cells , 19. KasIL1A polymorphisms are associated with several cancers [IL1A SNPs, rs3783550, rs3783546, rs1609682, and rs3783521, were associated with a significantly increased risk of renal cell carcinoma [IL1A rs3783553 polymorphism and a significantly decreased risk of gastric cancer  [IL1A rs1800587 polymorphism was related to a higher risk of multiple myeloma development [p = 0.036). After stratification by gender, we found that the recessive model of rs3783550, rs2856838, rs1609682, and rs3783521 were associated with increased CRC risk in females. Moreover, it should be noted that the incidence rate of CRC differs significantly according to gender [IL1A variants rs3783550, rs2856838, rs1609682 were located in the intron, and rs3783521 were in promoter. As both the intron and promoter play important roles in gene expression, the alterations of the SNPs might also influence gene via modulating the functions of these regions. In addition, gender may reflect the different levels of exposure to risk factors related to diet, occupational exposure, or lifestyle. However, due to the limitations of the collected data, it was only possible to study the effect of gender rather than the complete impacts of other factors contributing to the gene environment. Therefore, it is necessary to carry out a complete analysis on the interaction of these factors with genetic polymorphisms in CRC susceptibility to verify the results of this study.Increasing evidence has showed that  cancers . Fei Wanarcinoma . Xiaofen = 0.02) . Abazis-o gender . ConsideIL1A gene expression did not verify in our population and the underlying mechanism upon this regulatory process still need to be further elucidated. Further research will be design to investigate the influence of the promising SNPs on intron and promoter functions in gene regulation, and the gender will be considered as an important factor in future research. Secondly, although variations of age and gender were considered during our analysis, some living habits were not evaluated in this study, such as alcohol consumption and cigarette smoking, which might facilitate the progression of CRC.Several limitations should be considered in this research. Firstly, the influence of the selected SNPs on IL1A genetic polymorphism rs2856838 might contribute to the risk of CRC in Chinese Han population. In addition, rs3783550, rs1609682, rs3783521, and rs2856838 were susceptible pathogenic variants for CRC among females. These results could suggest new targets for CRC risk assessment, prevention and prognosis.Our study first provided objective evidence that the Additional file 1:Table S1. Primers used for identification of the IL1A polymorphisms. Table S2. Distributions of age and gender in CRC patients and controls. Table S3. Functional annotation of the selected variants provided by HaploReg 4.1. Table S4. eQTL analysis for the five IL1A SNPs provided by GTEx database. (DOCX 24 kb)


Hair-coloring products include permanent, semi-permanent and temporary dyes that vary by chemical formulation and are distinguished mainly by how long they last. Domestic temporary hair dyes, such as fuchsin basic, basic red 2 and Victoria blue B, are especially popular because of their cheapness and facile applications. Despite numerous studies on the relationship between permanent hair dyes and disease, there are few studies addressing whether these domestic temporary hair dyes are associated with an increased cancer risk. Herein, to ascertain the bio-safety of these temporary hair dyes, we comparatively studied their percutaneous absorption, hemolytic effect and cytotoxic effects in this paper. Furthermore, to better understand the risk of these dyes after penetrating the skin, experimental and theoretical studies were carried out examining the interactions between the dyes and serum albumins as well as calf thymus (CT)-DNA. The results showed that these domestic temporary hair dyes are cytotoxic with regard to human red blood cells and NIH/3T3 cell lines, due to intense interactions with bovine serum albumin (BSA)/DNA. We conclude that the temporary hair dyes may have risk to human health, and those who use them should be aware of their potential toxic effects. Given their widespread use, the impact of hair dyes on human health has been a topic of intensive study and discussion in recent years ,2. Hair-Despite significant research on permanent hair dye materials, the study on the relationship between cancer and temporary hair dyes is still in its infancy . EspeciaRecently, some toxicologists have challenged this viewpoint and begun to shine the light on the toxicity of these dye materials, since after cleavage many of the temporary hair dye molecules can also form toxic aromatic amines like PPD . Orange Herein, we examined the bio-safety of three domestic temporary hair dyes: Fuchsin basic (FB), Victoria blue B (VBB) and basic red 2 ) is often employed to analyze the quenching mechanism ,24:(1)F011 to 2.34 × 1012, which is much larger than the largest dynamic quenching constant for biological molecules, 2.0 × 1010  (Aobsd refers to the observed absorption at a given dye), the extinction coefficient for the free dyes and the extinction coefficient for the dyes in the fully bound form, respectively.For absorption experiment, to compare quantitatively the affinities of the dyes bound to DNA, the intrinsic binding constants K’http://autodock.scripps.edu/) and DS Visualizer software . The three-dimensional structural information of BSA and DNA was from http://www.rcsb.org/pdb/ . The 3D structures of the dyes were generated in Discovery Studio 4.5 Client. The grid center coordinates of the box along the x-, y-, z-axes were set to 126 Å, 126 Å and 126 Å for BSA–dye interactions and 88 Å, 86 Å and 126 Å for DNA–dye interactions, respectively. The Lamarckian genetic algorithm was employed to search the docking conformation.Molecular model docking simulations were based on the AutoDock program  downloaded from the In recent decades, whether temporary hair dyes do harm to human health has been controversial with limited data available to address this issue. For the first time, to the best of our knowledge, we have explored the bio-safety evaluation of three direct hair dyes , with ethically permitted strategies. In the present work, the percutaneous absorption, cytotoxic effect on RBC of human blood and NIH/3T3 cells, and the BSA/DNA interactions of these three hair dyes were comparatively studied. FB and BR2 can potentially be absorbed through human skin and all three dyes have cytotoxicity on human RBC and NIH/3T3 in very light concentration. Moreover, experimental and theoretical studies suggest that the three dyes have intense interactions with BSA and CT-DNA. This leads us to conclude that the three temporary hair dyes could result in significant potential risk to human health and those who use them should be aware of their potential toxic effects.


Climate change threatens coffee production and the livelihoods of thousands of families in Mesoamerica that depend on it. Replacing coffee with cocoa and integrating trees in combined agroforestry systems to ameliorate abiotic stress are among the proposed alternatives to overcome this challenge. These two alternatives do not consider the vulnerability of cocoa and tree species commonly used in agroforestry plantations to future climate conditions. We assessed the suitability of these alternatives by identifying the potential changes in the distribution of coffee, cocoa and the 100 most common agroforestry trees found in Mesoamerica. Here we show that cocoa could potentially become an alternative in most of coffee vulnerable areas. Agroforestry with currently preferred tree species is highly vulnerable to future climate change. Transforming agroforestry systems by changing tree species composition may be the best approach to adapt most of the coffee and cocoa production areas. Our results stress the urgency for land use planning considering climate change effects and to assess new combinations of agroforestry species in coffee and cocoa plantations in Mesoamerica. In Mesoamerica – the area comprising Panama to central Mexico – the productivity of Arabica coffee (Coffea arabica L.) is expected to drastically decline as suitable growing areas shift2, and pests and pathogens incidence increases under unfavourable climate conditions4.Adapting agricultural systems to climate change is particularly challenging for perennial crops that take long before farmers fully benefit from their management decisions. Yet, a sense of urgency has developed among farmers, scientists and policy makers across the tropics as climate warming and extreme weather events compromise the productivity of major perennial crops2 an ever growing number of news and blogs from private sector, NGO’s and research organisations are reporting the replacement of coffee by cocoa in zones under 600 m a.s.l. (above the sea level) mainly in Mesoamerica , and a scenario with high emissions RCP 8.5, which predicts an average temperature increase of 2.0 °C (1.4–2.6 °C) by 2050 (period 2046–2065). We focus on climate projections for the 2050 s to align with the United Nations framework of global challenges in agriculture and food security13. For simplicity, we focus the results in the intermediate scenario and included the variation between the two scenarios assessed here into the main text, the full results for climate change scenario with high emissions are available as supplementary information.To evaluate these two alternatives, shifting coffee-cocoa plantations or maintaining and promoting crops-agroforestry, we assessed the vulnerability of both coffee and cocoa under climate change and the potential impacts of climate change on the habitat suitability for 100 of the most common tree species in coffee and cocoa plantations across Mesoamerica. We modelled current and future climatic niches with ensemble modelling algorithms19. Our model projections show that 83–87% of current cocoa areas will remain suitable, especially in the humid areas along the Atlantic coast (0–300 m a.s.l.)  