Probiotic strains are incorporated into food substrates to contribute to fermentation process. The technological suitability of such strains to improve the flavor and nutritional value of fermented food is strain-specific. Potentially probiotic yeasts isolated from Nigerian traditional fermented foods were assessed for production of volatile compounds by gas chromatography-mass spectrophotometry. Phytases were characterized for activity and stability at different pH (3–8) and temperatures (25-50 °C).Pichia kluyveri LKC17, Issatchenkia orientalis OSL11, P. kudriavzevii OG32, P. kudriavzevii ROM11, and Candida tropicalis BOM21. They include alcohols (14), carbonyls (13), esters (10), and organic acids (8). Phenylethyl alcohol was the highest higher-alcohol in Issatchenkia orientalis OSL11 (27.51 %). The largest proportion of esters was detected in P. kudriavzevii OG32 (17.38 %). Pichia kudriavzevii OG32 and C. tropicalis BOM21 showed vigorous gowth in minimal medium supplemented with sodium phytate (2 g L−1). Extracellular phytases from P. kudriavzevii OG32 and Candida tropicalis BOM2 showed optimal activiy at pH 4.6 (104.28 U) and pH 3.6 (81.43 U) respectively.A total of 45 volatiles compounds were identified from intracellular cell-free extracts of Results obtained revealed species- and strain-specific potentials of the yeast strains to improve flavor and mineral bioavailability of fermented food products. Therefore, the application of these yeasts as starter cultures during food fermentation process is a very promising method to enhance the flavor profile and enhance mineral bioavailability in indigenous cereal-based fermented food products. Probiotics are live microorganisms which, when administered in adequate amounts, confer health benefits on the host . Indigenburukutu, cheese, fura, gowe, kunu-zaki, mawe, nunu, kefir, ogi, olives, and wines [Saccharomyces species, including strains of Debaryomyces hansenii, Issatchenkia orientalis, Galactomyces geotrichum, Kluyveromyces marxianus, K. lactis, Pichia farinosa, P. anomala, P. kudriavzevii, and Yarrowia lipolytica. Their robust sizes (approx. 5 × 10 μm), stress tolerance, antibiotic resistance that is not due to mobile genetic materials, non-pathogenic, non-toxigenic and non-allergenic nature, versatile enzyme profile, and production of several bioactive compounds contribute to the selection of yeasts as probiotics.Several authors have reported the probiotic potentials of yeast strains from several indigenous fermented foods and beverages: nd wines –11. MostSaccharomyces yeasts are gaining attention as excellent producers of flavor compounds. They include some species of Candida, Galactomyces, Geotrichum, Hanseniaspora, Pichia, Saccharomycopsis, Saprochaete, Starmera, and Wickerhamomyces [Technological suitability, including contribution to appealing flavor and improvement of nutritional value, are desirable for the selection of probiotic strains to be incorporated in non-dairy food systems, especially cereal models . Volatilamomyces –15. VOCsamomyces –18.Phytate , the main form of phosphorous and a complex with dietary minerals  in cereals, legumes, and nuts, is indigestible in the monogastric gastrointestinal tract , 20. PhyArxula adeninivorans, Hanseniaspora guilliermondii, I. orientalis, P. anomala, P. kudriavzevii, Saccharomyces cerevisiae, and S. pastorianus [Phytase-active probiotic strains have the potential to improve the in situ and in vivo bioavailability of the divalent minerals, during food fermentation and in the gastrointestinal tracts respectively , 24. Sevtorianus , 25–28. In a previous study, we determined the probiotic potentials of yeasts isolated from some cereal-based Nigerian traditional fermented food product . These ySolvents and other chemicals were of analytical grade. Sodium phytate was obtained from Sigma Chemical Co. .Pichia kluyveri LKC17, Issatchenkia orientalis OSL11, P. kudriavzevii OG32, P. kudriavzevii ROM11, and Candida tropicalis BOM21 by sequencing D1/D2 region of large subunit of 26S rDNA gene. The GenBank accession numbers are KJ472904, KJ472906, KJ472905, KJ472907, and KJ472908 respectively [Yeast strains used in this study were selected based on the demonstration of probiotic potentials in a previous study . They ha−1 and 4 °C for 10 min, the supernatant was discarded and yeast cells were washed twice with sterile saline water (0.85% NaCl).A 1% (v/v) of 24-h-old broth culture of each yeast strain was inoculated in YPD broth and incubated at 30 °C till exponential phase (24 h). A 10 mL broth culture was centrifuged at 7500 rev minExtraction of volatile compounds was done by liquid-liquid extraction . A suspe−1. The oven temperature was held at 100 °C for 6 min, heated at 4 °C min−1 to 150 °C, then at 8 °C min−1 to 220 °C and held at 220 °C until an approximate run time of 40 min.Volatile compounds in the concentrated extract were separated and detected by using a gas chromatography-mass spectrophotometer (GC-MS) . The separation of volatiles was carried out in an ELITE 1 non-polar capillary column (30 m X 0.25 mm (ID); 0.25 μm film thickness). One microliter of extract was injected (split ratio 1:10) into the injection port and carried along the capillary column by helium gas (99.9%) at a flow rate of 1 mL minThe mass spectrophotometer was operated in the electron impact mode and mass spectra were taken using an ionization voltage of 70 eV. The mass scan range was 40–400 AMU, with a scanning speed of 0.2 s. Data acquisition and generation of chromatograms and mass spectra were done with the TurboMass software .The identification of volatile compounds was performed by comparing the mass spectra with the standard spectra database from the NIST Ver. 2.1 2009 Mass Spectra Library. The proportion of each compound was calculated by comparing the peak area with the total area.