Euryarchaeota, Korarchaeota, and Nanoarchaeota being predominant while Thaumarchaeota and Crenarchaeota were predominant in the surrounding soils. Additionally, the observed nutrient pathways: phosphorus, nitrogen, and sulfur were all significantly more predominant in termite mound soils than in their comparative surrounding soils. Alpha diversity showed that archaea were not significantly different within termite mound soils and the surrounding soils. The beta diversity revealed significant differences in the archaeal taxonomic composition and their functional categories between the termite mounds and surrounding soils. Our canonical correspondence analysis revealed that the distribution of archaeal communities was likely dependent on the soil properties. Our results suggested that termite activities may promote the diversity of archaea; with some of our sequences grouped as unclassified archaea, there is a need for further research to unveil their identity.Recent studies involving microbial communities in termite mounds have been more focused on bacteria and fungi with little attention given to archaea, which play significant roles in nutrient cycling. Thus, we aimed at characterizing the archaeal taxonomic and functional diversity in two termite mound soils using the shotgun sequencing method with the assumption that termite activities could promote archaeal diversity. Our findings showed that termite mound soils have archaeal groups that are taxonomically different from their surrounding soils, with  Mound-building termites from an agricultural perspective are seen as pests, since they affect crops and farm structures, therefore leading to profit loss . NonetheCurrent studies have revealed that termite societies can increase the activity of soil bacterial  and fungMany studies that have been carried out so far on termite mound soil microorganisms are based on culture-dependent and sanger-sequencing methods. These methods only offer little data about microbial diversity . HoweverCoptotermes species. The distance between the termite mounds and their comparative surrounding soils in each location was 10 m. The soil samples were preserved in cooler boxes filled with ice blocks during the sampling, transported to the Microbial Biotechnology Laboratory under frozen conditions, and stored until analysis. The soil samples were divided into two parts: (1) for physical-chemical analysis (stored in a fridge at 4 °C) and (2) for DNA analysis (stored in a freezer at −80 °C) for 14 days. After the soil analysis, the mean values were used for a statistical analysis.We collected sixteen (16) soil samples from termite mounds and their surrounding soils in August 2017. That is four (4) soil samples from termite mounds (T1) and four soil samples from their comparative surrounding soils at Braklaagte (S1). We also collected four (4) soil samples from termite mound soils (T2) and four (4) soil samples from their comparative surrounding soils at Zeerust (S2). Braklaagte (25°26′13.5″ S 26°05′50.4″ E) and Zeerust (25°27′11.2″ S 26°07′33.8″ E) are located in North West Province, South Africa. The mean air temperature of the area ranges from 3 °C to 21 °C and 17 °C to 31 °C in winter and summer, respectively. The annual precipitation of the province is roughly 360 mm, with most falling in the middle of October and April [+) were analyzed by colorimetry and flame photometry, respectively [+2) and magnesium ions (Mg+2) were extracted using the 1 M ammonium acetate method at pH 7.0 and evaluated using an atomic absorption spectrophotometer. The determination of the organic carbon content was carried out using the dichromate digestion [The soil’s physical and chemical characteristics were analyzed using standard procedures. Soil particle size analyses were performed using the hydrometer method . The Uniectively . The totigestion ,20.® DNA isolation kit  following the manufacturer’s instructions. The concentration of the extracted DNA was measured by fluorescence using the Quant-iT PicoGreen dsDNA kit , which was assessed on a DQ 300 fluorometer . The extracted DNA was stored at −80 °C while awaiting sequencing.The metagenomic DNA was extracted from 0.25 g of each sample collected from termite mound soils and their corresponding surrounding soils using the PowerSoilWe used the Illumina MiSeq 2500 platforms  for sequencing the extracted DNA. All the datasets were created by whole metagenome shotgun sequencing at Molecular Research LP . An amount of 50 ng of DNA from each sample was used to construct library sequencing via the Nextera DNA Sample Preparation Kit (Illumina). The library insert size was assessed with an Experion Automated Electrophoresis Station (Bio-Rad). The insert size of the libraries alternated from 300 to 850 bp (average 500 bp). Each library was loaded to a 600 Cycles v3 Reagent cartridge (Illumina), and the sequencing was performed using a 2 × 250 base pair sequencing run. https://www.mg-rast.org (accessed on 10 July 2019) [https://kbase.us/) , which is a comprehensive stand-alone framework. The genomes were annotated using the Prokka annotation pipeline and non-redundant (NR) database. The predicted genes were used to hit the Kyoto Encyclopedia of Genes and Genomes (KEGG) and the non-redundant protein (NR) database via the KBase pipeline. The resulting archaeal table was agglomerated accordingly to each taxa level, and unclassified reads were retained for statistical purposes. Next, the abundances (The raw sequences of each of the metagenomes were uploaded to the metagenomics rapid annotation online server (MG-RAST) at ly 2019) . In the ly 2019)  against ly 2019) , which pundances  were trap < 0.05). Pielou Evenness and Shannon diversity indices were assessed for each of the samples and these indices were compared between habitats using a Kruskal–Wallis test. All these analyses were performed using PAST version 3.20 [The differences between physicochemical parameters were determined by a one-way analysis of variance (ANOVA) for the comparison of means with Tukey’s pairwise comparison test for significance level  and 6,949,161 (T2) sequence reads for the termite mound soil samples and 7,490,918 (S1) and 7,057,133 (S2) sequence reads for the surrounding soil samples. After quality control (QC) was executed in MG-RAST , the quaEuryarchaeota, Korarchaeota, and Nanoarchaeota were predominant  in the termite mounds, while Crenarchaeota and Thaumarchaeota predominated in the surrounding soils  more predominant in termite mound soils than their comparative surrounding soils  and their functional categories  , while the functional categories were not significantly different ), as depicted with the PCoA  of the archaeal community. The Shannon and Evenness indices showed that there were no significant differences in the alpha diversity of the archaeal taxonomic composition  . Howeverthe PCoA .Thermococcus and Sulfolobus. On axis 2, the vector length of P positively correlated with Cenarchaeum, Methanosarcina, and Methanosphaerula, but negatively correlated with Haloterrigena.To determine the effect of soil properties on the archaeal community distribution, a canonical correspondence analysis (CCA) was used . All theIn this research, we investigated and characterized the archaeal communities in termite mound soils relative to their surrounding soils using the shotgun metagenomics sequencing approach. Shotgun metagenomics has shown its importance and usefulness in evaluating the composition and abundance of microbial communities ,27. Thisp-value > 0.05) between termite mound soils and the surrounding soils  and functional categories (like carbohydrate and amino acids and derivatives) in each environment. Furthermore, we used the PCoA plot to depict the beta diversity, which is the diversity between the termite mound soils and their surrounding soils. The PCoA plot revealed that termite mound soil samples (T1 and T2) formed no distinct clustering with the surrounding soil samples (S1 and S2)  in the archaeal community taxonomic composition between the termite mounds and the surrounding soils, as shown by the analysis of similarity (ANOSIM), and the strength of the difference was R = 0.17. There was no significant difference in the functional categories that were considered between the mound soils and the surrounding soils. Differences in the bacterial community structure have been described by various studies [Firstly, to answer the question “are the taxonomic and functional diversity of archaeal communities in termite mound soils significantly different from their surrounding soils?”, Shannon and Evenness indices were employed to analyze the alpha diversity. The alpha diversity showed that archaea were not significantly different  a. This m studies ,12. TermCenarchaeum, Methanosphaerula, and Methanosarcina predominated the termite mound soil (T2), while Candidatus Nitrososphaera, Nitrosopumilus, and Haloferax predominated the surrounding soil (S2) (Methanosarcina (which was predominant in T2) can act as biofilter [To answer if each environment had distinguished archaeal communities, a principal component analysis (PCA) was plotted. Our PCA analysis showed, for example, that oil (S2) . The disiofilter ,33 and riofilter . This cop < 0.05) lower in termite mound soils than the surrounding soils . Making out plans to isolate and culture these archaea could give room for the discovery of novel archaeal species for industrial uses.Euryarchaeota, Korarchaeota, and Nanoarchaeota were more predominant in termite mound soils, while Thaumarchaeota and Crenarchaeota were more predominant in the surrounding soils. Additionally, all observed nutrient pathways were predominant in termite mound soils in contrast to their surrounding soils. Furthermore, our canonical correspondence analysis revealed that soil properties influenced the archaeal distribution, with P explaining most of the entire variation. The presence of sequences assigned to unclassified archaea in this study could suggest the need for further research to unveil their identity.This study, to the best of our knowledge, is the first of its kind to profile archaeal diversity in termite mound soils using the shotgun sequencing approach. The alpha diversity showed that archaea were not significantly different within termite mound soils and the surrounding soils. Furthermore, the beta diversity showed significant differences in the archaeal taxonomic composition, although the functional categories between the termite mounds and surrounding soils were not significantly different. We also reported that


In this study, the complete mitochondrial genome sequence of Niya chicken  was obtained by using the next-generation sequencing technology for the first time. The complete mitogenome sequence of Niya chicken is 16,784 bp in length, containing 2 ribosomal RNAs, 13 protein-coding genes, 22 transfer RNA genes and a D-loop region. Our work provides a valuable source of data for phylogenetic studies and helps us to understand the maternal origins of the native chickens. The existence of Niya chicken has not been reported until 2006 during a national livestock resources survey in China. Later in 2010, this newly discovered breed was listed on the National Livestock and Poultry Genetic Resources Protection List of China based on its unique characteristics. Niya chickens have uniformly black feathers and are believed to have been bred by the local people in the Taklimakan Desert region, giving them a long history of living in a hot, dry, harsh environment. To understand the maternal origin of the Niya chicken, we assembled its complete mitochondrial genome using high-throughput sequencing technology for the first time. The Niya chicken was collected from its core breeding center – Minfeng County (37.07 N and 82.70 E), Xinjiang Uygur Autonomous Region, China. The Niya chicken specimen (Voucher No. NY150159) was stored at −80 °C in the Museum of Hunan provincial key laboratory for genetic improvement of domestic animal, Changsha, China. We extracted the total genomic DNA of Niya chicken and used the genomic DNA to generate Illumina sequencing libraries. The libraries then sequenced on Illumina Hiseq 2500 sequencing platform. In total, we generated 11 Gb raw sequencing data and submitted the sequence reads to the NCBI Sequence Read Archive (SRA) with accession number SRR4302067. The assembled complete mitochondrial genome of Niya chicken has been deposited in Genbank with accession number MT635915.ND6) and the rest 8 tRNAs are encoded on the light strand. Twelve of 13 PCGs initiate with an ATG start codon except for COX1, which begins with GTG. The PCGs use four types of stop codons which are TAA, TAG, AGG and incomplete stop codon T, which dues to the 5′ terminal of the adjacent gene  phylogenetic tree by using Mega 7.0 (Kumar et al.