Fig. , Text S1Cocoa could potentially replace 85% of the vulnerable coffee areas under climate change in moist regions at elevations under 400 m a.s.l. and 53% at elevations between 400–700 m a.s.l. Areas to be replaced decrease sharply with altitude with no possibility beyond 1,200 m.a.s.l under RCP 4.5 and 1,600 m.a.s.l under RCP 8.5 Fig. .Figure 2N-fixing and timber in mid-altitudinal coffee areas (400–700 m a.s.l.) and lowland cocoa areas , Psidium guajava (guava) and Manguifera indica (mango) are among the most vulnerable species with average loss of 53% in suitable areas. Major gains (>15%), however, are found for species such as Spondias mombin (jobo) and Manilkara zapota (sapodilla) in coffee, Melicoccus bijugatus (mamon) in cocoa and Tamarindus indica (tamarind) in both coffee and cocoa areas , Inga oerstediana, I. ruiziana and I. jinicuil (guama) are the most vulnerable to expected climate change, with losses of 56% in suitable areas. Only two species, of the selected, may expand their suitability in >26% across cocoa areas, Inga laurina (guama) and Senna atomaria (vainillo), but only up to 4% in future coffee areas.High losses (>15%) are expected for 25 of the 30 eas Fig. . Most coCedrela odorata (cedar), as well as, the locally important timber species Perymenium grande (tatascán) and Pachira quinata (pochote), in both coffee and cocoa areas , Ceiba pentandra (ceiba) and Guazuma ulmifolia (guácimo) in both coffee and cocoa areas.In the case of timber trees, we estimate loses of >15% for 22 of 37 species in coffee and 12 tree species in cocoa areas. The most vulnerable timber species include the widely common eas Fig. . MarginaN-fixing species and 10 timber species). Most of these tree species are already present in coffee plantations but mainly in low densities and remain underutilised. Only 9% of coffee areas have very low tree species options (≤3 species).Despite the overall losses in suitability for some of the most popular tree species, our projections suggest that agroforestry could persist as a viable alternative to manage coffee and cocoa plantations in Mesoamerica under climate change. By 2050, approximately 72% of coffee areas  will be suitable for more than 30 tree species. This includes a portfolio of at least 10 species per main use  potentially available 25 stressing the comparative advantage of the region for cocoa production. Despite this large potential, currently Mesoamerica is a minor player in the global cocoa supply chain . In general, cocoa production systems in the region include smallholders, with low levels of input use, old plantations and low yields (60–328 kg ha−1 year−1)26. It is argued that this panorama could change substantially if, for instance, farmers used to the management of a specialised perennial crop such as coffee, turn their efforts to cocoa production.Large areas are highly suitable for cocoa production in Mesoamerica under current climatic conditions and this suitability remains under climate change in 2050, opposing to the trends reported for the current largest cocoa production countries in West Africa27 and the capacity to meet the strict existing quality standards. Still, the potential of the region remains large, but fuelling cocoa expansions will require well-structured efforts to i) reduce barriers to transformation, ii) ensure coupling of production to markets and iii) adequate land use planning to avoid expansion of cocoa into natural forests29 .Only considering the coffee vulnerable areas to climate change that will be suitable for cocoa in 2050 , there could be 7.5 M ha in Mesoamerica available for cocoa production. Even at the extremely low yields typical of the region, these potentially new producing areas could add 1.5 million tons of cocoa to the global supply. In reality the actual coffee areas that can be replaced by cocoa will be lower than these estimated areas, because farmers may lack financial capacities to transform their coffee plantations30. However, it seems highly probable that current agroforestry schemes will need to be modified in terms of species composition, since some of the most popular tree species are also vulnerable to future climate. It is particularly concerning the losses in habitat suitability of N-fixing trees such as E. poeppigiana (poró) and the majority of Inga species. These species make up the most abundant agroforestry trees in coffee and cocoa plantations in Mesoamerica32, and have a key role for the management of soil fertility and sustain more stable productivity34, especially in low-input and small farming plantations35. Therefore, our results anticipate a serious threat for future coffee and cocoa plantations if alternatives for N-fixing species are not promptly identified.Alternatively, by managing agroforestry systems, farmers could potentially maintain their current coffee and cocoa plantations using suitable trees to ameliorate microclimatic conditions. This alternative could also prevent the expansion of agricultural activities towards protected areas that are reported to be suitable in the futureC. odorata (cedar), E. poeppigiana (poró), Inga spp., M. indica (mango), P. americana (avocado) and P.guajava (guava), all widespread in agricultural fields or open areas and of easy regeneration and propagation. We found that some currently underutilised tree species in coffee and cocoa plantations could potentially maintain or even increase their suitable distribution ranges under future climate, such as the fruit trees M. sapota, S. dulcis, Brosimum alicastrum, and the timber trees Simarouba glauca and Ceiba pentandra. These species are present in low densities in coffee and cocoa plantations, and most of them are remnants of previous vegetation36.Rethinking current agroforestry species composition in coffee and cocoa landscapes requires the identification of the best tree species. Currently, farmers have a clear preference towards few species such as 39, farmers’ perceptions and local knowledge regarding management and utilisation of these tree species, as well as market incentives to facilitate their wider use. In our assessment, we employed a species distribution modelling (SDM) approach disregarding these aspects. Therefore, the interpretation of our results is driven by the expected changes in biophysical conditions characterised here as changes in extreme precipitation and temperature events. The evidence has shown that these changes are particularly important for agroecosystems in Mesoamerica, and other regions affected by El Niño Southern Oscillation, in which this phenomenon shapes the ecosystem productivity40, not only across dry regions but also in rainforests19.Expanding the use of underutilised species in agroforestry systems will require a deeper understanding of their agronomic performance considering other factors beyond just climate , ecological interactionsHere we show that coffee systems are more vulnerable than cocoa systems to climate change. Not only is coffee more sensitive than cocoa to future climate, but also the tree species commonly used in coffee plantations are more vulnerable to the expected climate change. Cocoa as an alternative to coffee could potentially occur in most of the vulnerable coffee areas, but this will require addressing other ecological constraints, the impacts of pest and diseases, costs of technological change and market requirements to determine the real potential of cocoa to replace coffee. Adapting coffee and cocoa to changing climates can benefit from agroforestry systems with a new set of currently underutilised tree species already present in coffee and cocoa plantations. The results of this study are a starting point to develop lines of research that support the re-design of agroforestry schemes and open new venues of research to adapt coffee and cocoa production systems in Mesoamerica.43; (ii) ecological and economic services identified by farmers45; and, (iii) availability of a minimum of 60 records to ensure accurate modelling results46.We selected 100 of the most commonly used tree species in cocoa and coffee plantations across Mesoamerica  are native of the neotropics; the others are economically important species and naturalised fruit trees in Mesoamerica  and 33 species mainly used as fruit trees47, MAPFORGEN48 and from the database of farm inventories used to select the tree species. No distinction was made between locations from natural forests or farms because this information was not always available in the original sources.We compiled presence location points of selected tree species (including coffee and cocoa) from the Global Biodiversity Information Facility (GBIF)49, removing the mismatches. Presence locations from 1959 or before were also removed to meet the current baseline climate used. Finally we reduced the possible effects of sampling bias and spatial autocorrelation through systematic sampling50. This approach consists in create a grid of a defined cell size (in our case 2.5 arc-min) and randomly sample one presence points per grid cell. In the Fourcade et al.50 assessment, the approach showed well performance among the other tested approaches irrespective the species and bias type, which is our case.Records with no geographic information or with obvious errors such as incomplete coordinates, locations in the ocean and mismatches between administrative data and coordinates were excluded from the analysis. For this, we compared the collected presence data and information on administrative boundaries with information from the DIVA-GIS database51 package dismo52.The final dataset with validated and unbiased presence locations comprised 130,480 occurrences for the 100 tree species combined 53, whereby the variables with the highest correlation (VIF > 10) were removed, resulting in nine bioclimatic predictors. Which were: (i) bio02, mean diurnal range; (ii) bio03, isothermality; (iii) bio08, mean temperature of wettest