−1 and 4 °C for 15 min) and cells were resuspended in 0.5 mL of sterile saline water. A 1% (v/v) of each cell suspension was inoculated into respective liquid growth media; phosphate-free minimal medium  as negative control, phosphate-containing minimal medium  as positive control and phytate-containing minimal medium  as test. The broth cultures were incubated at 30 °C for 48 h. Yeast growth was determined after gentle agitation and measurement of the optical density at 600 nm [Ao is the absorbance in phosphate-containing minimal medium and Ai is the absorbance in either phosphate-free or phytate-containing minimal medium.The test yeast strains were screened for phytase production by determining their ability to grow in a minimal medium with phytic acid as the sole source of phosphorus . Yeast ct 600 nm . This wa4NO3, 0.5 g MgSO4.7H2O, 2 g CaCl2, 0.5 g KCl, 0.01 g FeSO4.7H2O, 0.01MnSO4.H2O). The medium was incubated in a shaking water bath (1700 rev min−1) at 30 °C for 48 h. Culture supernatant with extracellular phytase was obtained after the centrifugation (7500 rev min−1 and 4 °C for 10 min) of the broth culture and used for extracellular phytase assay [A 1% (v/v) of an overnight culture of selected yeast strain was inoculated in a 250-mL Erlenmeyer flask, containing 100 ml of minimal salt medium  (0-5 mmol mL−1). One unit (U) of phytase activity was defined as that which liberated one micromole of phosphate per minute under the assay conditions [Phytase activity at different pH (3–8) was assayed by measuring the amount of inorganic phosphate liberated from sodium phytate in different buffer systems; 0.2 M citrate buffer (pH 3.0 and 6.0), 0.2 M acetate buffer (pH 3.5–5.5) and 0.2 M Tris–HCl buffer (pH 7.0–8.0). The reaction mixture consisted of 0.8 mL of the respective buffer containing 2 mM of Na-phytate and 0.2 mL of enzyme extract. Negative controls were prepared from enzyme extracts mixed with respective buffer without phytic acid. Reaction mixtures were incubated at 37 °C and stopped after 30 min by adding 1 mL of 10% trichloracetic acid (TCA). The blank was prepared by adding 10% TCA solution before the substrate was added. Determination of liberated inorganic phosphate was performed according to the ferrous sulfate-ammonium molybdate method [nditions .Phytase was incubated at pH 3-8 for 1 h at 4 °C and different temperatures (20-50 °C) for 1 h. The residual phytase activity was assayed and relative activity was calculated .Pichia kluyveri LKC17 produced eighteen volatile compounds and carbonyls accounted for the highest number (7) and largest proportion (36%). The highest variety of alcohol was produced by I. orientalis OSL11. Phenyl ethyl alcohol was noted to be the largest proportion of alcohol produced by I. orientalis OSL11, P. kudriavzevii OG32, and P. kudriavzevii ROM 11. The largest proportion of esters was detected in P. kudriavzevii OG32 and it accounted for 17.38% of the total volatile compounds produced by the strain  as sources of phosphorous for growth at 30 °C for 48 h. This was observed as increased cell density (600 nm) in respective broth cultures Fig. . The relC. tropicalis BOM21 and P. kudriavzevii OG32 respectively. These indicate that they are acid phytases. Considering extracellular phytase from P. kudriavzevii OG3, a decline in activity was recorded as the pH move toward extreme acidic and neutral pHs. However, another peak (70.3 U) was recorded at pH 5.6 for C. tropicalis BOM21. Extracellular phytases from both test strains retained approximately 60% of optimal activity over a wide range of pH (3–8) (Fig. The effects of pH based on three different buffer systems on extracellular phytase activity of both strains are shown in Fig. In addition to the probiotic potentials of yeasts, some strains produce valuable metabolites that contribute to the sensory quality and nutritional value of fermented food products . These aI. orientalis OSL11 and P. kudriavzevii OG32 and ROM11. It is one of the most abundant higher alcohols produced by yeasts, arising from the degradation of phenylalanine through the Ehrlich pathway [P. kudriavzevii OG32 is the most dominant ester producer. Prominent among the esters from the strain is benzyl butanoate. It has a fruity aroma, specifically bringing about tropical, pineapple, and apple reminiscence [P. kluyveri LKC17 and pentadecanoic acid 3 methylbutyl-acetate from I. orientalis OSL11, P. kudriavzevii OG32, and C. tropicalis BOM21 are important esters that were detected in significant proportions. Methyl 2-phenyl acetate is a high strength odor compound that is characterized by honey and jasmine aroma [P. kluyveri and P. kudriavzevii from some other traditional fermented food sources [Starmera caribaea and Hanseniaspora guilliermondii were reported to produce high levels of acetate esters with highly desirable flavor [The potentially probiotic yeasts evaluated in this study showed strain-specific potentials to impart pleasant taste and distinct flavor on fermented foods by producing volatile organic compounds such as organic acids, alcohols, and esters. Previous studies reported species and strain specificity in the profile of flavor compounds produced by yeasts . Among t pathway , 18, 37. pathway . Several pathway . Esters  pathway , 16, 18.niscence . In addine aroma . Similar sources , 39. Othe flavor .P. kudriavzevii OG32 and C. tropicalis BOM21 are in agreement with previous studies, being an indication of phytase production. Pichia kudriavzevii has been reported to produce cell-bound, intracellular and extracellular phytase [Phytase production has been previously detected and quantified with growth test in liquid medium supplemented with phytate salts as the sole source of phosphate , 27. The phytase , 40. TheC. tropicalis BOM21 and P. kudriavzevii OG32 in the acidic pH range of pH 3.6-pH 4.6 are similar to the optimum pHs of phytases from other yeasts [C. tropicalis BOM21 and P. kudriavzevii OG32 to remove phytate in diverse food fermentation models and the intestine.The maximum activity exhibited by phytases secreted by r yeasts , 41, 42.The volatile compounds identified to be produced by yeasts in this study are safe and possess relevant aroma for food use. In addition the phytase secreted by the yeast strains demonstrated activity and stability at conditions that prevail during food fermentation. Therefore, the application of these potentially probiotic yeasts as starter cultures during food fermentation process is a very promising method to enhance the flavor profile and enhance mineral bioavailability in indigenous cereal-based fermented food products.