Mycobacterium leprae, causes nerve damage, deformity and disability in over 200,000 people every year. Because of the long doubling time of M. leprae (13 days) and the delayed onset of detectable symptoms, which is estimated to be approximately 3–7 years after infection, there is always a large percentage of subclinically infected individuals in the population who will eventually develop the disease, mainly in endemic countries. piRNAs comprise the largest group of small noncoding RNAs found in humans, and they are distinct from microRNAs (miRNAs) and small interfering RNAs (siRNAs). piRNAs function in transposon silencing, epigenetic regulation, and germline development. The functional role of piRNAs and their associated PIWI proteins have started to emerge in the development of human cancers and viral infections, but their relevance to bacterial diseases has not been investigated. The present study reports the piRNome of human skin, revealing that all but one of the piRNAs examined are downregulated in leprosy skin lesions. Considering that one of the best characterized functions of piRNAs in humans is posttranscriptional mRNA silencing, their functions are similar to what we have described for miRNAs, including acting on apoptosis, M. leprae recognition and engulfment, Schwann cell (SC) demyelination, epithelial–mesenchymal transition (EMT), loss of sensation and neuropathic pain. In addition to new findings on leprosy physiopathology, the discovery of relevant piRNAs involved in disease processes in human skin may provide new clues for therapeutic targets, specifically to control nerve damage, a prominent feature of leprosy that has no currently available pharmaceutical treatment.Leprosy, which is caused by the human pathogen  Mycobacterium leprae, the causative agent of leprosy, is the only known bacterium that infects Schwann cells (SCs) of peripheral nerves. In addition to SCs, M. leprae is an obligate intracellular pathogen that infects macrophages and dendritic cells; it mostly affects the skin, mucosa and nerves but may be transported within these cells anywhere in the body1. Over 200,000 people have been diagnosed with leprosy every year for the last 10 years, and approximately half of these people are clinically recognized to have some physical disability that may result in prejudice and stigma2. It is likely that the number of cases is much higher than officially described, and it is thought that there may be up to 4 million hidden cases of leprosy today waiting to be diagnosed, mainly due to loss of medical expertise and lack of access to the health system4.5. Therefore, piRNAs may have high importance for clinical implications for a variety of diseases.Several piRNomes have been described, and most of these have been related to different forms of cancer. piRNAs are important for gametogenesis, embryogenesis and stem cell maintenance5. The piRDisease database 20196 cites 28 diseases with some proven piRNA interference, but none of them are caused by infectious agents. However, there have been reports of piRNA acting on viral diseases8. In addition, mapping piRNAs onto specific diseases may increase the availability of possible biomarkers, therapeutic targets9 and chemoresistance evaluation10, which are critical issues for modern leprosy control13.Posttranscriptional gene silencing by piRNA is mediated by two mechanisms as follows: (i) piRNAs target transposon sequences found in the 3-UTRs or 5-UTRs of mRNAs; and (ii) if the piRNA is derived from a pseudogene and antisense transcript transcribed from the opposite strand of the endogenous genes, then the piRNA targets the mRNA of the corresponding endogenous geneAfter quality control, alignment, and transcript quantitation, several small noncoding RNAs (sncRNAs) and other transcript fragments were identified. From these sequences, an average of 26,000 reads per sample were recognized as piRNAs, identifying a total of 337 differentially expressed (DE) piRNAs in leprosy patients. In total, 15, 86 and 69 piRNAs were exclusively expressed in skin lesions from tuberculoid (TT) tissue, lepromatous (LL) tissue and healthy subject  tissue, respectively. In addition, 139 piRNAs were expressed in all tissue samples irrespective of whether from healthy or leprosy patient skin tissue, and 28 piRNAs were significantly expressed in more than one group  to HS showed that 14 piRNAs were downregulated in leprosy skin , we examined potential piRNA targets using the miRandaWe identified over 5,000 putative target genes, and 955 of these target genes were targeted simultaneously in both their 5′ and 3′ regions. Using gene ontology . In silico analyses of targets and GO enrichment  revealed genes and pathways important in the leprosy phenotype and the mechanism of immunophysiopathology.15.A recent finding of an unexpected role for piRNA in somatic cells has revealed that the capacity to promote sensory axon regeneration after injury may be blocked by piRNA. The piRNA pathway acts against axon regrowth independent of its return to the nucleus and transcriptional silencing. However, with posttranscriptional gene silencing, piRNA inhibition of axonal regeneration is blocked, and sensory nerve regeneration occursM. leprae may be silent or expressed at low levels  and IL-6, and the pathways of these molecules are regulated by piRNAs as described in  and contribute to cell survival, invasion, migration, chemoresistance, and metastasis, and as a result, several classes of TAM inhibitors are being developed in clinical studies17. GAS6 is produced by quiescent fibroblasts, osteoblasts, and macrophages through GGCX with vitamin K as a coenzyme to the completion of GAS6 carboxylation16. Thus, the regenerative function of macrophages in SCs infected with M. leprae may depend on the expression of vitamin K-dependent genes (such as GGCX) that produce GAS6 within the microenvironment to promote regrowth of damaged nerves15.GAS6 is involved in the stimulation of cell proliferation and is associated with a variety of diseases19 and as a marker of neuropathic pain in patients with leprosy20. Therefore, IL-6 may be related to bette nerve regeneration in addition to the stimulation of a more efficient cellular response under the auspices of a downregulated piRNA profile.In the present study, the downregulation of piRNAs target to IL6R can allow the expression of IL6R in the membrane of pro-regenerative macrophages. This receptor is necessary for the effective action of IL-6 that has been described in patients with higher titers21. After a demyelinating injury, oligodendrocyte precursor cells (OPCs) become activated and subsequently proliferate and migrate to the lesion site22, but they have limited action related to corticosteroid treatment due to inhibition of pro-regenerative macrophage uptake24. Pro-regenerative macrophages persist at the site of the injured nerve and regulate SC maturation and conduction velocity postinjury by a complex GAS616-dependent transcriptional profile via GGCX and vitamin K pathways. Increased GAS6 has been described in type 2 reactional episodes of erythema nodosum leprosum (ENL)25. The downregulated expression of the piRNAs that control GAS6 production in LP may be related to higher levels of GAS6 in ENL.Corticosteroids are used to treat acute nerve damage in leprosy, but they have moderate efficacy in treating nerve function impairment. It has been shown that corticosteroids do not have a superior effect to placebo in improving nerve function28. These studies evaluated the daily injection of 50 mg of vitamin K into patients experiencing leprosy reaction, and they reported an extraordinarily rapid improvement within five days with a lowering of fever, alleviation of pain symptoms and full recovery of the patient. Within fifteen days of this treatment, all symptoms of the reactional episode had disappeared without any infiltration. After eight months, the skin lesions disappeared completely and did not persist, without any tingling. Later examination of nasal swab and skin biopsies for the presence of acid-fast bacilli were found to be negative. Based on the interaction of GAS6 and vitamin K pathways, these reports of relief from the often debilitating symptoms of ENL and rapid improvement of skin lesions prior to the use of MDT and corticosteroids suggest the possibility that piRNAs may be involved in posttranscriptional mechanisms promoting migration of pro-regenerating macrophages to the site of nerve injury followed by regrowth of damaged nerves.Three studies published between 1950 to 1953 showed regression of leprosy nodules in patients treated with vitamin K3 derivatives, which was decades prior to the use of multidrug therapy (MDT) to treat leprosy patients32. According to the present data, all DE piRNAs in leprosy lesions are downregulated, suggesting that nerve regeneration is not inhibited by piRNAs in leprosy patients. Furthermore, the present work discloses new mechanisms involved on leprosy physiopathology and reveals novel therapeutic targets involved in neurodegeneration and neuropathic pain.The absence of piRNAs allows peripheral nerve regenerationIn total, 17 lesion tissue samples were collected as follows: (i) 6 from healthy subjects (HS); (ii) 6 from lepromatous leprosy patients (LL), and (iii) 5 from tuberculoid leprosy patients (TT). All samples were obtained prior to MDT at URE Dr. Marcello Candia (URE) located in the city of Marituba . Immediately after collection, all samples were frozen and stored in RNAlater (SIGMA R0901). Informed consent was obtained from all individual participants.Total RNA was extracted using TRIzol . After isolation, total RNA was stored at − 80 °C until further analysis. Total RNA amount and integrity were determined using the Qubit R 2.0  and Agilent 2,200 TapeStation  according to the manufacturer’s specifications. Samples with concentrations above 100 μg/uL and RIN (RNA Integrity Number) > 5 were used for sequencing. The concentration of 1 μg/5 μL of sample was used as input to library preparation. We synthesized 17 libraries using the TruSeq Small RNA Library Preparation Kit  according to the manufacturer’s instructions. The pool of libraries was quantified using ABI 7,500 equipment  and a KAPA Library Quantification Kit . The libraries were sequenced using a MiSeq Sequencing System  and the MiSeq Reagent Kit v3 150 cycle .33. Htseq-count software34 with piRbase annotation35 was used to quantify transcripts. Before piRNA quantitation, we grouped colocalized piRNAs to avoid ambiguous counting. Differential expression analysis. To identify the differentially expressed (DE) piRNAs, the following four analyses were performed: (i) leprosy vs. HS samples, (ii) TT vs. HS samples, (iii) LL vs. SH and (iv) TT vs. LL samples. For these analyses, raw read count  were used with DESeq2 library34 in the statistical platform R . piRNAs satisfying the following criteria were tagged as differentially expressed: |Log2 (fold-change)|> 2 and p-value < 0.05. For graphical analysis of piRNAs, expression data were normalized to RPKM. The area under the curve (AUC > 0.9) from the Receiver Operating Characteristic (ROC) analysis was used to identify potential biomarkers among DE piRNAs. All statistical analyses were performed on the platform R.After sequencing, resulting reads were preprocessed and quality filtered (QV > 25). STAR Aligner (v. 2.4.0.1) was used to map the reads to the human genome (v. hg19)36 in R37 to identify all biological processes significantly associated (p 0.05) to piRNA-targeted mRNAs5. PCAs and heatmaps were created in R, and the piRNA-gene interaction network was constructed using Cytoscape software v 3.2.138.piRNA mRNA targets were identified by predicting the complementarity sequence between each piRNA and the 5-UTRs or 3-UTRs regions of all known human mRNAs  with miRanda [v3.3a], applying a stringent alignment score , energy threshold  and at least 80% of complementarity. Functional analysis was performed using cluster profiler libraryEthics statement. This study was performed in accordance with the recommendations of the Brazilian National Ethics Committee (CONEP) guidelines approved by Pará Federal University Ethics Committee number CAAE 26765414.0.0000.0018 with written informed consent from all subjects, in accordance with the Declaration of Helsinki. The protocol was approved by the Pará Federal University Ethics Committee.Supplementary information.