quarter; (iv) bio09, mean temperature of driest quarter, (v) bio13, precipitation of wettest month; (vi) bio14, precipitation of driest month; (vii) bio15, precipitation seasonality; (viii) bio18, precipitation of warmest quarter; and, (ix) bio19, precipitation of coldest quarter.We used bioclimatic predictors (baseline period of ~1960–1990) from WorldClim23. We selected the intermediate scenario RCP 4.5, which predicts an average temperature increase of 1.4 °C (0.9–2.0 °C), and a scenario with very high emissions RCP 8.5, which predicts an average temperature increase of 2.0 °C (1.4–2.6 °C) by 2050 (period 2046–2065). We focus on climate projections for 2050 to align with the United Nations framework of global challenges in agriculture13. For each selected scenario, we predicted species suitability using the 17 General Circulation Models (GCM) available for both RCP scenarios (Supplementary Table We based the projections of future distribution in 2050 s on two Representative Concentration Pathways scenarios (RCPs) of climate change from the Intergovernmental Panel on Climate Change (IPCC)51 using a consensus method for species distribution modelling (SDM) compiled by the package BiodiversityR21, which calculate ensemble suitability as a weighted average of probabilities predicted by 17 SDM algorithms  criterion computed by the R package PresenceAbsence56. Although some authors tend to criticise this method, the evidence57 has shown that AUC has strong correlation with the presence-absence threshold that makes sensitivity equal to specificity and remains a valid measure of relative model performance. Considering that, predictions from each of the 17 SDM algorithms were transformed to AUC weights by dividing each by the total of all AUC predictions. We selected the SDM algorithms with AUC weights >0.05, which means at least 5% of contribution to the consensus predictivity21, and recalculated weights to sum to one53. The AUC values for the selected SDM models are shown in supplementary information Fig. S7.For the model calibration, we performed a 4-fold cross-validation by randomly assigning (without replacement) location data to four bins. The performance of different SDM algorithms was evaluated for each bin separately after algorithms were calibrated with data from the other three bins. The SDM performance was assessed by the area under the curve  + specificity (true negative)60, which requires at least 66% of agreement among GCMs to keep the predicted presence or absence in a given grid cell.Since there are no criteria to assess which of the GCMs best predict future climate, by incorporating all 17 GCMs we included all plausible changes in the distribution of the focal species. The results of the 17 GCMs presence-absence layers were integrated into a single layer, using the criterion of likelihood scale61 and tidyverse62. Layers were processed using the R packages maptools63, raster64, rgeos65 and rgdal66. To produce Figs 67 and svglite68 were used.Organising the datasets relied on R packages magrittrSupplementary Information


Inflammation and oxidative stress are recognized as important contributors to Parkinson’s disease pathogenesis. As such, genetic variability in these pathways could have a role in susceptibility for the disease as well as in the treatment outcome. Dopaminergic treatment is effective in management of motor symptoms, but poses a risk for motor and non-motor adverse events. Our aim was to evaluate the impact of selected single-nucleotide polymorphisms in genes involved in inflammation and oxidative stress on Parkinson’s disease susceptibility and the occurrence of adverse events of dopaminergic treatment.NLRP3 rs35829419, CARD8 rs2043211, IL1β rs16944, IL1β rs1143623, IL6 rs1800795, CAT rs1001179, CAT rs10836235, SOD2 rs4880, NOS1 rs2293054, NOS1 rs2682826, TNF-α rs1800629, and GPX1 rs1050450. Logistic regression was used for analysis of possible associations.In total, 224 patients were enrolled, and their demographic and clinical data on the disease course were collected. Furthermore, a control group of 146 healthy Slovenian blood donors were included for Parkinson’s disease’ risk evaluation. Peripheral blood was obtained for DNA isolation. Genotyping was performed for IL1β rs1143623 C allele with the risk for Parkinson’s disease . CAT rs1001179 A allele was significantly associated with peripheral edema . Other associations observed were only nominally significant after adjustments: NOS1 rs2682826 A allele and excessive daytime sleepiness and sleep attacks , SOD2 rs4880 T allele and nausea/vomiting , IL1β rs1143623 C allele and orthostatic hypotension , and NOS1 rs2682826 A allele and impulse control disorders . We did not find any associations between selected polymorphisms and motor adverse events.We observed a nominally significant association of the CAT genetic variability and peripheral edema was observed as well. Therefore, the results of our study suggest some links between genetic variability in inflammation- and oxidative stress-related pathways and non-motor adverse events of dopaminergic treatment. However, the investigated polymorphisms do not play a major role in the occurrence of the disease and the adverse events of dopaminergic treatment.Apart from some nominally significant associations, one significant association between The online version of this article (10.1186/s12974-019-1439-y) contains supplementary material, which is available to authorized users. Inflammation and oxidative stress are recognized as important mechanisms in pathogenesis of Parkinson’s disease’ (PD) . HoweverNeuroinflammation is one of the main pathological hallmarks of PD . MicroglThree main processes lead to ROS production in PD brain . First, IL-1β rs16944 has been associated with PD risk [TNF-α rs1800629 has been associated with the disease risk [TNF-α SNPs [IL6 rs1800795 [NOS1. NOS1 rs2682826 has already been associated with levodopa-induced dyskinesia [NOS1 polymorphisms have also been connected to the disease risk [SOD2 rs4880 was not found to be associated with PD risk [Several studies evaluating the effect of genetic polymorphisms or alterations in proteins’ functions in the inflammation- and oxidative stress-related genes on the risk for PD in either cell culture, animal models, or human samples have already been performed, but conflicting results were presented. Some studies have already linked the NLRP3 function to PD risk , 30. Fur PD risk , 32. Alsase risk , but latase risk . MoreoveF-α SNPs , 35 and s1800795  showed tskinesia . NOS1 poase risk –39, althase risk . GPX1 [4ase risk –43 and C PD risk , 46.Activated inflammation pathways and increased oxidative stress contribute to death of dopaminergic neurons in SNpc and consequential dopamine depletion. Therefore, dopamine replacement strategy has a central role in PD management . DopaminAlthough the association of genetic variability of inflammation and oxidative stress genes with the risk of PD has already been explored to some extent, the association with the treatment outcome has not been studied yet. First, our aim was to evaluate the possible association between selected SNPs in inflammation and oxidative stress pathways with the risk of PD. Furthermore, it has been suggested that pathways involved in disease pathogenesis may also influence treatment outcome . The latA total of 224 PD patients were enrolled in this retrospective cohort study. Patients were recruited in succession and evaluated at the Department of Neurology, University Medical Centre Ljubljana, Slovenia between October 2016 and January 2018. Inclusion criteria were (1) diagnosis of PD according to the UK Parkinson Disease Society Brain Bank criteria  by an exPatients and their caregivers underwent a structured interview to obtain clinical and demographic data. Information was additionally obtained from medical records. We focused on eight AEs of dopaminergic treatment as main end points: motor fluctuations, dyskinesia, EDS and sleep attacks, VH, nausea/vomiting, OH, PE, and ICDs. The AE was defined as absent or present according to clinical examination, clinical documentation, and patients’ answers to specific questions. The time of the AE occurrence after treatment initiation was not taken into account.A control group of 146 healthy unrelated Slovenian blood donors, aged 50 to 65 years, were included in the study for the purpose of PD susceptibility evaluation.The study protocol was approved by the Slovenian Ethics Committee for Research in Medicine (KME 42/05/16). All subjects gave written informed consent in accordance with the Declaration of Helsinki.NLRP3 rs35829419, CARD8 rs2043211, GPX1 rs1050450, and SOD2 rs4880; five promoter SNPs IL1β rs16944, IL1β rs1143623, TNF-α rs1800629, IL6 rs1800795, and CAT rs1001179. Additionally, the SNP function prediction tool was used [NOS1 (rs2293054 and rs2682826) and one SNP in CAT (rs10836235). We selected SNPs with minor allele frequency at least 2% and with determined function based on literature and/or in silico prediction.Nine candidate genes were studied on the basis of their direct involvement in the signaling cascades of the inflammation and oxidative stress pathways , 51, 52,was used  to selecPeripheral blood samples were obtained for DNA extraction. Genomic DNA was isolated using the FlexiGene DNA Kit  according to the manufacturer’s protocol. Genotyping was performed for 12 SNPs. Nine of them were genotyped with KASPar assays  according to the manufacturer’s instructions. Three of the SNPs were genotyped with TaqMan genotyping assays  also according to the manufacturer’s protocol. Ten percent of samples were genotyped in duplicate as quality control, and all the results were concordant.U test was used for the assessment of the effect of numerical data on the AEs. Logistic regression was used to calculate odds ratios (ORs), and 95% confidence intervals (CIs) to examine the associations of selected SNPs and clinical data with the risk for AEs of dopaminergic treatment. Dominant, additive, and recessive genetic