Feather pecking (FP) in laying hens reduces animal welfare and leads to economic losses for the layer industry. FP is considered a heritable condition that is influenced by dysregulation of neurotransmitter homeostasis, the gut microbiome, and the immune system. To identify genes and biological pathways responsible for FP behavior we compared the brain transcriptomes of 48 hens divergently selected for FP. In addition, we tested if high feather peckers (HFP) and low feather peckers (LFP) respond differently to light since light has been shown to trigger FP behavior.Of approximately 48 million reads/sample an average of 98.4% were mapped to the chicken genome (GRCg6a). We found 13,070 expressed genes in the analyzed brains of which 423 showed differential expression between HFP and LFP. Genes of uncertain function and non-coding RNAs were overrepresented among those transcripts. Functional analyses revealed the involvement of cholinergic signaling, postsynaptic activity, membrane channels, and the immune system. After the light stimulus, 28 genes were found to be differentially expressed. These included an interaction cluster of core components of the circadian clock. However, differences in the response to light between HFP and LFP were not detectable.Genes involved in cholinergic signaling, channel activity, synaptic transmission, and immune response were found to be involved in FP behavior. We propose a model in which the gut microbiota modulates the immune system, which in turn affects cholinergic signaling. This might have an influence on monoamine signaling with possible involvement of GABA or glutamate signaling. Feather pecking (FP) is a serious problem of the layer industry causing economic losses and massive impairments of animal welfare. The propensity to perform this damaging behavior is a complex trait influenced by numerous environmental factors as well as a genetic component. Reported heritability estimates of around 0.15  indicateA longstanding theory explained FP as a redirected foraging behavior , 5, whicAs comprehensively reviewed by , it thusadj < 0.01)   to DE ncRNAs.The average number of reads per sample was 48,502,572  with an average mapping efficiency of 98.4% after alignment to the reference genome with TopHat  and KEGG pathway analyses with DE genes between HFP and LFP hens with clusterProfiler  are showARNTL, NFIL3, NPAS2, and PER2 are overrepresented in these GO terms. The category GO molecular function exclusively contains IDs related to gene expression.Concerning the comparison of DE genes between animals before and after light stimulation Table , the majGeneset analysis with STRING  was perfAnalysis of DE genes before and after light stimulation with STRING Table  led to tFIP1L1 as well as SSU72, CPSF2, CPSF3, CPSF4, WDR33, CDC5L, PCF11, CLP1, CSTF1, CSTF2, CSTF3, and PAPOLA, exclusively contains genes, which are responsible for mRNA processing as well as mRNA binding. The slightly smaller cluster with fewer connections exclusively contains genes, which are associated with the immune system - T-cell proliferation and regulation in particular. The immune cluster consists of the DE genes CD28 and LIF as well as CD274, CTLA4, CD80, CD86, IL2B, IL12A, IL6ST, and LIFR. Analysis of DE genes before and after light stimulation led to the discovery of one interaction cluster, which contains the DE genes ARNTL, PER2, PER3, NFIL3, NPAS2, and LONRF3, all of which, except LONFRF3, are core components of the circadian clock  to be associated with severe FP This gene plays a major role in immune response [IRF5 (padj = 0.0002), IRF6 (padj = 1.17E-06), and IRF8 (padj = 0.0002) to be DE between the lines. Wysocki et al. reported DE of genes related to monoamine signaling, namely MAOA (monoamine oxidase A) and HTRR1B (5-hydroxytryptamine (serotonin) receptor 1B). We were able to confirm MAOA (padj = 7.2E-05), but not HTRR1B. Instead, we found DE of HTR1A (padj = 0.04), HTR1E (padj = 0.001) and HTR6 (padj = 2.34E-05).In this study, we compared 24 full-sib pairs of laying hens selected for HFP and LFP in a transcriptomics approach. Besides the line comparison, we assessed the transcriptional changes before and after light stimulation. To our knowledge, this is the first brain transcriptomic study in FP that used RNA-seq. Comparison with the few other available transcriptome studies revealed only little overlap, which might be due to the fact that these studies are microarray-based and applied different designs. Hughes and Buitenhuis  used a mresponse  and mighCHRNA2, CHRNA9, CHRNB3, and CHRNB4 coding for subunits of nicotinic acetylcholine receptors (nAchR). Most other studies found serotonergic or dopaminergic signaling to be involved in FP [CD28 and LIF as well as CD274, CTLA4, CD80, CD86, IL2B, IL12A, IL6ST, and LIFR), all of which are linked to T-cell proliferation and regulation  for several hours until they clearly showed FP and were then sacrificed as well and brains were collected for RNA isolation. All brain samples were kept in RNA later and frozen at − 80 °C until further processing.Experiments were conducted in White Leghorn layer strains divergently selected for FP behavior , 55. StrWhole chicken brains were pulverized with a Retsch mixer mill MM400 in liquid nitrogen at a frequency of 30 Hz for 1 min and immediately frozen at − 80 °C. For RNA isolation 900 μl Qiazol (QIAGEN), 5 μl DX reagent and 20–30 ceramic beads were added to10–30 mg of pulverized brain. The mixture was placed in a Bead Ruptor (Omin Inc.) and homogenized with the following program: 4.5 m/sec, 3 times 15 s, 10s dwell. After 5 min incubation at room temperature 100 μl gDNA Eliminator solution (QIAGEN) was added and the sample was vortexed for 15 s. After addition of 180 μl chloroform the sample was vortexed for 15 s and incubated at room temperature for 3 min. Sample was centrifuged at 12,000 g and 4 °C for 20 min and aqueous phase was transferred to a fresh reaction tube. An equal amount of 70% ethanol was added to the sample and vortexed for 15 s. RNA was purified using the RNeasy Plus Universal Mini Kit (QIAGEN) according to the manufacturer’s protocol and stored at − 80 °C.Input total RNA was visualized on a TapeStation 4200 (Agilent) and all but three samples showed excellent RNA quality with RIN-scores > 8 . Samples were quantified on a Qubit 2.0 Fluorometer (ThermoFischer) and 500 ng of total RNA was used as input for the TruSeq stranded mRNA library kit (Illumina) following the manufacturers manual. Resulting libraries showed a fragment size distribution of around 300 bp and were 2 × 75 bp paired-end. Sequencing was conducted on the HiSeq 4000 (Illumina) with 10 samples / lane.Raw sequencing reads were processed with Trimmomatic version 0.36  for adapOne BED file was created containing DE ncRNAs and one BED file containing all protein coding genes annotated in chicken genome version GCF_000002315.5 . The closest coding gene to each respective ncRNA was identified with BEDtools (Version 2.27.1) .org.Gg.eg.db (Version 3.11.4) [p-value correction. Genes differentially expressed with an absolute LogFC ≥0.5 were analyzed for enrichment in GO terms and KEGG pathways against the background of all expressed genes in our data set. For the gene set analysis with STRING (Version 11.0) [p-value below 0.01 were used. Protein interaction networks for these genes were also computed with STIRNG using the following settings: evidence-based coloring of network edges, inclusion of all interaction sources, medium required interaction score 0.4, max number of interactions to show 1st shell: no more than 10 interactions, 2nd shell: no more than 10 interactions. For DE genes before and after light stimulation LogFC threshold was 0.5. Settings for protein interaction networks were as follows: evidence based coloring of network edges, inclusion of all interaction sources, medium required interaction score 0.4, max number of interactions to show 1st shell: none / query proteins only, 2nd shell: none.Gene set analyses were conducted using the R package clusterProfiler (Version 3.16.0)  along wi 3.11.4)  and usinon 11.0) , DE geneAdditional file 1. Transcriptome alignment summary.Additional file 2. FPKM values.Additional file 3. Results of differential expression analyses.Additional file 4. Detailed information on eRNAs.Additional file 5. Results of clusterProfile analyses.Additional file 6. Results of STRING analyses.