Quercus suber) leaves were determined for six samples with seeds of different geographical origins covering the natural distribution of the species. The leaves of all samples exhibited a hard texture and oval shape with a dark green colour on the hairless adaxial surface, while the abaxial surface was lighter, with numerous stomata and densely covered with trichomes in the form of stellate multicellular hairs. The results suggest an adaptive role of leaf features among samples of different provenance and the potential role of such variability in dealing with varying temperatures and rainfall regimes through local adaptation and phenotypic plasticity, as was seen in the trial site, since no significant differences in leaf traits among the various specimens were found, for example, specific leaf area 55.6–67.8 cm2/g, leaf size 4.6–6.8 cm2 and photosynthetic pigment . The leaves showed a substantial cuticular wax layer (154.3–235.1 µg/cm2) composed predominantly of triterpenes and aliphatic compounds  that contributed to forming a nearly impermeable membrane that helps the plant cope with drought conditions. These characteristics are related to the species and did not differ among trees of different seed origin. The major identified compound was lupeol, indicating that cork oak leaves may be considered as a potential source of this bioactive compound.The chemical composition of cuticular waxes and pigments and the morphological features of cork oak ( The extracellular surface of plant leaves is covered by a hydrophobic layer known as the cuticle, which is composed primarily of cutin, an insoluble polyester of hydroxyfatty acids, glycerol and complex mixtures of waxes that are deposited within and above the structural cutin matrix ,2,3. CutCuticular waxes confer properties upon the leaf surface such as nonstomatal water loss and gas exchange control, as well as protection from the external environment. Wax deposition is often a response to water stress and therefore stress resistant plants adapted to arid conditions often have thicker wax layers than those from more temperate locations or those that are susceptible to stress ,12,13,14Quercus suber L.) is an evergreen sclerophyllous tree species distributed in the western Mediterranean Basin, with a natural range including Algeria, France, Italy, Morocco, Portugal, Spain and Tunisia. Cork oak forests play an important ecological role in terms of carbon sequestration, soil protection, hydrological cycle regulation and ecosystem sustainability, while they are also of critical economic importance due to their production of cork that feeds a dedicated industrial chain ,62–3), rCuticular wax was extracted from whole fresh leaves with dichloromethane in a Soxhlet apparatus over 6 h using a sample of 20 leaves per tree. After extraction, the solvent was evaporated. This extraction method yields a total wax mixture containing both epicuticular and intracuticular waxes that cover both sides of the leaf. 2, obtained by digitalization).The amount of soluble cuticular lipids was determined from the mass difference of the extracted leaves after drying at 105 °C and was expressed on a leaf surface area and dry weight basis  and by comparing their fragmentation profiles with published data [The cuticular wax (obtained as dichloromethane extracts from 20 leaves per tree) was analyzed using gas-chromatography mass spectrometry (GC-MS). Two mg of each leaf extract was taken and derivatized in 120 μL of pyridine; the compounds with hydroxyl and carboxyl groups were trimethylsilylated into trimethylsilyl (TMS) ethers and esters, respectively, by adding 80 μL of bis(trimethylsily)-trifluoroacetamide (BSTFA). The reaction mixture was heated at 60 °C for 30 min in an oven. The derivatized extracts (1 μL) were immediately analyzed by GC-MS , with an ionization energy of 70 eV and the MS source was kept at 220 °C under the following GC conditions: Zebron 7HGG015-02 column , with injector at 280 °C. The column temperature was initially held at 50 °C for 1 min, raised to 150 °C at a rate of 10 °C minhed data ,64. Two For scanning electron microscopy (SEM), small pieces of fresh- and air-dried leaves were fixed to sample holders and observed with a scanning electron microscope (TM 3030 Plus Hitachi).The cuticular membrane was observed in a thin leaf cross section by optical microscopy. The leaves were impregnated with DP 1500 polyethylene glycol and cross sections of approximately 10 µm thickness were cut with a rotary microtome (Medite M530). The sections were stained with safranin and astral blue and were mounted in Euparal. Observations were made using a light microscope (Leica DM LA) and the photomicrographs were taken with a Nikon Microphot-FXA.p < 0.05. All statistical analyses were performed using the Sigmaplot® .To compare leaf functional traits among provenances, one-way analysis of variance (ANOVA) was performed. Duncan’s post-hoc tests were used to analyse pairwise differences between provenances. Statistical significance was set at Quercus suber trees of different provenance. Using PCA, the hyperspace was defined by the original variables and dimension reduction was carried out using the significant principal components (new axes). These new axes were correlated with the original variables. Thus, the samples were plotted onto a reduced space where similar samples could be grouped. Agglomerative hierarchical CA, using the Euclidean distance and single-linkage method, was carried out to assess the existence of groups of samples suggested by PCA [TM software, version 6, from Statsoft .Principal component analysis (PCA) and cluster analysis (CA) were performed to analyse the chemical composition of cuticular wax from leaves of d by PCA . PCA andQuercus suber leaves from six different provenances suggest the adaptive value of leaf features under a Mediterranean climate, namely, specific leaf area, leaf size and photosynthetic pigment, highlighting their potential role in dealing with varying temperatures and rainfall regimes through local adaptation and phenotypic plasticity. Cork oak leaves possess a substantial cuticular wax layer that forms a nearly impermeable membrane which is a tool to cope with adverse drought related with environmental conditions. The composition of the cuticular wax also favours the hydrophobicity of the layer by inclusion of very long-chain alkanes and alkanoic acids . These characteristics are species specific and did not differ across provenances.The results obtained for Q. suber.Triterpenes are the major component of the cuticular wax complex and contain a high proportion of lupeol. Thus, the leaves of cork oak represent a potential source for this interesting bioactive compound. The chemical composition of the cuticular wax did not differ among provenances, which may be ascribed to similar water stress conditions in the Mediterranean region, which probably has a stronger effect than genetic variability in


IFITM3 polymorphisms in influenza A(H1N1)pdm09 severity is not yet conclusively determined. This study aimed to evaluate such polymorphisms frequencies and IFITM3 levels in an infected Brazilian cohort of 314 influenza A(H1N1)pdm09 cases and its putative association with clinical, epidemiological and virological data. Individuals were clinically classified into mild, severe and fatal cases. IFITM3 polymorphisms were detected by specific Taqman probes in real time PCR reactions. IFITM3 levels were determined by quantitative real time PCR. Thus, the different clinical groups presented similar distribution of rs34481144 and rs12252 genotypes and allelic frequencies. There was no significant association between the polymorphisms with severity of disease by using distinct genetic models. Additionally, geographic distribution of mutants showed that rs34481144A allele was more predominant in Brazilian Southern region. In contrast, rs12252C allele presented similar frequencies in all regions. Individuals with the distinct rs34481144 and rs12252 genotypes showed similar levels of IFITM3 and viral load in their respiratory specimens. Furthermore, IFITM3 levels were comparable in the distinct clinical groups and were not correlated with influenza viral load in analyzed samples. Thereby, rs34481144A and rs12252C polymorphisms were not associated with severity or mortality of influenza A(H1N1)pdm09 infection nor with IFITM3 transcript levels and influenza viral load in upper respiratory tract samples in a Brazilian cohort.Influenza is a major public health problem that causes acute respiratory infection in humans. Identification of host factors influencing in disease outcome is critical for recognition of individuals with increased risk. Investigations on the role of rs34481144A and rs12252C  Influenza viruses cause acute respiratory infections in humans, which are correlated to annual epidemics and occasional pandemics, representing a relevant public health problem pdm09 virus into the human population. During this period, healthy young adults were severely affected and counted for a significant portion of the fatalities  is a proinflammatory cytokine belonging to the group of interferon (IFN) stimulated genes (ISGs), which are induced after viral infection. The process of induction of ISGs starts following the fusion of viral envelope with the cell endosomal membrane, when the viral genetic material is released into the cytoplasm and allows its recognition by cellular pattern recognition receptors (PRRs) pdm09 infection pdm09 positive cases, whose samples were collected during the 2012–2018 period, further classified according to their clinical presentation as mild, severe and fatal infections. Furthermore, we explored putative relationships between these SNPs with clinical, epidemiological and virological variables, in addition to IFITM3 expression levels.In this study, we report the distribution of Our laboratory is a National Reference Laboratory part of the Influenza Surveillance System (ISS) for the Brazilian Ministry of Health and is one of the National Influenza Centers (NICs) of the World Health Organization (WHO) WHO, . Thus, wTotal RNA was extracted from clinical respiratory samples by using the QIAmp Viral RNA Mini kit , according to the manufacturer's instructions. For influenza A(H1N1)pdm09 detection, extracted RNA was tested in a one-step real time RT-PCR assay using specific primers and probes , as recommended by the WHO. Influenza viral load was indirectly determined by real-time RT-PCR at the moment of diagnosis as the CT (cycle threshold) number during its detection   in a real time PCR assay performed in the Step One Plus equipment  with 5–20 ng DNA, per sample, according to manufacturer's instructions. Cycling parameters were as follows: 95°C for 10 min, followed by 50 cycles of 95°C for 15 s and 60°C for 90 s. In this methodology, each TaqMan probe presents a distinct fluorescent signal and is specific for amplification of a different nucleotide at each position. In the end of the run, the software groups the samples according to their specific unique (homozygotes) or mixed (heterozygotes) fluorescence signal, as previously described , as previously described  and were carried out in duplicates. Cycling conditions consisted of the initial