models were used for analysis depending on the genotype frequencies. All statistical tests were two-sided. Bonferroni correction was used to account for multiple comparisons to prevent false positive results. For genetic data, p values up to 0.0042 (0.05/12) were considered statistically significant, while p values between 0.0042 and 0.0500 were considered nominally significant. For clinical data, p values up to 0.0063 (0.05/8) were considered statistically significant, while p values between 0.0063 and 0.0500 were considered nominally significant. The study power was calculated for each of the eight AEs separately due to their different frequencies. Three allele frequencies were used for power calculations for each AE, namely minimum polymorphic allele frequency of 6%, average polymorphic allele frequency of 30%, and maximum polymorphic allele frequency of 69%. Power calculations were conducted by the PS Power and sample size calculations, version 3.0, and are presented in the Additional file Median and 25th to 75th percentile range were used to describe central tendency and variability of continuous variables, while frequencies were used to describe the distribution of categorical variables. The agreement of genotype frequencies with Hardy-Weinberg equilibrium and univariate analyses of the individual effects of categorical variables on the AEs were conducted by chi-squared test. Nonparametric Mann-Whitney 042 0.05/2 were coWe evaluated the possible associations of tested SNPs with PD susceptibility. The control group consisted of 146 healthy blood donors with the median age of 56 years. There were 113 men and 33 women in the control group.p < 0.001).General demographic and clinical characteristics of PD patients are presented in Table NLRP3 rs35829419 and NOS1 rs2293054 in the control group where the frequencies did not meet the HWE requirements. Moreover, they also deviated from the genotype frequencies reported for the European population within the 1000 Genomes project. Consequently, these two SNPs were excluded from the PD susceptibility analysis.Frequencies for the 12 investigated SNPs in both the patient and the control group are presented in the Table IL1β rs1143623 and the risk for PD. The carriers of at least one C allele and also heterozygotes had lower odds for developing PD. The association remained nominally significant even after adjustment for age and sex . The results are presented in the Table When PD susceptibility analysis was performed, univariate logistic regression analysis showed no statistically significant results. The only nominally significant result was the association between p = < 0.001). Furthermore, patients ever being treated with DAs also had more than three times higher odds for developing nausea/vomiting  and 12 times higher odds for developing ICDs . Regarding motor AEs, tremor-predominant PD decreased odds for the development of motor fluctuations and dyskinesia for more than twice , while ever being treated with DAs increased odds for the development of these two AEs for more than six and five times, respectively . Also continuous clinical data  showed some significant associations with certain AEs  on the occurrence of AEs Table  showed tCAT rs1001179 A allele carriers . Furthermore, heterozygotes had significantly lower odds for PE development  as well. Carriers of the NOS1 rs2682826 A allele had nominally significant higher odds for developing EDS and sleep attacks . Under additive genetic model, only carriers of the AA genotype had nominally significant higher odds for this AE development . Heterozygotes for the GPX1 rs1050450 had nominally significant higher odds for VH development . Nausea and vomiting were also less likely to occur in carriers of the SOD2 rs4880 T allele , but the association was only nominally significant. The IL1β rs1143623 T allele showed a nonsignificant trend towards association with lower odds for developing OH , while heterozygotes had nominally significant lower odds for developing OH . The investigated SNPs were not associated with ICD development or with the motor AEs after univariate analysis. Results of the univariate analyses under dominant and recessive models for genetic data are presented in Table Univariate analysis of the effect of genetic variability on the occurrence of AEs also showed some significant and nominally significant associations. Significantly lower odds for the PE occurrence were detected in the polymorphic Adjustments of statistically significant and nominally significant genetic associations for clinical data were carried out in the multivariate logistic regression analysis. Since all four continuous clinical parameters  appeared to be mutually correlated, which was checked by the Spearman’s correlation coefficient, only age at diagnosis was used for adjustments. Additionally, significant and nominally significant genetic associations . The association of NOS1 rs2682826 A allele with higher odds for developing EDS and sleep attacks remained nominally significant . Furthermore, IL1β rs1143623 C allele retained a nonsignificant trend towards association with lower odds for developing OH . Furthermore, the SOD2 rs4880 T allele association with nausea/vomiting was adjusted for sex, ever being treated with DAs, and age at diagnosis. The genetic association was still nominally significantly associated with lower odds for developing nausea/vomiting even after the adjustment . Results are presented in Table Associations of the NOS1 rs2682826 A allele carriers had higher odds for developing ICDs after adjusting for age at diagnosis and ever being treated with DAs   sleep phase and non-REM sleep cycle during the night, which could lead to EDS and sleep attacks during the day [NOS1 gene expression and consequently cause daytime wakefulness disturbances by the mechanism similar to the above mentioned mouse model.EDS and sleep attacks affected 35.5% of patients in our cohort, which is in agreement with previous data stating that this AE manifests in up to 50% of patients , 57. We  the day , 62. As  the day , it coulSOD2 rs4880 may play a role in this AE’s occurrence. Carriers of the SOD2 rs4880 T allele had nominally significant lower odds for developing nausea/vomiting. This SNP has been reported to decrease enzyme’s function [SOD2 rs4880 might play a role in the nausea/vomiting development as also suggested by our study. Although nausea/vomiting may also result from the direct effect of levodopa on the area postrema of the brain stem [In total, 29.6% of patients in our cohort experienced nausea/vomiting, and according to the literature, this is the most common AE of dopaminergic replacement therapy . We detefunction . There ifunction . Aggregafunction . ENS is function . It has function . As aggrfunction , the latain stem , there iIL1β rs1143623 C allele, which decreases the promoter activity [Another common AE of dopaminergic treatment is OH. It affected 37.7% of patients in our cohort, which is comparable to the data in literature . Howeveractivity . Dopaminactivity , 71. Somactivity , 74, butCAT rs1001179 A allele significantly lowers odds for developing the edema for more than two times. This SNP has been reported to increase gene’s expression [Rates of peripheral edema vary from 5 to 16% of patients treated with DAs , which ipression . The mecpression .NOS1 rs2682826 A allele with higher odds for ICDs after adjustment for significant clinical parameters. SNPs of NOS1 were already associated with several psychiatric disorders such as obsessive compulsive disorder, anxiety, and depression [Furthermore, ICDs appeared to be significantly associated with DAs and with age at diagnosis. DAs have already been strongly associated with this AE , which ipression , 77.NOS1 rs2682826 with levodopa-induced dyskinesia [Our study did not confirm the results of Santos-Lobato et al., which showed an association of the skinesia . We alsoskinesia , 32, 36.Although our study presents novel findings and is designed with a different pathway-based approach to clarifying AEs’ mechanisms, some limitations have to be considered. The control group in the risk analysis is not matched by sex and age. However, we have considered this in the statistical analysis by adjusting for these two parameters. The patient cohort is of moderate size, although it is comparable to the sample sizes of similar PD pharmacogenetics studies. The time of AEs’ occurrence in relation to medication initiation was not taken into account. All of the AEs were analyzed as categorical variables, but with the use of clinical scales to evaluate the severity of various AEs, we could look into possible associations in more depth. Furthermore, prospective study would have a greater chance to detect even subtler and timely relations between treatment and AEs. It should also be noted that our results should be validated in a different independent patient population, before they could be applied in a clinical practice.Nevertheless, even though it is known that AEs of dopaminergic therapy can be alleviated to some extent by appropriate treatment modifications in many PD patients, AEs still impact their quality of life . In lighIL1β with the risk for PD or NOS1, SOD2, IL1β, and CAT with certain non-motor AEs of dopaminergic replacement therapy; however, the evidence presented here is limited. Further association and functional studies are warranted.To the best of our knowledge, this is the first study using pathway-based approach to address the relationship between inflammation and oxidative stress polymorphisms and risk for PD or AEs of dopaminergic treatment. We were able to detect some indication of possible associations of genetic variability in NOS1, SOD2, IL1β, and CAT present possible candidates for future studies on predictive biomarkers of non-motor AEs.The results of this study confirm the possible association of the inflammation pathway with the risk for PD. Furthermore, the results of this study are the first indication that inflammation and oxidative stress pathways may be involved in the pathogenesis of non-motor AEs of dopaminergic treatment in PD, although they may not play a major role in the process. Further studies on independent samples are warranted to confirm the involvement of genetic variability in these pathways on the occurrence of AEs of dopaminergic treatment in PD. Furthermore, functional studies would possibly lead us to new knowledge on AEs’ pathogenesis and their possible management. For now, SNPs of Additional file 1:Table S1. Power calculations. Table S2. SNPs included in the study with their predicted and experimentally determined functions. Table S3. Excessive daytime sleepiness and sleep attacks, visual hallucinations, and nausea and vomiting. Table S4. Orthostatic hypotension, peripheral edema, and impulse control disorders. Table S5. Motor fluctuations and dyskinesia. (DOCX 56 kb)