Data from three different techniques that describe global and regional connectivity differences within functional resting-state networks in transwomen and transmen with early-in-life onset gender incongruence are provided: (1) we obtained spatial maps from data-driven independent component analysis using the melodic tool from FSL software; (2) we provide the functional networks interactions of two functional atlases’ seeds from a seed-to-seed approach; (3) and global graph-theoretical metrics such as the smallworld organization, and the segregation and integration properties of the networks. Interpretations of the present dataset can be found in the original article,  Specifications table•These data provide structural and resting-state functional MRI scans of transgender individuals with gender incongruence. This condition is rare with prevalence rates around 1% in the population •These data can be valuable for researchers who aim to explore the underlying neurobiology of different gender variants from cisgender identities to transgenderism by means of MRI.•replication crisis.These data can be used as an independent sample for studies that aim to characterize and decompose spatial and temporal components into functional group-networks using resting-state imaging in young adults. The use of an independent sample in MRI studies can also be helpful to deal with the 1https://doi.org/10.17632/hjmfrv6vmg.2Data include the original T1-weighted and T2*-weighted images of the 87 participants in the original article https://doi.org/10.17632/dn82xj4bft.3https://doi.org/10.17632/zt27ykdrgr.4movement_parameters.txt file in https://doi.org/10.17632/ts8c7fm8dj.1The processed resting-state images that were used for the independent component analysis (ICA) approach were published in https://doi.org/10.17632/ts8c7fm8dj.1The general linear model (GLM) comparing groups of transgender people with cisgender groups can be found in 1.1https://doi.org/10.17632/rw2yhtpj96.3https://doi.org/10.17632/bgyzz94mz9.3https://doi.org/10.17632/ts8c7fm8dj.1For the seed-to-seed and graph-theory approaches, unsmoothed images  after deleting connections with negative values. Graph theory measures can be found in the Mendeley repository (doi: 22.1Twenty-nine transmen (TM) and 17 transwomen (TW) with gender incongruence according to the ICD-11 with an identification with the other gender; and 22 ciswomen (CW) and 19 cismen (CM) underwent MRI evaluation.2.2T1 and T2*-weighted images were acquired in a SIEMENS MAGNETOM TrioTim syngo MR B19. T1-weighted images were acquired in the sagittal plane using a MPRAGE iPAT GRAPPA (PAT 2), TR = 2,300 ms, TE = 2.98 ms, TI = 900 ms, echo spacing = 7.1 ms, flip angle = 9°, matrix size = 256 × 256 mm, 240 slices, resolution = 1 mm isotropic, bandwidth = 240 Hz/pixel, total scan time of 7.48 minutes. T2*-weighted gradient-echo echo planar imaging was acquired in the resting-state: TR = 2,500s, TE = 28ms, echo spacing = 0.48 ms, flip angle = 80°, matrix size = 256 × 256, 40 slices, resolution = 3 mm isotropic, bandwidth = 2404 Hz/pixel, volumes = 240 , no in-plane GRAPPA, through-plane multiband or any Partial Fourier were used.2.3http://afni.nimh.nih.gov/afni) tools, included: discarding the first five volumes to allow magnetization stabilization, despiking, motion correction, grand-mean scaling, linear detrending, and temporal filtering (maintaining frequencies above 0.01 Hz).Basic functional image preprocessing, using AFNI , we created spheres of 5 mm with the FSL software as previously described .We also used the 200 ROIs of the functional Craddock's atlas 2.4To control for head motion, an exclusion cut-off was established for mean interframe head motion at ≥ 0.3 mm translation or 0.3° rotation; and for maximum interframe head motion at ≥ 1 mm translation or 1° rotation.https://github.com/maartenmennes/ICA-AROMA).To remove the effects of head motion and other non-neural sources of signal variation from the functional data, we used an ICA-based strategy for Automatic Removal of Motion Artifacts (ICA-AROMA) As a quality control measure to assess the efficacy of ICA-AROMA in reducing relationship between signal variation and motion, we performed correlations between framewise head displacement and overall signal variation after regressing the ICA-AROMA components.2.5https://fsl.fmrib.ox.ac.uk/fsl/fslwiki/) was used to obtain temporal-concatenated spatial maps based on an ICA approach. Temporally and spatially coherent patterns of signal variation were extracted from functional images with a predetermined dimensionality of 25. The SN, the DMN and the bilateral ECN were considered to be the networks of interest.Melodic from FSL v5.0.10  into the subject's 4D space-time dataset. This ends in a set of subject-specific timeseries, one per group-level spatial map. Next, those timeseries are regressed  into the same 4D dataset, resulting in a set of subject-specific spatial maps, one per group-level spatial map. We then tested for group differences using the FSL permutation-testing tool  with threshold-free cluster enhancement (TFCE). A binarized mask for each network was applied.2.6fslmeants command (https://fsl.fmrib.ox.ac.uk/fsl/fslwiki/Fslutils). The connectivity between two ROI was estimated using Pearson's correlation between their time series. Therefore, a 56 × 56 matrix and a second 200 × 200 matrix were obtained for each of the 87 subjects. Seed-to-seed intergroup differences in the strength of the edges were obtained with the threshold-free network-based statistics, TFNBS The first eigenvariate of the BOLD signal temporal series was extracted for the ROI form the two atlases with the 2.7A graph-theory approach was applied using the same matrices (56 × 56 × 87 for the Stanford atlas and 200 × 200 × 87 for the Craddock's atlas). Networks were constructed using only positive r values, i.e., setting negative values to 0. We used a sparsity threshold to create a set of undirected graphs  using the r correlation values as edge weights for each pair of seeds for each subject. Sparsity is a measure of network density that ensures that all subjects’ networks would have the same number of edges to facilitate group comparisons. The range of sparsities was 5 to 25% with incremental steps of 2.5%. These percentages mean that, in the first threshold, 95% of the weakest connections will be deleted, 92.5% for the second and so on. The global graph theory measurements computed were:clustering coefficient, which quantifies the number of connections that exist between the neighbors of a node as a proportion of the maximum number of possible connections. As a global measurement, it is the average of the clustering coefficient of all nodes. This measurement was normalized by 1,000 random networks .The The characteristic path length of a node, which is the average of the minimum number of edges that must be traversed to go from this node to any other network node. As a global measurement, it is the average of the characteristic path length of all nodes. This measurement was normalized by 1,000 random networks.Modularity, indicates the degree to which a network can be subdivided into well-delineated modules, each containing several densely interconnected nodes with relatively few connections between nodes in different modules.Small world coefficient, defined as the ratio of the average clustering coefficient to the characteristic path length divided by the ratio of the same measurements obtained from equivalent random networks. Networks that have this small-world property usually have coefficients >1.