steps of 50°C for 2 min and 94°C for 5 min, followed by 40 cycles of 94°C for 30 s, 50°C for 30 s and 72°C for 45 s. A final melting curve analysis was performed to determine amplification specificity (60–95°C with a heating rate of 0.2°C/s and continuous fluorescence measurement). Expression data were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression and analyzed by using the 2CT−ΔΔ method.After total RNA extraction, we proceeded to reverse transcription with oligo (dT) primers and Superscript III Reverse Transcriptase . Quantitative real time PCR was carried out in a real time PCR Step One Plus equipment  with the following specific primer sequences for http://www.oege.org/software/hwe-mr-calc.shtml)  for the allele frequency and genotype distribution was assessed by using the web program (n = 92), SARI (n = 140) or fatal (n = 82) cases, as mentioned in the methodology section. Of note, influenza A(H1N1)pdm09 virus was the most prevalent subtype among the Brazilian cases from this period . Therefore, 51.6% of the patients were male and the median age of the individuals was 39 years old (p < 0.01), as already expected, since the higher the age, the greater the risk of disease complications , considering that comorbidities are also risk factors for complications. Regarding demographical origin of the samples, most of them were collected at the Brazilian Southern region, followed by the Southeastern and the Northeastern regions pdm09 infection during the 2012–2018 period. Individuals were classified according to their infection severity as ILI (ears old . MoreoveIFITM3 rs34481144A and rs12252C genotypes were in accordance with HWE (p > 0.05). The overall IFITM3 rs34481144A allelic frequency was 31.0% . The rs3448114-AA recessive genotype was always transmitted with the rs12252-TT dominant genotype, and the rs12252-CC recessive genotype was ever inherited with the rs34481144-GG dominant genotype in our influenza cases. By contrast, both dominant genotypes (GG and TT) could be inherited with any of the other genotypes. These results suggest that their presence at homozygosity does occur simultaneously or consists of a rare event.Intriguingly, the presence of rs34481144-AA and rs12252-CC homozygous mutant genotypes concomitantly was not detected in any of the influenza cases in our cohort (p = 0.05). In contrast, rs12252C allele presented similar patterns of predominance in the Southern, Southeastern and Northeastern regions .In Brazil, distinct geographical regions present dissimilar patterns of genetic ancestry due to specific migratory currents from distinct continents pdm09 positive cases during the post-pandemic period, from 2012 to 2018. Although the frequency of rs34481144A in our influenza cohort  was substantially higher than those previously described for healthy subjects from the Americas region (23.3%; Auton et al., IFITM3 expression levels and influenza viral load in our specimens. In our study, the rs12252 CC genotype was not detected in patients in the fatal group, probably due to the low frequency of this genotype in our cohort. Earlier studies had detected a positive correlation between rs12252C and disease severity, mainly in Chinese cohorts (Zhang et al., Similarly, we found no association between the presence of rs12252C and clinical outcomes of influenza infection, loci could be affected by natural selection, genetic drift, recombination and mutation (Slatkin, IFITM3 expression levels in our analysis, further investigations need to be performed to better elucidate the functional correlation of these two loci inheritance patterns.A very provocative finding of this report was that the recessive genotypes of both SNPs were not detected together and were always inherited with the dominant genotype of the other locus. This outcome was also previously described by others (Randolph et al., Slatkin, . HoweverIFITM3 expression, we further noted that IFITM3 transcript levels, in our respiratory specimens, were not correlated with influenza viral load nor with clinical status of the influenza cases. This observation would likewise suggest that IFITM3 levels, in the moment of diagnosis, in this type of samples, are not related to the disease outcome and could not be used as a molecular biomarker for the disease progression.Considering the lack of connection between the genotypes and IFITM3 transcript levels and influenza viral load in upper respiratory tract samples in the Brazilian infected cohort studied. Subsequent functional studies would add valuable information on the role of these SNPs during influenza infection.In conclusion, our results showed no association of rs34481144A and rs12252C with severity or mortality of influenza infection nor with The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.The studies involving human participants were reviewed and approved by Fiocruz-IOC Ethics Committee. Necessity of written informed consent was avoided in this study because the participants were evaluated under the Brazilian ISS, as approved by the local Ethics Comittee.JM and AM designed the experiments. JM, CG, and AM conducted the experiments. JM, MO, and AM performed the statistical analysis. JM, MO, CG, MS, and AM contributed to the writing and editing of the manuscript. AM and MS contributed with funding acquisition. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The following indices were used: modified contamination factor (mCf), modified contamination factor degree (mCdeg), mobility factor (MF), reduced partition index (IR), potential ecological risk factor , modified potential ecological risk factor , potential ecological risk index (RIZ) and modified ecological risk index (RIm). For mCf,mCdeg and IR own classification scale was proposed. It was proven that most useful indices for assessment of soil pollution with metals were mCf and mCdeg. The mCf together with the IR allow to simultaneous assessment of soil pollution and stability for individual metals. These indices were appropriate for soil contaminated with different concentrations of metals, washed under both hydrodynamic conditions using various washing agents and different effectiveness of metals removal. Thus, they may be considered as most useful for evaluation of remediation method, feasibility of washing agent and assessing soil quality after washing.Remediation of soils contaminated with metal must ensure high efficiency of metals removal, reduce bioavailability of residual metals and decrease ecological risk. Thus, for comprehensive environmental soil quality assessment, different indices must be used. In this study, suitability of 8 indices was used for soil highly contaminated with Cu (7874.5 mg kg An important sources of anthropogenic metals emissions are mining and metallurgy, which cause a severe environmental contamination in their immediate vicinity. Among the various elements of the environment, soils are those in which the effects of metals contamination persist for the longest time and remains even after removing the pollution source. Soil contamination with metals affects individual organisms and entire ecosystems, which is why they should be removed.1. The decrease in capillary forces that occurs during the washing increases the mobility of the pollutants and the rate of their movement2.Among the available methods allowing the removal of metals there are soil washing (both in batch and dynamic conditions), with water or with washing agent (WA) of a high affinity for contaminant removal. Soil washing can remove contaminants adsorbed on soil particles by reducing the strength of bonding between pollutants and soil, increasing the hydraulic conductivity of the soil and the availability of pollution3. The broad application of soil washing results from the possibility of using different washing agents dedicated to removal a specific pollutant and the ease in controlling the operational conditions. During soil washing, the contaminated soil is vigorously mixed with washing solution, which in turn reduces the remediation time and provides high pollutant removal. Another advantage of soil washing includes reducing the volume of soil to be treated by concentrating pollutants into fine particles, which reduces the remediation cost. Among different remediation methods, soil washing characterizes with a high public acceptance4.Soil washing is well-established technology in many countries to remove permanently a variety of pollutants from highly contaminated soils5. For effective washing technology it is recommended to determine firstly the optimal parameters in the laboratory studies. This approach reduces the risk of failure and gives the opportunity to view and to resolve potential difficulties that may appear in the full-scale operation6. Such tests are conducted under batch and/or dynamic conditions (column leaching mode). In these methods, however, different mechanisms of metals transport occur7. In batch experiments, sorption/desorption of metals is determined under equilibrium conditions, whereas in column washing under non-equilibrium conditions, which run slower than under batch washing8. The efficiency of metals removal via soil washing in batch and dynamic conditions can vary widely. The efficiency of washing at the level of 70–80% is considered to be substantial. However, the efficiency above 50%, is still considered as cost-effective, and possible to use on a pilot and technical scale6.This method requires soil excavation and soil transportation to a place of disposal. In practice, in situ methods, i.e. carried out directly in contaminated sites are also preferred13. Natural and environmentally friendly WAs, such as dissolved organic matter or soluble humic substances, can be also used to remove metals from contaminated soils with high efficiency18. Despite the process efficiency, some metals still remain in soil after treatment. Therefore, the quality of treated soil must consider several aspects, i.e. pollution level, metal stability and environmental risk. The key to assess soil contamination with residual metals can be using soil pollution indices. They are regarded as a tool and guide for an extensive evaluation of the state of the soil environment19. The indices are widely used and can be developed on different criteria, e.g. individual metals contamination 22 and multi-metals contamination 23. Other indices are based on the concentration of metals in their individual chemical forms, along with having great importance in determining the mobility of metals  and strength of metal binding with mineral-organic soil components 25.During the past decades, studies have evaluated the effectiveness of removal of metals from the soil with various WAs, such as acids, salts and chelating agents26. There are some attempts to use them for assessment soil quality after remediation, but only with the use of selected, single indices, mainly concerning environmental risk28. However, as these indices were originally used for sediments, they are based on comparison between current metal concentrations with their geochemical background levels. Although, some of these indices can be used for urban and agricultural soils with elevated metal concentrations19, their using for highly contaminated soils can be inconclusive. For