Scarpa’s fascia preservation plays a great role in decreasing the volume of wound drainage and reducing seroma after abdominoplasty. This study aimed to assess the role of Scarpa’s fascia preservation in patients with ventral hernias associated with abdominal wall deformity who underwent concomitant hernio-abdominoplasty in terms of early and late postoperative outcome and quality of life.Patients with ventral hernia and abdominal wall deformity underwent combined hernio-abdominoplasty. Patients were randomly allocated to 1 of 2 equal groups: group I underwent Scarpa’s fascia preserving hernio-abdominoplasty and group II underwent hernio-abdominoplasty with removal of Scarpa’s fascia. Volume of drainage, time to remove drains, return to work, and complications were recorded.P = 0.36). The mean total volume of postoperative drainage was significantly lower in group I than group II . Group I had earlier drain removal  and earlier return to work  than group II. There were no recorded cases of hematoma or hernia recurrence after repair.Fifty patients  were included to the study. Both groups had comparable operation time, pain score, and complication rate (24% versus 40%, Scarpa’s fascia preservation in combined ventral hernia repair and abdominoplasty was associated with significantly lower volume of postoperative drainage, earlier removal of drains, and similar recurrence rate to hernio-abdominoplasty with removal of Scarpa’s fascia. Repair of ventral hernias combined with abdominoplasty has been described to improve the contour deformity of the whole musculofascial layer, especially in the waist area.6 To avoid seroma formation, subcutaneous drains are usually used; however, long-term drainage is not advisable, as it may increase patients’ discomfort.10One of the most frequent complications after both ventral hernia repair and abdominoplasty is seroma formation, with a reported incidence reaching up to 20%.11 A prospective trial12 reported a significant decrease in the volume of wound drainage and earlier removal of drains in patients who underwent Scarpa’s fascia preserving abdominoplasty.Preservation of Scarpa’s fascia helps preserve the arterio-veno-lymphatic system and improve the reabsorption of the fluid released from interstitial spaces.The present study aimed to assess the role of Scarpa’s fascia preservation in patients with ventral hernias associated with abdominal wall laxity and deformity who underwent concomitant on-lay prosthetic repair of ventral hernia and abdominoplasty in terms of volume of drainage, time to drain removal, complications, recurrence of hernia, and quality of life. Abdominal wall deformity was defined as excess skin and subcutaneous tissue associated with laxity of the abdominal wall musculature.This was a randomized, single-blinded controlled trial (NCT03721575) on patients with ventral hernias and abdominal wall deformity who underwent on-lay mesh hernioplasty and abdominoplasty in the General Surgery Department, Mansoura University Hospital in the period of January 2016 to January 2018. Ethical approval from the institutional review board of Mansoura Faculty of Medicine was obtained.13Patients included to the trial were adult patients below 60 years with ventral hernias and abdominal wall deformities of class III or IV according to Pitanguy’s classification of abdominal deformities.2 and American Society of Anesthesiologists class I-II were included.Only patients with body mass index (BMI) ≤40 kg/m14 patients with uncontrolled chest problems, uncontrolled diabetes mellitus, or coagulopathy.We excluded patients with (1) major abdominal wall defect warranting abdominal wall reconstruction; (2) recurrent incisional hernias after mesh hernioplasty; (3) complicated hernias defined as inflamed, incarcerated, and strangulated hernias; and (4) heavy smokers who smoke ≥25 cigarettes per day,Patients were randomly allocated to 1 of 2 equal groups: group I underwent Scarpa’s fascia preserving hernio-abdominoplasty and group II (control group) underwent classical hernio-abdominoplasty with removal of Scarpa’s fascia. Both groups underwent on-lay mesh repair of the ventral hernia.www.randomization.com). Allocation concealment was undertaken by sealed envelope method. The study was single-blinded, as the patients were aware of the nature of the study, yet not aware of the group they were allocated to. The operating surgeons were aware of treatment group allocations and the nature of the study.Randomization was conducted by an online software  at the night before surgery.Written informed consents to participate in the trial were obtained from the patients before enrollment to the study. Patients with high risk for thromboembolism according to Geneva risk score for venous thromboembolism17Preoperative pictures were taken in the anterior and lateral views in the anatomical position to compare with postoperative results. The intended sites of surgical incisions were marked while the patient was standing according to Le Louarn.18 whereas Scarpa’s fascia preservation was done according to Le Louarn.17 The procedures were performed by a team of general surgeons that included one of the study authors (A.E.) who had prior training and experience in plastic surgery.Procedures were done under general anesthesia. Two grams of ceftriaxone were given on induction. Classic abdominoplasty without Scarpa’s preservation was done according to Regnault,Lower transverse incision was made first by scalpel, then deepened down to the external oblique aponeurosis and rectus sheath using electrocautery. Dissection was deepened laterally down to the Scarpa’s fascia and the flap was dissected just above Scarpa’s fascia in group I Fig. , whereasThe flap was elevated till the level of the umbilicus, then an inverted V-shaped periumbilical incision was made to separate the umbilicus from the surrounding skin and the whole umbilical stalk was dissected using scissors down to the level of the anterior rectus sheath. The lower part of the upper abdominal flap below the umbilicus was split longitudinally to facilitate subsequent flap elevation above the level of the umbilicus then the hernia sac was dissected off the flap Fig. .The central part of the flap and the Scarpa’s fascia was elevated above the umbilicus using electrocautery till the xiphoid process, dissecting immediately above the anterior rectus sheath Fig. . The disAfter complete flap elevation, the hernia sac was opened and the contents were reduced back to the peritoneal cavity. Pre-taken full-thickness polypropylene 1 sutures were taken before closure of the defect Fig.  about 5 The linea alba was plicated using monofilament polyamide loop 1 suture starting from the xiphoid process in a downward direction reaching just above the umbilicus, then continued downward to the symphysis pubis, making sure that the closed abdominal defect was not included in the midline imbrication. Afterward, a microporous, heavy-weight, uncoated polypropylene mesh was placed above the rectus sheath using the on-lay technique Fig.  with opeIn group I, the central part of Scarpa’s fascia, where the mesh is to be placed, was excised, whereas the lateral parts of the Scarpa’s fascia and sub-Scarpal tissue in the infraumbilical region were preserved. The lateral edges of the Scarpa’s fascia were advanced medially for 2–3 cm to cover the peripheral parts of the mesh and then were fixated to the anterior rectus sheath by 2/0 polyglactin sutures Fig. . No progThe location of the new umbilicus was marked midway between the xiphoid process and symphysis pubis. An inverted V-shaped incision was made and the umbilicus was brought outside the incision and sutured using 4/0 polypropylene sutures. In the cases where the umbilical stalk was excised, an artificial umbilicus was created.After adequate hemostasis was confirmed, 2 passive tube drains of size 24 Fr were brought out at the lateral edges of the wound and the wound was closed in 2 layers; Scarpa’s fascia and deep dermis then the skin using subcuticular 2/0 polypropylene sutures.Follow-up was done in the outpatient clinic at 4, 8, 14, 21, and 30 days postoperatively then every 3 months for 1 year.The wound was inspected for infection, hematoma, or dehiscence. Wound healing and time to stitch removal were recorded. Pain was evaluated with Visual Analog Scale from 0 to 10 where 0 implied no pain and 10 indicated the worse severe pain. Patients were asked to record the volume of drain output on daily basis and the output was assessed by the investigators with regard to the amount and quality during follow-up visits until the drains were removed. Drains were removed when their output was below 30 ml/d.19At 1 month after surgery, the abdominal scar, quality of life, and drain output were assessed and photographs of the abdominal scar were taken. During follow-up, patients were assessed regarding any abdominal wall deformity, excess skin, abdominal wall sensation, body contour, recurrence of hernia, and quality of life as assessed by the Carolinas equation for quality of life.After drain removal, seroma was evaluated in the next visit. Clinical signs of seroma included