Streptococcus pyogenes  causes a variety of infections that include life-threatening, severe invasive GAS infections, such as streptococcal toxic shock syndrome (STSS), with > 30% mortality rate, despite effective antibiotics and treatment options. STSS clinical isolates highly express streptolysin O (SLO), a member of a large family of pore-forming toxins called cholesterol-dependent cytolysins (CDCs). SLO is an important toxic factor for GAS and may be an effective therapeutic target for the treatment of STSS. Our aim was to identify a monoclonal antibody (mAb) that reacts with SLO and has therapeutic potential for STSS treatment.We focused on mAbs that had originally been established as neutralizing reagents to perfringolysin O (PFO), another member of the CDC family, as some cross-reactivity with SLO had been reported. Here, we confirmed cross-reactivity of an anti-PFO mAb named HS1 with SLO. In vitro analysis revealed that HS1 mAb sufficiently prevented human neutrophils from being killed by STSS clinical isolates. Furthermore, prophylactic and therapeutic injection of HS1 mAb into C57BL/6 mice significantly improved the survival rate following lethal infection with an STSS clinical isolate. These results highlight the therapeutic potential of HS1 mAb for STSS treatment. Streptococcus pyogenes, also known as group A Streptococcus (GAS), is one of the most common human pathogens, and causes a wide spectrum of diseases ranging from pharyngitis and skin lesions to streptococcal toxic shock syndrome (STSS) and necrotizing fasciitis. The first two are generally self-limiting, while the latter two are severe and life-threatening, with mortality rates of 30–70% even with immediate antibiotic therapy and intensive care [ive care –4. Thereive care . The preive care , includiive care .+-glycohydrolase [Severe invasive GAS clinical isolates often express virulence factors more abundantly than non-invasive GAS clinical isolates. For example, STSS clinical isolates highly express streptolysin O (SLO), which belongs to a large family of pore-forming toxins called the cholesterol-dependent cytolysins (CDCs) –13. SLO ydrolase –25. TakeClostridium perfringens-derived perfringolysin O (PFO) in the CDC family [Several hybridomas that produce monoclonal antibodies (mAbs) specific for C family  have beeC family . The aimThe experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment.Hybridomas that produce mAbs (mouse IgG1) reactive with PFO were recovered from liquid nitrogen. 3H10 and 2C5 hybridomas were re-cloned by the limiting dilution method at 0.3 cell/well in a 96-well plate, resulting in 7 and 11 colonies, respectively. 4D8 hybridoma was re-cloned at 1 cell/well in a 96-well plate, resulting in 5 colonies. The re-cloned hybridomas 3H10, 4D8, and 2C5 that showed the most potent binding activities against PFO were renamed HS1, HS2, and HS3, respectively.ο-Phenylenediamine dihydrochloride (Sigma-Aldrich) in 0.05 M citrate–phosphate buffer (pH 5.0) was used for detection. Absorbance was read at a wavelength of 490 nm with an iMark Microplate Reader .First, 96-well plates were coated with 50 µL (1 μg/ml) of PFO  or SLO . The plates were washed four times with 0.1% Tween20 in sterile PBS (PBS-T), and then blocked with 1% BSA in PBS-T for 2 h at room temperature; the diluted HS1, HS2, and HS3 were incubated for 2 h at room temperature. After the samples were washed with PBS-T, they were incubated with anti-mouse IgG conjugated to horseradish peroxidase  for 2 h at room temperature. After washing the samples with PBS-T, 0.5 mg/ml Equal amount (1 μg/ml) of PFO  or SLO  was suspended in SDS sample buffer (Bio-Rad), and boiled for 5 min. The proteins (10 ng/10 μL) were separated by SDS-PAGE, and then transferred to polyvinylidene fluoride membranes . Membranes were then probed with HS1, HS2, and HS3 mAbs at a concentration of 1 μg/ml as the primary antibody. Subsequently, membranes were labeled with anti-mouse IgG conjugated to horseradish peroxidase , and visualized using the ECL Western Blotting Detection System .emm3 genotype) and NIH230 (emm49 genotype) were isolated by the Working Group for Beta-hemolytic Streptococci in Japan [The invasive strains NIH34  in 24-well plates containing 600 µl medium, or 100 nM interleukin (IL)-8 solution ; these were incubated with or without 3 × 106 bacteria in the absence or presence of HS1, HS2, HS3 mAbs, and control mouse IgG  for 60 min at 37 °C prior to the assay. After 60 min incubation, cells in the lower wells were collected and 104 10 μm microsphere beads  were added. Cells were stained with propidium iodine (Sigma-Aldrich) for flow cytometry to quantify viable neutrophils, and were analyzed using a FACSCalibur .Human neutrophils were isolated from the venous blood of two healthy volunteers, in accordance with a protocol approved by the Institutional Review Board for Human Subjects, National Institute of Infectious Diseases, Japan (Permit number: 756). This study complies with the guidelines of the Declaration of Helsinki. A modified chemotaxis assay was performed as previously described , 16. Bri7 colony-forming units in 0.5 ml PBS) was inoculated intraperitoneally into 6-week old C57BL/6 male mice (5 or 6 mice per group). 1 h before or after infection, mice were administered an intraperitoneal injection of HS1, HS2, HS3 mAbs, and control mouse IgG (Rockland) (1 mg per mouse). Mouse survival was monitored daily for the indicated time period. At the end of the experiment and the humane endpoint, mice were euthanized by cervical dislocation under anesthesia. The humane endpoint was applied when abnormal appearance over a prolonged period with no visible indications of recovery was observed.All animal protocols were approved by the Animal Experiments Committee at the National Institute of Infectious Diseases , and were compliant with the Guide for Animal Experiments Performed at the National Institute of Infectious Diseases, Japan. Six-week old C57BL/6 male mice, 20–25 g, were purchased from Japan SLC , and maintained in specific pathogen-free conditions. Mice were kept in collective cages (5–6 mice/cage) under a 12-h light/dark cycle and received water and feed ad libitum. GAS isolate (4.0 × 10Data are expressed as mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism 8 . Statistical significance was determined by the log-rank test. A value of p < 0.05 was considered statistically significant. *p < 0.05. The number of animals used in each experiment was determined on the basis of previously obtained results with the experimental model system , 28. FigHybridomas that produce neutralizing mAbs 3H10, 4D8, and 2C5 against PFO  were re-We next investigated the neutralizing capacity of HS1, HS2, and HS3 mAbs for the cytotoxic activity of