industrial objects connected with metal smelting, however, estimation of soil quality based on pollution indices is missing26.Historically, most of these indices were used mainly for assessment of pollution level in sediments, then were adopted to soils contaminated with different metals and of different level of contamination2EDTA) and next-generation WAs .In highly contaminated soils, despite a considerable decreasing of metal concentration after soil washing, the difference between residual metal concentrations in soil and geochemical background levels can still be high, indicating high pollution level, the same as for soil before remediation. However, taking into account that the purpose of remediation is decrease metal concentration to legislative level, regarding the current and, if possible, planned use of the land, more reliable for soil pollution assessment after remediation can be using the indices, at which current metal concentration will be referred to permissible levels in remediated soil instead of geochemical background. This was done in this study: modification of existing indices, based on soil pollution with single- and multi-metals, as well as own classification of the indices was proposed. Moreover, suitability of these modified indices, as well as indices based on strength of metal binding in soil and on ecological risk of metals, were assessed for soil after remediation. In order to check the indices at various levels of metal contamination and different remediation efficiency, all indices were calculated for: (1) soil of different degree of contamination by various metals , (2) washed in batch and dynamic conditions, and (3) using both conventional (Na3)2, Pb(NO3)2 and Zn(NO3)2, according to the procedure of Chaiyaraksa and Sriwiriyanuphap29 and incubated for three months at room temperature. Metals values were assumed according to concentrations in soils around the Legnica smelter in Poland18. Because the aim of this study was to check the suitability of different indices to assess degree of soil contamination (and subsequently soil remediation), it was assumed that soil must be contaminated with metals of different concentration: from very high  to low (below permissible concentration). Such concentration range is necessary to select universal indices and to propose own classification scale for these indices. So, in this study soil was very highly contaminated with Cu, moderately with Pb and weakly with Zn.Soil taken from the top layer of agriculture area was used for the study. After air drying, the samples were ground and sieved through a 2 mm sieve. To obtain metals contamination similar to soils from the metallurgical area, the soil was spiked with a mixture of Cu waste-derived in form of soluble organics from municipal sewage sludge (SS_WAs) and (2) commercially available Na30. Briefly, WAs were extracted (1:10 ratio (w/v)) with water (DOM) and 0.1 M NaOH (HLS and SHS) from the dried sewage sludge (105 °C). In addition, before the extraction of SHS, dissolved substances (i.e. sugars and proteins), waxes, and bitumen were removed from the sludge, as described in paper by Klik et al.30.As SS_WAs, three different washing solutions were used: DOM, HLS and SHS. The previous study presented a detailed extraction procedure and characteristics of the used washing solutions2EDTA and HLS was extremely effective in removal of all metals, DOM was extremely effective in Cu removal and with low efficiency in Pb removal, SHS was effective in Pb removal etc.). Such scope of experiments allows to select the most universal indices of environmental quality of soil and to propose own scale for classification of these indices.The methods of soil remediation and WAs were selected in such way to indicate various efficiency of metals removal from soil, e.g. Na−1. The concentration of Na2EDTA was typical used for soil treatment with the chelator 31. In all WAs, the pH was adjusted to 4 and the contact time was 2 h (established in the previous study based on preliminary tests). After washing, samples were collected, centrifuged, and filtered through Whatman filter papers 42. The supernatants were acidified with HNO3 and kept at 4 °C before Cu, Pb, and Zn analysis.The washing experiments under batch conditions were conducted in polyethylene tubes (50 mL) with soil to solution ratio of 1/40 (w/v). The samples were shaken in a mechanical shaker at 150 rpm at room temperature  and two layers of rinsed gravel (2–4 mm and 1–2 mm) , for each washing solution for 24 h. All column experiments were conducted at room temperature, under laboratory conditions.The tests were carried out at concentration and pH of WAs the same as in batch washing. Washings solutions were added to the reactor from the top and the eluate was collected from the bottom of the reactor by the automatic sampling device over time. Samples were taken every hour. Before proper soil washing procedure, distilled water was applied for 1 h to saturate the soil. Pore volume for the column reactor was 56.8 ml. The research was conducted at 2 different flow rates , originally proposed by Hakanson21 for sediments, now is commonly used to assess soil contamination with metals35. For Cf calculation, mostly preindustrial or geochemical background concentrations of metals are used. The use of such reference values can be not reliable for assessment of metal pollution in highly contaminated before and after remediation. This is because in remediated soil achieving metal concentration on the level of geochemical background is impossible and it is not legally required. According to Polish Environmental Protection Law36, remediation is based on the removal or reduction the amount of substances causing the risk from soil and groundwater to the level of limit values, their control and limitation of migration, so that the contaminated area ceases to pose a threat to human health or the state of the environment, taking into account the current and the planned land use. Therefore, in this study for the assessment of metal pollution in contaminated and post-remediated soil, original Cf factor was modified to mCf, taking as reference of metal concentration, not preindustrial/geochemical background, but the limit values for metals in industrial soil given in a national regulation (here the Ordinance of the Minister of Environment37). The mCf was calculated according to the following formula: s is the concentration of metals in soil sample, Cr is the limit value for a given metal in soil according to OME37: 600 mg kg−1 for Cu and Pb and 2000 mg kg−1 for Zn. The mCf was categorized into five levels: mCf Cf Cf  Cf Cf > 4 means extremely contaminated soil.(b)multi-metal soil pollution.The contamination factor (21 as a contamination degree (Cdeg). The Cdeg is a sum of the Cf for individual metals. Like the Cf, the Cdeg was also used to assess soil contamination with metals35. Because in the present study, the Cf was changed for mCf, consequently the Cdeg was changed for mCdeg and the mCdeg equation was modified as follows:Cf is the modified contamination factor calculated in Eq.  is expressed by the following equation:F1, CF2, CF3, and CF4 are the concentrations of metal in exchangeable/acid soluble, reducible, oxidizable and residual fractions, respectively. With the MF, metal can be immobile (< 1%), weakly mobile (1–10%), moderately mobile (11–30%), highly mobile (31–50%) and very highly mobile (> 50%).The IR informs about the strength of metals binding with mineral-organic soil components. When IR is close to 0, metal occurs in an easily soluble and exchangeable form, while IR close to 1 means that a given metal dominates in stable forms, mainly in the residual form25.i is the index number of the sequential extraction step, for the weakest to the strongest (k) , and Fi is the percentage content of the considered metal in a i-fraction. Because the IR corresponds with the MF, the following classification was proposed: lack of stability (IR ≤ 0.1), low stability (0.1 < IR ≤ 0.3), medium stability (0.3 < IR ≤ 0.5), elevated stability (0.5 < IR ≤ 0.7), high stability (0.7 < IR ≤ 0.9) and very high stability (IR > 0.9).The Er assesses the impact of individual metal including their various toxicological effects21. The Er is also used to evaluate the ecological risk for sediments and soils39. In the present study, the Er was calculated and compared according to the formula given by Hakanson21 and used in the study by Zhu et al.40  and according to our modification  using mCf instead of Cf:Tr is the toxic-response factor for a given metal , mCif is the modified contamination factor.The 40, the metals with the highest Tr  were not analyzed, we assumed the same classification of soil ecological risk as Zhu et al.40: low risk . Because in the present study Eir,Z and Eir,m were established, therefore two RI were also calculated according to the Eqs.  using a spectrometer equipped with an automatic sample introduction and dilution system (SIPS). The samples were mineralized in a mixture of concentrated HCl and HNO3 before determination of total metals content. Mineralization was carried out in a microwave oven . Metals distribution was determined according to a modified BCR procedure given by Pueyo et al.43, the following fractions were determined: exchangeable and acid-soluble fraction (F1); reducible fraction (F2); oxidizable fraction (F3); and a residual fraction (F4).The equilibrium pH of soil was measured with a pH meter (Hanna Instruments) in distilled water . The soil texture was determined using a Mastersizer 2000 particle size analyzer. The content of organic matter was determined by the Tiurin method and CEC was measured according to the Kappen’s methodCF1, CF2, CF3, and CF4 is the concentration of metal in a given fraction, Ctotal is the total metal concentration.The obtained results are averages of three replicate measurements. The statistical analyses were performed with Statistica 13.1 . One-way analysis of variance (ANOVA), followed by Tukey’s HSD test was employed to indicate significant differences between means for metal removal efficiencies and environmental indices in soil treated with different WAs under batch and dynamic soil washing. Validation of the data was carried out through comparing total metal content obtained by total digestion with sequential extraction of metals. The metal recovery ratio (R) was calculated as follows:46, our intention was to show the different efficiency of metals removal and their different fractional distribution in batch- and dynamic-washed soil in terms of the possibility of assessing the usefulness of modified environmental indices for soils with different remediation efficiency.Comparing the metals removal under different soil washing conditions, the batch washing was more efficient compared to dynamic washing. This may be due to the way of contact between the soil and WAs, which is more vigorous under batch conditions and a higher ratio of soil weight to volume of washing solution in comparison to dynamic washing. Although higher metal removal under batch than dynamic soil washing is typical phenomenon37. Therefore, Cu removal mostly affected the overall efficiency of soil remediation.In the present study, the soil was