swelling, discomfort, erythema, pain, and skin edema. Radiologic assessment by ultrasonography was done for clinically suspected seromas.The primary outcome of the study was the total volume of drain output in milliliters. Secondary outcomes included the mean volume of drain output at 2, 4, 8, and 11 days after surgery, time to remove drains, operation time, hospital stay, viability of the flap and umbilicus, and complications including recurrence, postoperative pain, waist circumference, patients’ satisfaction, and quality of life.www.clincalc.com).The sample size of the study was calculated by a power analysis of the primary endpoint of the study  using online software  of 0.80. To compensate for loss to follow-up and dropouts (estimated to be around 20%), 50 patients were ultimately included to the trial.In light of the results of a previous trialt test for paired samples was used to detect differences in the means of continuous variables and Fisher exact test or chi-square test was used for processing categorical variables. P values <0.05 were considered to be significant.Statistical analysis was performed using SPSS software version 17 . Continuous variables were described as mean ± SD. Categorical variables were reported using percentages. Student’s After initial assessment of 61 patients, 11 patients did not meet the inclusion criteria of the study and were excluded and 50 patients were ultimately enrolled to the trial as illustrated in the Consort flow chart . The mean preoperative waist circumference was 114.6 ± 4.6 cm (range 103–121 cm).Patients were 49 (98%) female and 1 (2%) male. The mean age of patients was 40.1 ± 7.7 years (range 28–58 years) and the mean BMI was 36.5 ± 1.6 kg/mTen patients (20%) had previous surgery for ventral hernia repair and 44 patients (88%) had previous abdominal surgery for indications other than hernia .Twenty-seven (54%) patients presented with umbilical hernia, 14 (28%) with epigastric hernia, and 9 (18%) with incisional hernia. The incisional hernias were following Pfannenstiel incision (n = 6) and lower midline incision (n=3). Twenty-five patients had associated medical comorbidities (15 diabetes mellitus and 10 hypertension) and 3 patients were smokers who quit smoking for 6 weeks before surgery.There were no significant differences between the 2 groups in terms of patients’ age, gender distribution, weight, height, BMI, waist circumference, previous abdominal surgery, associated comorbidities, type of ventral hernia, and Pitanguy class as shown in Table No significant difference in the operation time or hospital stay of both groups was observed. The median size of the abdominal wall defect was 9 cm (range 7–17 cm) as measured intraoperatively with the patient relaxed after tissue dissection. The median size of defect for umbilical hernias was 7 cm (range 7–9 cm), for epigastric hernia was 7 cm (range 7–10 cm), and for incisional hernia was 11 cm (range 9–17 cm).P = 1).Three patients (6%) experienced minor ischemia of the abdominal flap with no significant difference between the 2 groups regarding flap viability .In 13 patients, the hernia sac was found encroaching on the umbilical stalk and the umbilicus was excised, whereas in the remaining 37 patients the umbilicus was preserved. None of the patients with preserved umbilicus experienced umbilical gangrene on follow-up. Time to stitch removal in group I (18.4 ± 2.1 days) was similar to group II (18.3 ± 2.5 days) with no statistically significant difference (P = 0.27). The mean postoperative waist circumference in group I was significantly smaller than group II . Patients in group I required significantly shorter time to return to work than group II .Both groups had comparable postoperative pain scores . Drains were removed earlier in group I compared with group II  complications were recorded. Complications were all surgical site occurrences in the form of superficial surgical site infection of the skin and subcutaneous tissue that did not warrant mesh explantation or drainage (n = 5), minor flap disruption defined as partial dehiscence of the flap from its skin attachment due to ischemia or infection (n = 5) and seroma (n = 6). There were no recorded cases of hematoma or recurrence of ventral hernia after repair. No cases of skin necrosis were recorded. There was no significant difference between the 2 groups in regard to postoperative complications  reported by other authors.28 The chief advantage of preserving the Scarpa’s fascia during abdominoplasty is reducing the incidence of seroma. Although the present trial found lower rate of seroma formation in the Scarpa’s fascia preservation group compared with the control group (8% versus 16%), this difference was not statistically significant. Similarly, in the study by Shahin et al.23 none of the patients with preserved Scarpa’s fascia developed seroma compared with 15% of patients in whom the Scarpa’s fascia was removed.In classical abdominoplasty, up to 30% of patients may develop seroma.29 The medial advancement of the preserved Scarpa’s fascia on both sides results in medial traction of the whole superficial fascial system of the lower abdomen with tightening effect on the flanks, improving the waist, and obliterating the lower midline dead space.30Scarpa’s fascia preservation also resulted in a significantly smaller waist circumference, in line with a previous study that concluded enhanced waistline in abdominoplasty with Scarpa’s fascia advancement.None of the patients in either groups experienced recurrence of ventral hernia on follow-up. Nonetheless, because the primary endpoint of the study included short-term outcomes such as the volume of postoperative drainage and incidence of seroma formation, the effect of Scarpa’s fascia preservation on hernia recurrence cannot be ascertained, as it requires longer follow-up.Limitations of the present study include being a single-center study with relatively small number of patients in each group. Moreover, larger, multicenter trials including high-risk patients and patients with major abdominal wall defects warranting abdominal wall reconstruction are needed to reproduce the results of the present trial and examine the feasibility and outcome of Scarpa’s fascia preservation in other patient groups.Preservation of Scarpa’s fascia in combined ventral hernia repair and abdominoplasty was associated with significantly lower volume of postoperative drainage, earlier removal of drains, and smaller waist circumference compared with classical hernio-abdominoplasty with removal of Scarpa’s fascia.


Nasonia vitripennis in Europe and a correlated haplotype frequency for the circadian clock gene period (per). To evaluate if this correlation is reflected in circadian behaviour, we investigated the circadian locomotor activity of seven populations from the cline. We found that the proportion of rhythmic males was higher than females in constant darkness, and that mating decreased rhythmicity of both sexes. Only for virgin females, the free running period (τ) increased weakly with latitude. Wasps from the most southern locality had an overall shorter free running rhythm and earlier onset, peak, and offset of activity during the 24 h period, than wasps from the northernmost locality. We evaluated this variation in rhythmicity as a function of period haplotype frequencies in the populations and discussed its functional significance in the context of local adaptation.Many physiological processes of living organisms show circadian rhythms, governed by an endogenous clock. This clock has a genetic basis and is entrained by external cues, such as light and temperature. Other physiological processes exhibit seasonal rhythms, that are also responsive to light and temperature. We previously reported a natural latitudinal cline of photoperiodic diapause induction in the parasitic wasp  The daily rotation of the Earth around its axis causes oscillating photoperiods that have led to the evolution of a large variety of activity patterns of organisms. Many behavioural and physiological activities, like mating, feeding, and resting, show distinct oscillating rhythms with a peak of activity at a specific moment during the light–dark (LD) cycle. These are driven by an endogenous clock that is reset daily (entrained) by the prevailing LD cycles and runs with an intrinsic period of approximately 24 h in constant darkness (DD) . The lenDay length (photoperiod) also oscillates seasonally and is an important cue for season-dependent behaviours, like migration in birds, hibernation in mammals, and diapause in insects (reviewed in ). AdditiNasonia vitripennis is maternally induced following a certain number of days (switch point) at a given critical photoperiod (CPP) and shows a robust clinal photoperiodic response [period (per) follows the observed cline in photoperiodic diapause induction [Larval diapause of the parasitoid response ,12. Apparesponse . Interesnduction ,14. per haplotypes with seasonal response is reflected in natural variation in circadian activities, that are known to be regulated by the gene period [τ and then analysed the timing and level of locomotor activity for the most southern and most northern populations. Our data indicate that activity timing and average free-running rhythm differ between southern and northern lines of N. vitripennis, suggesting a latitude-dependent effect on the circadian clock, consistent with clinal per haplotypes.To investigate if the observed correlation of e period , we analNasonia vitripennis, we used isofemale lines established from natural field collected populations [Calliphora spp. pupae as hosts in mass culture vials under LD 18:06, 20 ± 1 °C, to minimise diapause induction. For establishing free running periods under constant darkness (DD), 17–25 isofemale lines from each location and 4–8 individuals from each isofemale line were used . As some individuals died before all data were collected, only 1072 individuals could be used for locomotor activity analysis: 548 females (163 virgin and 