SLO against human neutrophil survival in the presence of an STSS clinical isolate. We have previously established a sensitive in vitro assay for GAS virulence based on SLO activity, by measure of neutrophil viability using a modified chemotaxis assay , 16; humTo verify the effect of the neutralizing mAb HS1 on infection in vivo, a 7-day survival rate was undertaken, using a mouse model intraperitoneally infected with an STSS clinical isolate. Pre-treatment with HS1 mAb, but not control IgG, HS2, and HS3 mAbs, significantly improved the survival rate of mice infected with the STSS clinical isolate  therapy in STSS has been evaluated . A high In this study, the ability of an STSS clinical isolate to kill neutrophils was inhibited by the addition of HS1 mAb, but not HS2 and HS3 mAbs. Furthermore, in the mouse model, pre- and post-treatment with HS1 mAb, but not HS2 and HS3 mAbs, significantly increased the survival rate of mice infected with an STSS clinical isolate. These results suggest that HS1 mAb, which prevents neutrophil death in GAS infection, may be used as an adjunctive protective antibody for STSS.Bacillus, Listeria, Lysinibacillus, Paenibacillus, Brevibacillus, Streptococcus, Clostridium, Gardnerella, Arcanobacterium, and Lactobacillus [Clostridium bifermentans-derived bifermentolysin and Clostridium sordellii-derived sordelliolysin [SLO belongs to a large family of pore-forming toxins called CDCs –13. CDCsbacillus . Notablyliolysin , 31. If We did not identify the specific conformational epitope of SLO and PFO recognized by HS1 mAb. In vivo protection assays were performed using only one strain, single dose, and intraperitoneal route of mAb administration.Additional file 1. Immunoblot analysis of HS1, HS2, and HS3 mAbs binding to PFO and SLO.


Bjerkandera adusta T1 was applied to investigate gene expression and regulation. A total of 132, 300, 422, and 823 differentially expressed genes (DEGs) were identified at 6, 12, 24, and 48 hr, respectively. Most DEGs exhibited high endopeptidase activity, cellulose catabolic process, and transmembrane transporter activity by using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Many decomposition genes encoding endopeptidases were induced a little later in B. adusta T1 when compared with previously investigated algicidal fungus Trametes versicolor F21a. Besides, the accumulated expression of Polysaccharide lyases8 (PL8) gene with peptidoglycan and alginate decomposition abilities was greatly delayed in B. adusta T1 relative to T. versicolor F21a. It was implied that endopeptidases and enzymes of PL8 might be responsible for the strong algicidal ability of B. adusta T1 as well as T. versicolor F21a.Fungal mycelia can eliminate almost all cocultured cyanobacterial cells within a short time. However, molecular mechanisms of algicidal fungi are poorly understood. In this study, a time‐course transcriptomic analysis of algicidal fungus  Bjerkandera adusta T1 was investigated by a time‐course transcriptomic analysis. The results showed that most differentially expressed genes exhibited high endopeptidase activity, cellulose catabolic process, and transmembrane transporter activity. Comparison of gene expression between B. adusta T1 and Trametes versicolor F21a during the algicidal process suggested that endopeptidases together with enzymes of the polysaccharide lyases family 8 might be responsible for the strong algicidal activity of B. adusta T1 as well as T. versicolor F21a.Algicidal process of fungus  Then, crude RNA was digested via 10 U DNase I  at 37°C for 30 min, and then, mRNA was isolated using Dynabeads® Oligo (dT) 25  following the manufacturer's instructions. One hundred ng mRNA of each sample was used to construct a sequencing library using NEBNext® UltraTM RNA Library Prep Kit . Paired‐end sequencing of cDNA fragments (~300 bp) was performed using Illumina HiSeq 4,000 platform at BGI‐Shenzhen, China.Mycelia of 2.4B. adusta T1 at 6, 12, 24, and 48 hr were analyzed. The quality of 150‐bp reads was assessed using the FASTQC program (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The paired‐end raw reads from RNA sequencing were trimmed using the pipeline Trimmomatic (v0.33) with parameters (LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 HEADCROP:12 MINLEN:36)  using STAR software (v2.5.3a)  , 0.5 μl of each primer (10 μmolL−1), and 120–150 ng cDNA . A total of 63,437,015 pairs of raw reads (SRA accession: PRJNA543936) were generated . Multi‐dimensional scaling (MDS) showed that the gene expression in mycelia cocultured with cyanobacterial cells was distinctly separated from that of mycelia without cyanobacterial cells . The GO terms of DEGs were enriched in the extracellular region, cell wall, signal recognition particle, proteasome core complex, prefold in complex, ribosome, and other cellular components categories  was higher than that of T. versicolor F21a (312 CAZyme genes). Seventy CAZyme modules were detected in B. adusta T1, compared to 43 CAZyme modules in T. versicolor F21a in the previous study  could decompose xyloglucans and β‐1,3‐glucans into xylose and glucose, respectively. The enzymes of GH128, AA7, AA6, and GH109 were less efficient in cyanobacterial cell disruption. It is noteworthy that the accumulated expression of Polysaccharide lyases genes, particularly the PL8 module was highly up‐regulated during the later stage of the algicidal process of B. adusta T1, which was much delayed when compared to T. versicolor F21a   of PL8 and alginate lyase (EC4.2.2.3) of PL14 were able to decompose peptidoglycan and alginate  were highly up‐regulated during the later stages of cocultivation. Proteomic analysis of P. salina also implied that the PL7 and PL8 enzymes, abundantly secreted together with enzymes of P.salina, were necessary for degradation of laminarin, cellulose, lipids, and peptides of brown algae  located sequentially in a gene cluster in the genome of Sphingomonas sp. ACM‐3962 strain were identified for MC biodegradation  that was slightly up‐regulated in the later stage. In consideration bacterial MlrA encoding a protease, fungal aflatoxin‐detoxifizyme could be a possible candidate enzyme involving in MC degradation. In order to investigate the mechanism for MC degradation in fungi, there is more work need to be done.The production of microcystins (MC) by cyanobacterial blooms often severely threatens human and ecosystems health ; Software (lead); Writing‐original draft . Hui Ma: Investigation . Shenrong Ren: Investigation (supporting). Xueyan Gao: Investigation (supporting). Xiaolong He: Investigation (supporting). Suwen Zhu: Resources ; Validation (supporting). Ruining Deng: Validation (supporting). Shihua Zhang: Conceptualization ; Writing‐review & editing . None required.