contaminated to the largest extent with Cu, which exceeded over 13 times permissible limit for industrial soils according to Polish legislative2EDTA). Under dynamic conditions, the higher efficiency of Cu removal, and consequently lower residual Cu concentration in soil, was at 1 ml min−1 than at 0.5 ml min−1 flow rate , while under dynamic conditions, DOM and HLS were the most effective for Cu removal (the efficiency > 70%). Similarly, higher Pb removal was obtained in batch than dynamic conditions. However, in both hydrodynamic conditions, most effective in Pb removal were HLS and Na2EDTA. In contrast to Cu, DOM was able to remove only 24% of Pb under batch washing and only 3% (on average) under dynamic conditions. As in contaminated soil, Zn concentration was the lowest, compared to Cu and Pb, and its removal efficiency with DOM, HLS, SHS and Na2EDTA were high in both washing conditions. However, batch conditions were also more favourable than dynamic conditions. For example, the efficiency of Zn removal with HLS was 90% under batch conditions (in washed soil Zn remained mainly in F4 fraction) and 70–78% under dynamic condition at both flow rates.Under batch conditions with all tested WAs, the average efficiency of Cu removal was 88%  and amino  groups readily interact with metals47. In all washing agents derived from sewage sludge, carboxyl (dissociated and undissociated) and unprotonated amino groups were the most abundant and the intensities in the infrared spectra for these groups decreased in the order DOM > HLS > SHS30. This can explain higher DOM efficiency in metal removal, especially Cu and Zn. In contrast, higher Pb removal with HLS and SHS than DOM suggests that its removal depends more on the presence of functional groups in macromolecular organic compounds in washing agents, especially humic acids than on functional groups in low-molecular-weight organics.The difference in metal removal with tested washing agents was related to their characteristics and affinity to individual metals. The washing agents extracted from sewage are composed of multiple organic compounds and functional groups. DOM contains mainly low-molecular-weight organics and fulvic acids. HLS contains both low-molecular-weight organics and macromolecular compounds (fulvic and humic acids), whereas SHS are mainly consist of fulvic and humic acids−1 were selected.Because better removal of Cu, Pb and Zn under dynamic conditions was at higher flow rate, to compare the suitability of the many commonly used indices for assessment remediation efficiency and soil quality, the results from batch and dynamic washing at flow rate of 1.0 ml min48, metals distribution should be determined in soil from each remediation action. The results on the changes in metals concentrations in individual fractions are shown in Table 50.Total content of metal measured in raw or treated soils is not a direct indicator of hazardous ecological consequences. Thus, each metal in the contaminated soil and soil after batch and dynamic washings was fractionated into different chemical fractions. According to Minkina et al.The results clearly demonstrated that the distribution patterns for individual metals depended on the soil washing conditions and the type of WA. The results have shown that the most mobile F1 fraction was considerably removed with the most of tested WAs under batch and dynamic conditions Table .2EDTA  and DOM , and the largest in soil washed with SHS . Under dynamic conditions, despite decreasing of total Cu, its distribution in all washed soils was similar to that in unwashed soil with the same order of fractions as F1 > F2 > F4 > F3 . In contrast, distribution patterns of Zn in dynamic-washed soil were more differential. Fraction F4, representing the lowest potential environmental risk, constituted the largest share in HLS-washed soil and in SHS-washed soil. However, the most mobile F1 fraction of Zn prevailed in DOM- and Na2EDTA-washed soil, followed by SHS- and HLS-washed soil    . In soil washed under dynamic conditions with all WAs, the mCf for Cu was above 3, reflecting strong to extreme contamination  , only soil washed with Na2EDTA under batch washing met the quality standards for industrial areas. In other soil samples, mCdeg was greater than 3 (from 5.0 to 18)  given by Hakanson21 resulted from the fact that these indices were dedicated to sediments. Moreover, the classification of Cf and Cdeg reflects the lower values of metal concentrations in contaminated sediments. Thus, the use of this classification for highly contaminated soils can be confused. For example, in this study Cu concentration in contaminated soil was 7874.5 mg kg−1 (13.1 times higher than OME37 limit), while Zn concentration was 566.1 mg kg−1 (3.5 times lower than OME37 limit). Calculated values of the original Cf for Cu and Zn were 787.4 and 6.4, respectively and in both cases, they indicated very high contamination (Cf ≥ 6). Another example is for soil washed in batch and dynamic conditions with Na2EDTA, for which the residual Cu concentration was 529.1 mg kg−1 (Cf = 52.9) and 2659.5 mg kg−1 (Cf = 265.9), respectively, indicating very high contamination (Cf ≥ 6) in soil washed in both conditions.The necessity of modification of original pollution indices (MF < 86%)  fraction. Metals in this fraction are bound to soil constituents with weak bonds and thus they are highly mobile51.In the present study, all metals in contaminated soil were highly mobile . In the case of HLS and SHS, the MFs were still high . After soil washing under dynamic conditions, the MFs for Cu were higher for DOM, HLS and Na2EDTA than after washing under batch conditions, and they did not differ statistically  , however, in soil washed under dynamic conditions it was 10 times higher (MF = 55.4%). In the case of HLS, the difference in MFs for both washing conditions was 9.5%, and Pb. For DOM and SHS, the conditions of soil washing did not affect the MF values of Pb. In soil washed with DOM, the MF of Pb was comparable as in unwashed soil  under batch soil washing. Despite decreasing the MF of Zn under dynamic conditions, the Zn mobility was still high for DOM-washed soil (MF = 67.1%) and Na2EDTA-washed soil (MF = 52.2%). The use of HLS and SHS led to decrease Zn mobility to low and medium level, respectively , i.e. 73.1 and 70.6%, respectively. Cheng et al.52 found high MFs for Cu, Ni and Zn, i.e. 68%, 64% and 53%, respectively in soil from the electroplating factory . The values of the MF for Cu, Ni and Zn were significantly decreased after soil batch washing with mixture of EDTA and oxalic acid to 22%, 25% and 23% for Cu, Ni and Zn, respectively. Decrease in metal mobility in soil after remediation is typical phenomenon. Gusiatin and Klimiuk25 demonstrated that significant decreasing of the MF values can be obtained through multiple batch soil washing. In soil spiked with Cu, Cd and Zn, the metals presented very high mobility (61% < MF < 84%), which was decreased below 10% for Cu and Cd and below 20% for Zn after triple soil washing with saponin biosurfactant. Also, Wang et al.53 showed that batch washing was very effective in decreasing Pb mobility with N,N-bis (carboxymethyl)-l-glutamic acid in mine soil, from 80 to 17%.High values of the IR index based on the metal distribution patterns25. Originally, the index was applied for assessment of metal stability in soil amended with different additives. Nowadays, it can be used also for soil remediated with soil washing25. An increase of the IR indicates metals redistribution in soil, from less to more stable fractions. In the case of BCR sequential scheme of metal fractionation, the F1 fraction is mobile, the F2 and F3 fractions are potentially mobile, and the F4 fraction is stable. Thus, the considerable increase of the IR is favoured by the increase of metal share in the most stable fraction.One of the tools to evaluate the stability of metals in soil is the IR index the changes in metal stability were captured. The values of the IR index for unwashed soil and soil washed with different WAs are shown in Fig. IR values in unwashed soil were relatively low (IR = 0.12–0.22) indicating low stability of all metals. Soil washing increased the IR for all metals, except for Pb in DOM-washed soil (IR < 0.3). In general, metals in soil from batch washing showed higher bonding intensities compared to soil from dynamic washing. In additions, metals differed in bonding intensities in washed soil. The differences were strongly related to metal distribution patterns (“2EDTA resulted in high stability of Cu (IR = 0.61 for both agents), whereas the stability of Cu in soil from dynamic washing was 2.6 times lower (IR = 0.26 for DOM and 0.22 for Na2EDTA). For Pb, the greatest differences in the metal stability between batch- and dynamic-washed soil (by IR = 0.47) was indicated for Na2EDTA. Medium Pb stability was in soil washed with SS_WAs under batch and dynamic conditions .With the use of the atterns “” sectionIR > 0.8). In contrast, the Zn stability in soil after dynamic washing ranged from low for DOM (IR = 0.28) to elevated for HLS (IR = 0.66).Batch soil washing was especially effective for Zn removal from mobile and potentially mobile fractions by all WAs, which resulted in high stability (IR) in soils after washing were conducted by Gusiatin et al.54. The authors have found that application of different biosurfactants as WAs  significantly increased the stability of Cd, Cu, Ni, Pb and Zn (based on the IR) in spiked soil. Similar observations were made by Cheng et al.52, who noticed an increase in the IR index in soils after washing with a mixture of EDTA and organic acids. The increase was associated with the effective removal of Cu, Ni, and Zn, mainly from F1 fraction, and increasing the share of the most stable fractions, which were removed less efficiently.Studies evaluating the metal stability . Similar discrepancy was demonstrated for Cu removed under dynamic soil washing. For Er,m, its discrepancy was noted for Pb in contaminated soil and in soil after washing. In both soils, Pb has shown the same potential ecological risk. This means that both Er,Z and Er,m cannot be considered as universal for ecological risk assessment in soil contaminated with metals and soil remediated in batch and dynamic soil washing with SS_WAs and Na2EDTA under specific operational conditions.In this study, calculated values of mCf and mCdeg were useful to identify soil pollution with metals. The mCf together with the IR characterize individual metals in terms of their pollution and stability degree in soil. The indices clearly distinguished the environmental quality of soil after remediation under different hydrodynamic washing conditions with Na2EDTA and SS_WAs. Thus, they may be an useful tool for assessment of soil remediation with different WAs. Their usefulness requires further verification in case of treatment of other types of soils contaminated with different levels of metals and/or metalloids.Considering legislative purposes of soil remediation, in this study, the use of current metal concentration in relation to permissible levels in soil instead of geochemical background/pre-industrial level was proposed for calculation the indices of soil pollution after remediation. In addition, own classification scale for soil pollution indices and metal stability in soil was proposed. The