385 mated) and 544 males (122 virgin and 402 mated). To study variation in locomotor activity in ulations . These lDrosophila activity monitors 2 (DAM2) (www.trikinetics.com) were used for activity registration, with 32 wasps per monitor. All assays were performed in light-tight boxes in temperature-controlled environmental chambers at 20 °C and 50% humidity. The light source in the box consisted of a white light with a maximum light intensity of about 200 lum/sqf. The Trikinetics system monitored how many times per minute each individual wasp interrupted an infrared light beam that passed through the center of the glass tube. To determine free-running period under constant darkness (DD), wasps were first entrained under LD16:8 for 4 days and then subjected to 10 days of DD. To compare daily activity profile under long and short photoperiods, adult wasps were recorded for 10 days in either LD 16:08 or LD 08:16 regime.To quantify animal movement over time, individuals were collected one day after emergence (mated group) or collected as pupae and allowed to develop into adults at room temperature (virgin group) and kept either at LD 16:08 or LD 08:16 based on experimental group. Adults were then individually transferred, without anaesthetization, to glass tubes (diameter 5 mm × height 70 mm) that were half filled with an agar gel containing 30% sugar. Trikinetics http://actogramj.neurofly.de. Double-plot actograms obtained with this software were eye inspected and dead animals were omitted from further analysis. Under constant darkness (DD) it was possible to measure the period of activity (τ) with periodogram analysis available in ActogramJ, which incorporated the chi-square test [tau, with the isofemale line nested into location as random effect (package nlme). The assumptions for parametric statistics were tested through diagnostic QQ plots. Only rhythmic individuals were included in these tests. All statistical analyses were performed with R statistical software . The raw locomotor activity data were first visualised with the program ActogramJ ; availabare test . Linear Under LD conditions the average activity was calculated as described by . The firp < 0.001), with small but significant differences between locations (p < 0.01). There was no correlation with latitude (p = 0.18), either for the overall data or for the sexes separately ((p < 0.01), but no latitudinal cline of rhythmicity was detected when mating status was taken into account (p = 0.20)  to 84% (COR). When considering the complete dataset, males were more rhythmic (90%) than females (68%) (parately . Virgin  = 0.20) . These rτ, which ranged from 22 h in the southern Corsica lines to 27.5 h in the northern Oulu lines. Overall, the free running period was shorter for individuals of southern latitudes and increased towards the north with a shallow but significant latitudinal cline in τ detected only for virgin females (p < 0.001)  and in virgin individuals compared to mated individuals .Among all tested individuals there was a large variation in < 0.001) . The avep < 2−16). Under LD16:08, both southern and northern wasps displayed a unimodal activity pattern . In agreement with the results obtained for the isofemale lines, the southern and northern isogenic lines differed in τ under constant conditions . The aveNasonia vitripennis has a broad distribution and it is thus expected to exhibit natural variation in biological rhythms [per, has already been reported by [N. vitripennis. We observed significant differences between females and males: males were more rhythmic than females and had shorter free-running periods (τ), and this difference was more apparent in mated individuals. Similar differences between sexes were observed by [N. vitripennis AsymC [Camponotus compressus, in which ovipositing queens exhibited arrhythmic locomotor activity during the egg laying phase and restored rhythmicity afterwards [Drosophila montana, in which the proportion of rhythmic individuals was higher at a lower temperature [ rhythms . Latitudorted by ,14. Hereerved by  and in tis AsymC . Interesterwards . In addiperature . More stτ) between sexes and locations. Towards the south, wasps had a faster clock, with τ close to 24 h, whereas wasps from northern latitudes had a slower clock with τ longer than 24 h. In virgin females, we observed a weak, but significant, latitudinal cline for τ, increasing towards higher latitudes. The presence of a positive latitudinal cline from south to north in DD rhythm was previously reported for Drosophila species [Arabidopsis thaliana the free running period under DD increases towards a northern latitude, and correlates with clinal variation in seasonal flowering time, regulated by photoperiodic cycles [τ towards northern latitude) were reported from the mosquito Culex pipiens [Pyrrhocoris apterus [We also found differences in the free running period . Southern and northern wasps displayed profound differences in their daily locomotor activity. Southern wasps were mainly active in the morning, with an increase in activity before the light turned on during the short photoperiod, whereas northern ones presented a unimodal evening activity, with a prolonged evening peak at the shorter photoperiod. This shifted activity pattern between southern and northern wasps can reflect local adaptation. In the south, temperatures are known to become high in the middle to late afternoon, and shifting the activity to the coolest part of the day (the morning) might be a response of insects that live in a hot environment [Nasonia lines have a reduced morning activity with their activity peak in the second part of the day when temperatures are higher. Similar differences in activity patterns between southern and northern populations have been reported for Drosophila, albeit between Drosophila species rather than populations within species [N. vitripennis was rather broad compared to the more precisely timed behaviour of Drosophila melanogaster, possibly reflecting a stronger selection on activity phase in D. melanogaster than N. vitripennis. On the other hand, Nasonia exhibited a stronger photoperiodic response [The period of the circadian clock of ironment ,23. In cironment . Norther species ,25,26,27response . per polymorphisms and circadian phase of activity has also been observed in Drosophila by [per splicing, which determines the phase of the seasonal activity peak [per alleles in Nasonia differ in splicing efficiency or posttranslational modification, but our data are consistent with the scenario reported in Drosophila. Moreover, the observed, albeit weak, cline in τ for virgin females follows that of photoperiodic diapause induction [per participates in photoperiod measurement by fine-tuning critical day length to latitude-dependent requirements, this would suggest an involvement of this clock gene in the photoperiodic timer of Nasonia. Consequently, the cline in free running period would reflect a mere “side effect” of the selection pressure on seasonal rhythms. In agreement with this, recent work by [Nasonia circadian clock that allows wasps to entrain to a wide range of light–dark cycles, including the northern, more extreme, photoperiods, without negative effect on fitness. The association between phila by ,29, whernduction ,14 and cnduction ,18. If pN. vitripennis lines. Many traits related to circadian activity showed a high level of plasticity, which allows flexibility in daily activities, depending on internal conditions  or external environmental conditions . Nevertheless, variation between geographic locations was maintained even in the plastic response to different stimuli, suggesting that natural selection acts on the response of the circadian system to the environment and not on the circadian clock, per se. Clearly, more detailed functional experiments are required to reveal the exact molecular mechanism underpinning circadian clock, photoperiodic timer, and their mutual connections.In conclusion, we described natural variation in the period and phase of daily rhythms between southern and northern


These plants are widely used in the Moroccan traditional folk medicine. The cytotoxic effect was explored against two cancer cell lines, Vero and RD, using the colorimetric MTT assay. The obtained results showed that the cytotoxicity differed according to the used extract with an efficient effect of Cotula cinerea extracts compared to Salvia verbenaca. A potent cytotoxicity was thus observed for the Cotula cinerea hexane extract which inhibited the growth of RD cell line at the lowest IC50 value (57.21±3.43 µg/mL). This was followed by the ethyl acetate extract and the essential oil with moderate effects against RD cell line and showed IC50 values of 187.52±6.27 µg/mL and 173.05±4.46 µg/mL, respectively. On the other hand, different results were obtained and Cotula cinerea essential oil was the most cytotoxic with the lowest IC50 value (72.72±2.18 µg/mL) against Vero cell line. In the same conditions, higher concentrations were needed in the case of Salvia verbenaca extracts. The results of this study showed thus that Cotula cinerea essential oil and hexane extract showed significant cytotoxic effects against RD and Vero cell lines, respectively, and could be considered as novel source of antitumor agents. This study is expected to be beneficial for clinical and traditional applications for Cotula cinerea as a remedy against cancer and opens new perspectives for further investigations on other types of cancer cell lines.The objective of this work was to investigate the cytotoxicological effect of the extracts (hexane, ethyl acetate, and Cancer is a complex disease caused by several factors such as genetic deregulation, environmental influencing agents, and epigenetic modification factors , 2. AccoTraditional forms of medicine, especially the herbal products deployed for centuries in many parts of the world, could be good alternative for scientific investigation due to their attributes against tumor cell lines. Indeed, plants