Cannabis sativa L. (C. sativa), a crop with increasing global importance, are almost entirely unknown. In an indoor controlled factorial experiment involving a type-II chemovar , the effects of increasing NaCl concentrations (1–40 mM) was tested in hydroponic and aquaponic solutions during the flowering stage. Growth parameters , plant physiology , and solution physicochemical properties  was measured throughout the experiment. Upon maturation of inflorescences, plants were harvested and yield (dry inflorescence biomass) and inflorescence potency (mass-based concentration of cannabinoids) was determined. It was found that cannabinoids decreased linearly with increasing NaCl concentration: -0.026 and -0.037% THCA·mM NaCl-1 for aquaponic and hydroponic solutions, respectively. The growth and physiological responses to NaCl in hydroponic—but not the aquaponic solution—became negatively affected at 40 mM. The mechanisms of aquaponic solution which allow this potential enhanced NaCl tolerance is worthy of future investigation. Commercial cultivation involving the use of hydroponic solution should carefully monitor NaCl concentrations, so that they do not exceed the phytotoxic concentration of 40 mM found here; and are aware that NaCl in excess of 5 mM may decrease yield and potency. Additional research investigating cultivar- and rootzone-specific responses to salt-induced stress is needed.The effects of salt-induced stress in drug-type  Soil water potential is first reduced by salt-induced drought, followed by toxicity from uptake of Clhiometry . Areas whiometry ; howevermM NaCl) . As watemM NaCl) , or disrng crops .+ and Cl-, as observed by Pythium and Fusarium spp.  has received minimal previous research attention. C. sativa is an annual herbaceous crop of increasing economic importance for a multitude of uses: hemp-type cultivars are a significant source of bast and woody fibers and drug-type cultivars produce a trove of secondary metabolites (namely Δ9-tetrahydrocannabinolic acid ~ THCA and cannabidiolic acid ~ CBDA) which are of significant medicinal value  ; howeveral value . Hemp-tyC. sativa growth, physiology, yield, and potency (secondary metabolite production), in a controlled environment, increasing concentrations of NaCl from 1 to 40 mM was applied in conventional hydroponic and aquaponic solutions during the flowering stage of growth. It was hypothesized that plants would have concentration-dependent responses to NaCl, predicting a trend of declining growth, physiological performance, yield, and potency as concentrations of NaCl increase. It was also expected that NaCl tolerance would vary by solution due to contrasting biogeochemistry between hydroponic and aquaponic solutions.To determine the influence of NaCl on C. sativa, located in Ontario, Canada. The C. sativa cultivar “Nordle”, a type-II chemovar , all plants were grown in a complete mineral fertilizer solution with an electroconductivity (EC) of 1.0 mS·cm-1 and a pH of 5.7 , to allow sufficient roots to develop into the solution. At 12 DFS, after a minimal root meristem length of 10 cm emerged from the exterior of the pot, plants were subjugated to their respective aqueous treatment solutions.The experiment was conducted at an indoor license holder of medical -2·s-1 at a 12 h photoperiod. During the light period conditions at the canopy level were: 29.7 ± 1.37°C, relative humidity of 41.8 ± 3.69% and CO2 concentration of 436 ± 38.4 ppm. During the dark period conditions at the canopy level were: 26.2 ± 0.98°C, relative humidity of 44.0 ± 4.59% and CO2 concentration of 479 ± 42.2 ppm.During the flowering stage, light was supplied from light emitting diodes  at a 2:1:7 ratio of blue, green, and red wavelengths which provided PAR at 655 ± 68.7 µmol·mThe experiment was a completely randomized design with two factors: solution type and NaCl concentration. Solution was either hydroponic or aquaponic. Each solution type had five different concentrations of NaCl. Hydroponic solutions had NaCl concentrations of (in mM) 1, 5, 10, 20, and 40; and aquaponic solutions had NaCl concentrations of (in mM) 4, 8, 10, 20, and 40. Each solution and NaCl concentration combination was replicated six times, with each replicate being an individual plant grown in one of the 10 NaCl and solution combinations (2 solution x 5 concentrations x 6 replicates = 60 plants). All solutions were replaced with 8 L of fresh treatment-based solution when any of the following occurred for any single plant: pH changed by more than 0.5, more than of 50% of solution was depleted, or EC changed by more than 20% in any given solution. Solution was also changed for all plants every seven days, if none of the prior criteria was met. Each week plant locations were randomized to mitigate minor environmental variation in the air .C. sativa , according to the manufacturer’s recommendations. The nutrient concentrations and EC for the primary hydroponic solution and aquaponic solution are presented in + and Cl- concentrations than the source solution (7.7 mM Na+ and 7.8 mM Cl-), aquaponic solution was mixed 1:1 with reverse osmosis filtered water. A fertilizer containing (in mM) 2.7 N, 1.6 P, and 1.6 K  was also added at one third concentration of the recommended rate to compensate for the dilution based on EC. Aquaponic solution was sourced from the deep water culture basin of a mature (five year operating) commercial coupled aquaponic system  stocked with 1,189 adult Oreochromis niloticus (Nile Tilapia) each approximately 1.0 kg, 11 months old and stocked at a density of 98 fish·m-3. Fish were fed 1,600 g of 4 mm “Floating Feed”  per day. The system was a single loop recirculating system with the same design as The University of the Virgin Islands presented in 2) deep water culture basins to grow plants. Aquaponic solution was maintained at 23 ± 0.1°C and a dissolved oxygen concentration of 11 ± 1.7 mg·L-1. Aquaponic