The B cell chemoattractant CXCL13 is a promising biomarker in rheumatoid arthritis (RA), with a plausible role in supporting diagnosis, monitoring disease activity and as a prognostic value. It is a key chemokine driving the formation of lymphoid follicles within the inflamed synovium. The objective of this systematic review was to evaluate the role of CXCL13 as a viable biomarker in RA.We conducted a systematic literature review of all published cohort and randomised controlled trials evaluating the role of CXCL13 in RA. The primary outcomes were; i) CXCL13 levels in RA patients compared to healthy controls, ii) the correlation between CXCL13 and markers of disease activity, and iii) the association between CXCL13 and treatment response.The search produced 278 articles, of which 31 met the inclusion criteria. Of the 12 studies evaluating CXCL13 expression in early or established RA, all reported higher levels than that seen in healthy controls. Twelve of sixteen studies reported a weakly positive correlation between CXCL13 and markers of disease activity including DAS28 and swollen joint count, with rho values between 0.20–0.67. In 2 studies, CXCL13 levels correlated with ultrasonographic evidence of synovitis. Eighteen studies assessed CXCL13 in response to therapeutic intervention. The majority signified a fall in levels in response to treatment including biologics and Janus kinase (JAK) inhibition. In some, this reduction was only seen in treatment responders. High CXCL13 levels predicted failure to achieve disease remission with csDMARDs. The evidence for treatment prediction with biologics was conflicting.Despite evidence to suggest a role in diagnosing RA and in detecting synovitis, the heterogeneity of studies included in this review limit our ability to draw robust conclusions. At present there are inadequate results to justify the routine use of CXCL13 as a biomarker in RA routine clinical practice. Rheumatoid arthritis (RA) is a chronic, systemic immune-mediated disease characterised by synovial inflammation and progressive joint destruction . AlthougExisting biomarkers in RA, including acute phase reactants have their limitations. C-reactive protein (CRP) is a sensitive measure of inflammation but lacks disease specificity for joint inflammation and elevated levels are seen in intercurrent illness or with obesity . The eryCXCL13, also known as B-lymphocyte chemoattractant (BLC) or C-X-C motif chemokine 13, is a key chemokine involved in the positioning and activation of cells at lymphoid and extra-lymphoid sites. It binds to its cognate receptor CXCR5, and attracts CXCR5-expressing B cells and follicular T-helper cells . OverexpCXCL13 is of interest as a biomarker in RA, with a profound effect on shaping local synovial architecture and may provide a signature of a specific RA disease subset. Several studies have investigated the link between CXCL13, disease activity, serological markers (such as RF and ACPA) and change in response to treatment. To our knowledge, these studies have not been analysed collectively. Our objective was to evaluate the current existing literature in RA, to assess how CXCL13 correlates with disease activity, treatment response, and to determine its use as a potential biomarker in clinical practice.The literature was searched systematically by two investigators (CS and AD) using MEDLINE and EMBASE databases. The search terms used were ‘rheumatoid arthritis’ OR ‘inflammatory arthritis’ AND ‘CXCL13’ OR ‘Chemokine Ligand 13’. The search was undertaken in February 2018 and re-run prior to the final analysis to identify new studies that could be retrieved for incorporation in the systematic review.We identified English language publications of cohort or randomised controlled trials (RCTs). Publications were included if they met the following eligibility criteria: 1) the study evaluated CXCL13 levels 2); the study included patients diagnosed with RA based on the American College of Rheumatology (ACR) criteria. Studies presenting duplicate data were excluded. Titles and abstracts were screened independently by two investigators (CS and AD). The full text of the potential studies for inclusion were retrieved and assessed for eligibility. Data was extracted independently (by CS and AD). Disagreements over study eligibility were resolved through discussion with a third reviewer (KB). The primary outcome of interest was i) the level of CXCL13 in RA patients compared to healthy control (HC) and ii) the correlation between CXCL13 levels and markers of disease activity and iii) the association between CXCL13 and treatment response.The search identified 278 articles. Of these, 228 were excluded based on title and abstract. A further 19 studies were excluded after full paper review. Figure CXCL13 was measured in serum in 25 studies, in synovial tissue in 4 studies, and in both serum and synovial tissue in the remaining 2 studies  [r = 0.26–0.69) [Sixteen studies evaluated how well levels of CXCL13 correlated with other measures of RA disease activity (Table  = 0.27) , 31 and 26–0.69) , 29, 31.26–0.69) , 19, 32,26–0.69) . Two stun = 4), TNF inhibitors including adalimumab, etanercept, infliximab and golimumab (n = 7), IL-6 cytokine and receptor blockers including tocilizumab, vobarilizumab, sarilumab (n = 6), anti-CD20 B cell depleting therapy rituximab (n = 2), JAK inhibitors tofacitinib and filgotinib (n = 2) and anti-lymphotoxin alpha agent pateclizumab (n = 2).Nineteen studies evaluated changes in CXCL13 levels with treatment Table . The theIn csDMARD treated patients, high baseline levels of CXCL13 were associated with a reduced likelihood of achieving low disease activity. Bugatti reported a high negative predictive value of 80% for remission after 12 months of methotrexate therapy . With anReduction in levels were also noted with rituximab, tofacitinib and filgotinib and pateclizumab. With rituximab, CXCL13 levels significantly decreased in all patients coinciding with B cell depletion irrespective of responder status , 36, 44.To our knowledge, this is the first systematic review exploring whether CXCL13 is a clinically viable diagnostic marker in RA. CXCL13 is raised in RA, both in early and established disease. Serum levels may predict active joint inflammation and synovitis over current biomarkers. Although levels fall in response to certain RA therapies, the prognostic value of CXCL13 in treatment response remains unclear.CXCL13 is a marker of synovial inflammation in patients with RA. Levels are significantly higher than seen in healthy controls, present in very early RA (even in cases of less than 6 weeks disease duration)  and in bSerum CXCL13 levels can be used as a surrogate marker for synovitis. CXCL13 is produced by multiple cell types within the synovium, including T cells, monocytes/macrophages, endothelial cells and fibroblast . A numbeHowever, the evidence surrounding the correlation between CXCL13 and markers of disease activity is conflicting. Some studies report associations with DAS28, CRP, ESR and swollen joint count, whilst other studies have found no such correlation. There are several possible reasons for this. The DAS28 score does not solely reflect active disease driven by inflammation. The more subjective components of the score  could be driven by non-inflammatory mediators, which may explain the lack of association with CXCL13. Unlike serum CXCL13 which is primarily produced by the inflamed synovial tissue, CRP is produced by hepatocytes in response to interleukins released during inflammation and may not directly reflect synovial inflammation. This may explain the absence of correlation between these two markers . There iRA is a heterogeneous disease with a variety of clinical presentations and differences in responsiveness to treatment modalities. Considerable molecular heterogeneity has been reported and different patient subgroups proposed on the basis of gene expression patterns and predominant synovial cell infiltrates . The lymMultiple studies reported that CXCL13 levels reduced in response to treatment with biologics including anti-TNF, anti-IL6, B cell depletion and small molecule JAK inhibitor. In some studies, the reduction in CXCL13 was only seen in treatment responders. These finding were not duplicated in all studies. Three studies found no change in levels with treatment. With methotrexate initiation, in contrast to the early reduction in acute phase markers, CXCL13 levels did not significantly change, reflecting a more persistent pattern of synovial inflammation .As a predictor of treatment response, evidence is also conflicting. In patients receiving csDMARDs, CXCL13 levels predicted failure for disease remission, especially when combined with autoantibody positivity , 34. WitThere are limitations to this review. The most significant of these is the heterogeneity of the research questions and results between the studies reviewed. Each employed a different study design, with diverse study populations and analyses of an assortment RA therapies. The samples size across the studies ranged from 18 to 1135. Without a meta-analytical approach, it is impossible to incorporate the impact from a large study into the results. Disease activity, severity and treatment response were measured by a variety of different outcomes, limiting the ability to make direct comparisons.A further limitation relates to the variability in CXCL13 assays. It is important to recognise that CXCL13 assays are primarily used in the research setting and are not accredited clinical laboratory assays. There is significant potential variation across technologies  and laboratories. We have presented the differences in methodology across the studies to make sure this aspect of variation is fully appreciated. The timing of biomarker collection, e.g. at time of assessment or at disease flare, was also not matched across the studies. In addition, as mentioned earlier, a number of studies used binary thresholds for CXCL13 rather than presenting absolute values which needs to be considered when interpreting the results presented.In summary, CXCL13 expression is raised in a subset of patients with early and established RA, and levels may fall in response to therapy. It may have a discrete role as a biomarker in selected synovial pathotypes as a surrogate indicator of lymphoid organogenesis and active joint inflammation. In our opinion, there is not yet evidence for CXCL13 to predict disease progression or treatment response when used alone, partly because of the heterogeneity of the research questions and results from the studies reviewed here. We conclude that at present, the results are insufficient to justify the routine use of CXCL13 as a biomarker in the clinic setting. More research is required. For example, a study designed to recruit patients with new onset inflammatory arthritis, defined phenotypically and stratified to receive therapy based on serum CXCL13 levels would be of considerable interest. Without such a trial to test the hypothesis that CXCL13 is a useful biomarker, it is difficult to make a firm recommendation regarding its use in RA.Additional file 1.