are rich sources of new compounds from which some secondary metabolites have been shown to possess anticancer properties –7. Furth Cotula cinerea and Salvia verbenaca from Morocco. Cotula cinerea which is of the Asteraceae family is a xerophytic plant widely distributed in sandy and desert grounds [ Salvia verbenaca is a Mediterranean plant currently belonging to the Lamiaceae family. The plant is widely distributed in Morocco and is widely used in Moroccan traditional folk medicine as a cholagogue, antiseptic, diuretic, and astringent [ Cotula cinerea and Salvia verbenaca. This work was therefore initiated in order to evaluate the in vitro antiproliferative effect of Cotula cinerea and Salvia verbenaca extracts and essential oil against tumor RD and Vero cell lines.In this work we were interested in exploring the potential cytotoxicological effect of grounds . Popular grounds . Salvia tringent . However Cotula cinerea were collected in March 2014 in the south of Morocco from the region of Dakhla-Oued Ed-Dahab (province of Aousserd). Those of Salvia verbenaca were collected on March 2015 at the locality called Ain Aouda, in the Rabat region, Morocco. The plants were identified by Professor Fennane from the Scientific Institute, Mohammed V University in Rabat. Voucher specimens of both plants were deposited in the herbarium of the Botany Department of this institution. The collected aerial parts were left under shade at room temperature until complete drying.The aerial parts of Cotula cinerea and Salvia verbenaca were crushed and 200 g of each plant material was macerated and stirred for 24 hours at room temperature in an ethanol/water  mixture with frequent stirring. The maceration process was repeated twice during 24 h each in order to achieve an exhaustive extraction. After filtration and evaporation of ethanol, the obtained aqueous solution was successively fractionated with hexane, ethyl acetate, and n-butanol. Then, solvents of the different obtained extracts were evaporated and the dried residues were used to explore their cytotoxic effect. The hexane, ethyl acetate, and n-butanol extraction yields were, respectively, 1.00, 3.00, and 4.50% for Cotula cinerea and 1.58, 4.64, and 5.28 for Salvia verbenaca.The dried aerial parts of Cotula cinerea aerial parts was extracted by hydrodistillation using a Clevenger-type apparatus. The extraction was achieved during six hours and the obtained oil (yield 0.92%) was stored in a glass vial until use.The essential oil of2 and 100% relative humidity atmosphere.The two tumor tested cell lines used in this study were RD  and Vero (Monkey kidney cancerous cell lines: ATCC N°CCL-81). These two species were obtained from the National Institute of Health, Rabat, Morocco, and were cultivated as previously described by Aneb et al., 2016 . BrieflyμL medium MEM containing 4.106 cells/mL were placed in each well containing MEM and incubated at 37°C in 5% CO2/humidified air for 24 h. After 24 h incubation, cells were then treated with the essential oil of Cotula cinerea or its extracts  in addition to those of Salvia verbenaca. Briefly, 100 μL of each extract at concentrations ranging from 31.25 to 500 µg/mL was dissolved in DMSO (1%) and was added to each well in triplicate. The microplates were then incubated for 48 h at 37°C in air condition of 5% CO2. Sterile PBS and 1% DMSO (vehicle) were used as negative controls.Before evaluating the antiproliferative activity, the cellular density of each species was determined using light microscopy. When cellular density reached a subconfluence threshold, cells were attached by trypsin-EDTA, washed twice with phosphate buffered saline (PBS), and centrifuged at 150 rpm for 15 min (4°C) and were then suspended in fresh culture medium (MEM). The enumeration of cells was made using Malassez cell count. Before treatment with extracts, 100 µL of MTT (5 mg/mL) was added to each microwell and incubated for 3 hours at 37°C in 5% CO2. Tetrazolium salts were cleaved to formazan and the reaction was stopped by addition of 100 µL of 50% (v/v) isopropanol-10% (w/v) sodium dodecyl sulfate (SDS) mixture to each well in order to dissolve insoluble formazan formed after tetrazolium dye reduction [50) were obtained by modeling the percentage of cytotoxicity versus concentration of extracts. Experimental data were achieved in triplicate and the obtained results were expressed as means ± SD.The evaluation of cell viability has been achieved through the use of MTT 3- assay. The test is based on the biotransformation of MTT into formazan crystals by mitochondrial dehydrogenases of living cells. The absorbance of the formazan will then be determined by spectrophotometry at 550 nm. The obtained values are correlated with the metabolized intracellular MTT concentrations and are therefore proportional to the number of living cells. Briefly, 20 Cytotoxicity (%) = 100 × (absorbance of untreated cells − absorbance of treated cells)/absorbance of untreated cells. Cotula cinerea and Salvia verbenaca. The three extracts  of each plant in addition to the essential oil of Cotula cinerea have then been assayed for their cytotoxic activity against RD and Vero cell lines using MTT assay.In an ongoing program aimed at the exploration of the phytochemical compounds and the biological activities of medicinal plants of Morocco, we were interested, in this work, in the cytotoxic effect ofThe obtained findings for the two cell lines RD and Vero are summarized in Figures  Cotula cinerea compared to Salvia verbenaca. Comparison of the obtained results for both cell lines showed that Cotula cinerea extract activity was higher than its corresponding Salvia verbenaca one. This was observed for both cell lines.For both lines, an increase of the cytotoxicity with the concentration of the essential oil or the used extracts was observed indicating a dose dependent effect. An examination of the obtained results showed that the cytotoxic effects of the investigated plants extracts were different according to the tested cell line with an efficient activity of Cotula cinerea hexane extract showed the higher cytotoxic effect followed by the ethyl acetate, the essential oil, and the n-butanol extract. In the same conditions and for the Vero cell line, the best cytotoxic effect was observed with the essential oil followed by the hexane, the ethyl acetate, and the n-butanol extracts.In the case of the RD cell line, the50 in µg/mL) and the obtained results are gathered in 50 value (57.21±3.43 µg/mL) was observed with the Cotula cinerea hexane extract indicating an important antiproliferative effect. The essential oil and the ethyl acetate extract showed moderate cytotoxic effects . Higher IC50 values (greater than 500 µg/mL) were observed in the case of Cotula cinerea n-butanol and most of the Salvia verbenaca extracts which presented then the weakest cytotoxicity activity.The cytotoxic activity was also expressed in terms of concentration required to inhibit 50% of the tested tumor cell lines  followed by the hexane, the ethyl acetate, and the n-butanol extracts . Higher concentrations were needed in the case of Salvia verbenaca extracts which showed the weakest cytotoxic effects.In the case of the Vero tumor cell line, The noticed differences in the observed cytotoxicity of the investigated mixtures could obviously be due to the phytochemical composition of each extract. The exact mechanism through which the explored extracts act on each tumor cell line is obviously difficult to highlight. Indeed, the explored extracts are complex mixtures containing several phytochemical compounds which could have different biological activities. In addition, these mixtures could act in a synergic or in an antagonism manner. Cotula cinerea and Salvia verbenaca extracts showed the presence of polyphenols such as flavonoids and other secondary metabolites. These compounds could explain the cytotoxic effect of this extract on RD tumor cell line. Indeed, the antitumor properties of phenolic compounds and flavonoids against several cell lines representing numerous human cell lines have been previously reported [A preliminary phytochemical screening of thereported . The exareported . Cotula cinerea essential oil against the Vero tumor cell line could be due to presence of cytotoxic molecules in the oil. Essential oils are complex molecules containing various and several terpenoids compounds belonging mainly to monoterpene and sesquiterpene chemical groups and presenting different functional groups such as alcohols, phenols, ketone, etc. [The antiproliferative activity ofne, etc. . These cne, etc. . Moreove Cotula cinerea essential oil is very complex involving several targeting pathways as previously reported [ Cotula cinerea essential oil can act through various mechanisms. Among these are the activation of the apoptosis via the caspases' family proteins expressions, the modifications in signaling pathways, the interruption of the life cell cycle, the modification in DNA methylation, and histones acetylation of tumor cell lines [ Cotula cinerea in cytotoxic activity.The action and mechanism ofreported . Due to ll lines , 15. The Cotula cinerea essential oil and hexane extract showed significant and encouraging cytotoxic effects against RD and Vero cell lines, respectively, and could then be considered as source of novel antitumor agents. The obtained results raise then the possibility of a potential use of Cotula cinerea as a source of potent anticancer products. This study is expected thus to be beneficial for clinical and traditional applications for Cotula cinerea as a remedy against cancer and opens new perspectives for further exploration on other types of cancer cell lines. Further investigations regarding the identification of bio-actives molecules responsible for these effects are needed. Examination of the cytotoxic activity of the Cotula cinerea individual compounds would give crucial information regarding the exact mechanism of this plant in antitumor activity.Our results indicated that