Our aim was to characterize the effects of calorie restriction on the anthropometric characteristics and physical performance of sportsmen and to evaluate the effects of calorie restriction and acute exercise on mitochondria energetics, oxidative stress, and inflammation. Twenty volunteer taekwondo practitioners undertook a calorie restriction of 30–40% on three alternate days a week for one month. Eleven volunteer sportsmen participated as controls. Both groups performed an energy efficiency test to evaluate physical performance, and samples were taken before and after exercise. The total weight of participants significantly decreased (5.9%) after calorie restriction, while the efficiency of work and the contributions of fat to obtain energy were enhanced by calorie restriction. No significant differences induced by acute exercise were observed in individual non-esterified fatty acid percentage or oxidative stress markers. Calorie restriction downregulated the basal gene expression of nitric oxide synthase, antioxidant enzymes, mitochondrial uncoupling proteins, and repairing stress proteins, but it enhanced the expression of sirtuins in peripheral blood mononuclear cells. In conclusion, one month of calorie restriction decreases body weight and increases physical performance, enhancing energy efficiency, moderating the antioxidant and inflammatory basal gene expression, and influencing its response to acute exercise. Calorie restriction is a dietary strategy usually based on decreasing the calorie intake (about 20–40% of the ad libitum diet) without challenging the intake of essential nutrients . This inIn humans, calorie restriction is also associated with decreased oxidative stress and inflammatory markers ,15. At tCalorie restriction strategies include fasting periods in which fatty acids are mobilized from intracellular stores to provide energy to working muscles. In this context, the fatty acid composition of the diet is known to influence the fatty acid composition of plasma/serum lipids ,21,22,23ClinicalTrials.gov (NCT02533479).Thirty-one well-trained male taekwondo practitioners agreed to participate voluntarily in this study. The inclusion criteria were age (18–50 years old), gender , non-smokers, and performing physical activity 3 days a week. All athletes who participated in the study, were professional athletes from the High-Performance Center of Barcelona . All participants followed a very strict training program, training between 3 and 5 days per week for 2–3 hours. Training seasons included workout on fitness, technique, and combat. This sport was chosen because athletes often have to lose weight acutely and are more likely to follow such demanding calorie restrictions. Most of participants competed at the national and international level. All the subjects were informed regarding the objectives, requirements, and possible risks of the study before giving their written consent. Before being accepted to participate in the research, each subject passed a complete medical examination, which included a medical history and resting electrocardiogram (ECG). The study protocol was in accordance with the Declaration of Helsinki for research on human beings and was approved by the local Ethics Committee of the Consell Català de l’Esport . The project was registered at Participants were distributed between the control group and the calorie restriction group. The control group included 11 healthy sportsmen who followed a complete diet during the entire study. The calorie restriction group was formed by 20 healthy sportsmen who followed a diet with 30–40% calorie restriction for 1 month (4 weeks). The macronutrient composition of both diets was similar at the beginning of the calorie restriction period . ControlAll of the participants were informed of the purpose and demands of the study before providing their written consent to participate. The protocol complied with the Declaration of Helsinki for research on human subjects and was approved by the Clinical Research Ethics Committee at the Direcció General de l’Esport of the Catalonian Sports Council (Ref. 07033C0301).Each participant was interviewed regarding their dietary, lifestyle, and training habits. The nutritional value of their daily diet was assessed using a 7-day food record, including all foods and beverages consumed, specifying the servings, cooking techniques, and daily distribution. From this information, a diet analysis was performed using a computer program based on food composition tables, CESNID .Over a period of six weeks, the participants practiced an every-other-day fasting calorie restriction (CR) program, decreasing calorie intake by 33% with respect to their usual diets. The participants restricted their habitual diets for three alternate days each week and on the other four days participants’ dietary intakes were the same as they were at the beginning of the study without changes to caloric intake or distribution of meals. Adherence to the nutritional intervention program was assessed using a 7-day dietary record during the last week of the intervention. All foods and fluids consumed, portion sizes, how foods were prepared, and how consumption habits were distributed throughout the day were recorded. Athletes resided for 24 hours at the High-Performance Center of Barcelona, and they strictly followed the diet prescribed by medicals services from the center and the research team, with whom they were in continuous contact.2max) using a computerized metabolic cart . A fixed treadmill rise (3%) was maintained throughout the test. The treadmill running rate was initially set at 6 km/h and increased by 0.5 km/h each minute until maximum sustainable effort (muscle fatigue or stabilization/decline in VO2max) [2max and corresponding to the upper anaerobic threshold (if this was not under the 70% of maximum) was calculated by linear interpolation of data from the maximal exercise test. Each participant performed a maximal exercise test on the treadmill after overnight fasting at the beginning and at the end of the nutritional intervention. The athletes spent five consecutive minutes at 50%, 60%, and 70% of their VO2max running rate, and at the upper anaerobic threshold running rate until exhaustion. Continuous determination of O2 consumed (mL/min), CO2 expired (mL/min), and expiratory volume (L/min) was performed using a computerized metabolic cart  and the respiratory quotient, which enables the determination of the individual contribution of fatty acids in the aerobic metabolism, was calculated [Each participant completed three standardized maximal treadmill exercise tests . The fir VO2max) ,29. The lculated .g, 30 min, 4 °C) of the blood.Four venous blood samples were obtained from the antecubital vein of each subject (pre- and post-exercise) at the beginning and at the end of the study with suitable vacutainers containing EDTA (ethylenediaminetetraacetic acid) as anticoagulant. Venous blood was collected in overnight fasted conditions (pre-exercise), and 2 h after finishing the exercise test (post-exercise) as this is coincident with an increment in circulating immune cells, with changes in antioxidant enzymes activities and in markers of oxidative damage . Plasma ® 10 Capillary GC column, 30 m × 0.53 mm, df = 0.50 µm.Plasma NEFAs were determined by a column chromatographic method followed by derivatization with reagent Meth-Prep™ II (GRACE) and subsequent gas chromatography as previously described ,32. The Catalase (CAT) activity was measured in plasma by the spectrophotometric method of Aebi . Superoxv/v). 12 N HCl was added and the samples were incubated for 1 h at 45 °C. Absorbance was measured at 586 nm. This method is specific for MDA determination.Malonyldialdehyde (MDA), a marker of lipid peroxidation, was analyzed in plasma using a colorimetric assay kit . Briefly, samples and standards were placed in Eppendorf tubes containing n-methyl-2-phenylindole (10.3 mM) in acetonitrile:methanol  and nitrotyrosine (150 µg) were determined by immunological methods using the OxiSelect™ Protein Carbonyl Immunoblot Kit  and OxiSelect™ Nitrotyrosine Immunoblot Kit  following the manufacturer’s instructions. Total protein concentrations were measured by the Bradford method . Samplesg, 30 min, 4 °C) in 10 K filters  to remove proteins. Supernatants were recovered and used to measure nitrite and nitrate concentration by detecting liberated NO in the gas-phase chemiluminescence reaction with ozone, using an NO analyzer .Plasma samples were centrifuged . Gene expression were normalized to the invariant control 18S rRNA. mRNA levels at the beginning of the study were arbitrarily referred to as 1. The primers and conditions used are listed in mRNA expression was determined by multiplex real-time PCR based on incorporation of a fluorescent reporter dye and using human 18S rRNA as reference. For this purpose, total RNA was isolated from PBMCs by Tripure extraction following manufacturer instructions . RNA (1 µg) from each sample was reverse transcribed using 50 U of Expand Reverse Transcriptase  and 20 pmol oligo (dT) for 60 min at 37 °C in a 10 µL final volume, according to manufacturer instructions. The resulting cDNA (3 µL) was amplified using the LightCycler FastStart DNA Master PLUS SYBR Green I kit . Amplification was performed at 55 °C and 45 cycles. The relative quantification was performed by standard calculations considering 2p < 0.05 was considered statistically significant. Two-way ANOVA analyzed the statistical factors exercise (E) and calorie restriction (CR). Not normally distributed data was analyzed by the Friedman and Kendal test, and when significant differences between groups were found, these were identified by the Wilcoxon test.Statistical analysis was carried out using the Statistical Package for Social Sciences (SPSS v.21.0 for Windows). Results are expressed as mean ± standard error of the mean (SEM). The Shapiro–Wilk test was applied to verify the normality and homogeneity of variance. The statistical significance of data normally distributed was assessed by two-way analysis of variance (ANOVA) and Student’s t-test for unpaired data to determine differences between different groups, and 2 (mL/min), VCO2 (mL/min), and the respiratory quotient during the exercise test performed at 50%, 60%, and 70% of maximal speed and at threshold, but it did not affect VO2 (mL/kg·min) when corrected by body weight, except for the test performed at 70% of maximal speed. The control group maintained the initial values of expiratory volume (L/min), VO2 (mL/min), VCO2 (mL/min), and respiratory quotient during the exercise tests performed at 50%, 60%, and 70% of maximal speed and at threshold , monounsaturated fatty acid (MUFA), or polyunsaturated fatty acid (PUFA) fraction . The NEFOxidative damage markers and antioxidant parameters were not significantly influenced by calorie restriction or exercise . The marMnSOD and Cu/Zn SOD), enzymes related with the NO synthesis (iNOS), mitochondrial uncoupling proteins (UCP3), and repairing stress protein , as shown in SIRT3) and the vitamin E carrier protein alpha-tocopherol transfer protein (α-TTP), which was additionally upregulated by exercise (HO-1), or the basal expression of NFκB, PGC1α, PPARγ, TFAM, and p53. Acute exercise enhanced the gene expression of MnSOD, UCP3, and α-TTP only after the calorie restriction intervention. On the contrary, acute exercise without calorie restriction maintained the basal expression of these genes but increased the expression of the inflammatory protein IL6 and SIRT3. Acute exercise in both calorie restriction and control conditions enhanced the expression of the repair stress proteins HSP72.Calorie restriction significantly downregulated the gene expression of antioxidant enzymes  and physical activity (three days a week) during one month on physical performance with respect to a non-calorie restriction diet. Calorie restriction is associated with weight loss and improved health outcomes such as lower cardiometabolic disease risk and improved functional status . The calCalorie restriction induces mitochondrial proliferation in rodents ,44, and In spite of the changes in gene expression, the exercise tests realized before calorie restriction and at the end of the calorie restriction intervention do not seem to compromise the plasma antioxidant/oxidant balance of the participants, as the markers of oxidative damage and the activity of antioxidant enzymes remained unchanged. Our results show the maintenance of lipid and protein oxidative damage markers and plasma antioxidant enzyme activities after the maximal exercise test performed by participants both before and after the calorie restriction period. A study performed in male non-trained adolescents with obesity showed that 4 weeks of exercise training and dietary restriction reduces body weight, increases basal antioxidant enzyme activities (SOD and glutathione peroxidase), and decreases basal protein-carbonyls . We founThe calorie restriction period that reduces fatty acid availability from diet does not alter the NEFA composition of plasma, including SFAs, MUFAs, and PUFAs. Overweight and obese people, who are highly sensitive to calorie restriction, have an accelerated reduction in adiposity due to greater lipid mobilization and oxidation . Acute eIn conclusion, calorie restriction increases energy efficiency without modifying the NEFA profile or the plasma oxidative balance. The short-term application of this diet decreases body weight and increases physical performance. Calorie restriction also decreased the PBMCs’ basal gene expression of the antioxidant MnSOD and Cu/ZnSOD and iNOS enzymes, HSP72, and mitochondrial UCP3, whereas acute exercise induced their expression reaching values similar to those of no caloric restriction in well-trained athletes. Finally, calorie restriction attenuated the expression of the proinflammatory cytokine IL-6 after acute exercise and potentiated the expression of SIRT3 and TTP.