and hydroponic solutions were adjusted to a pH of 5.70 ± 0.025 using H3PO4. NaCl treatments were applied by mixing pre-weighed laboratory grade inorganic NaCl  to each individual plant solution upon change. Treatment-appropriate Na+ concentration for each solution was verified for each solution using a portable ion selective electrode meter .Hydroponic solution was prepared by mixing reverse osmosis filtered water with “Plant-Prod MJ™”, a commonly used commercial fertilizer for 1 × width2) ÷ 300]. Growth index has been previously used as a canopy volume metric for C. sativa by Each week, plant height, growth index, number of branches, and leaf chlorophyll content index (CCI) was measured. Plant height was measured from the base of the substrate to the apical meristem to the nearest cm. Growth index was measured using plant height, and two perpendicular canopy width measurements, using markers on the pot as reference points and calculated according to 2 assimilation rate (A), net CO2 assimilation rate at light saturation (Asat) and stomatal conductance (sg) was measured on the center leaflet of the newest fully matured leaf of each plant, using a portable photosynthesis machine . The conditions of the chamber were maintained at: CO2 of 500 ppm, temperature of 28.0°C, ambient relative humidity (41.8 ± 3.69%), a flow rate of 500 µmol·s-1 and PAR at 350 µmol·m-2·s-1 and 1,500 µmol·m-2·s-1 for A, and Asat, respectively. A PAR of 1,500 µmol·m-2·s-1 was used for Asat as it was predetermined by measuring leaf light response curves on the experimental plants (data not shown) and a PAR of 350 µmol·m-2·s-1 was used for A as it was the lowest measured intensity at the canopy height.At 21, 35, and 56 DFS, leaf net CO-2·s-1 light pulse. Water use efficiency, in terms of dry inflorescence (at 13% moisture content) produced per L of solution absorbed over the experiment, was calculated as: dry inflorence biomass (g) ÷ total solution uptake (L).At 21, 35, and 56 DFS chlorophyll fluorescence parameter Fv/Fm using a portable fluorometer  was measured on the center leaflet of the most recently expanded leaf of each plant. Fv/Fm was measured by dark adapting leaf tissue for 20 min using detachable leaf clips. The fluorometer was then attached to the leaf clips and fluorescence was measured before and after the fluorometer emitted a 2,100 µmol·m+, Ca2+, and K+ removal (measured in mg) was calculated by subtracting the total element weight three days after solution exchange, from the initial total element weight. Element weights were calculated by multiplying the solution volume (L) by the concentrations of the element (mg·L-1)—using portable ion selective electrode meters . Solution pH and EC was measured at each solution exchange with a portable pH/EC meter .Solution uptake and changes in pH and EC were measured at every solution exchange. Solution uptake was calculated using the height differences of the solution in the container right after the change and before the next change of nutrient solution. Solution loss due to evaporation was minimal but was standardized through by measuring loss in aerated solution without a plant, over seven days. Na2PO4, NH4-N, NO3-N, SO4, B, Ca, Cu, Fe, K, Mg, Mn, Mo, Na, P, Si, and Zn, at an independent commercial laboratory . Metals were analyzed with inductively coupled plasma optical emission spectrometry. For each nutrient analyzed, the solution volume in L (V2) and nutrient concentration in mg·L-1 (N2) after three days, was compared to initial nutrient concentrations (N1) in the stock solution and the initial solution volume (V1), to calculate nutrient removal through the following equation: Nutrient removal = (V1 × N1) – (V2 × N2).Nutrient removal was measured at 35 DFS, a time point previously shown to be the time at which vegetative growth  ceases and only reproductive growth continues under a 12 h photoperiod . Nutrienth, 6th, and 7th days in the drying room through thermo-gravimetric loss , the dry weight was measured and adjusted to 13% moisture content based on the measured moisture content of the treatment sample, following the current commercial C. sativa industry’s common practice. After drying, three 10 g composite inflorescence biomass samples from the apical meristems of each treatment were analyzed for concentrations of Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), cannabinol (CBN), cannabigerol (CBG), cannabichromene (CBC), tetrahydrocannabivarin (THCV), cannabidivarin (CBDV), the acid forms of the previous cannabinoids, cannabigerovarin acid (CBGVA) and total terpenoids at a commercial cannabis testing laboratory . Cannabinoids were analyzed using ultra-high-performance liquid chromatography and mass spectrometry detection, while terpenoids were analyzed using gas chromatography and mass spectrometry detection. The mean cannabinoid content (amount) per plant was calculated as: dry inflorescence biomass (g·plant-1) × mean cannabinoid concentration (%).At 68 DFS plants were harvested and inflorescence biomass (yield) was separated by manually trimming entire inflorescences from the plant. Inflorescences were wet weighed and let to dry in paper bags in a drying room maintained at 15 ± 0.8°C and a relative humidity of 40 ± 2.9% for seven days. Once inflorescence biomass reached an average moisture content of 13 ± 2.3% determined through measuring moisture content of composite treatment samples at the 5C. sativa growth, physiology, yield, potency, and solution physiochemical properties, data for each solution was analyzed separately using JMP Statistical Discovery Version 14.0 . For all tests, a Type-I error rate of α = 0.05 was used to determine the significance of the results. For solution physiochemical properties, growth and physiological performance parameters measured through time—i.e., Na+, K+, Ca2+ removal, plant height, canopy growth index, number of branches, CCI, leaf-gas exchange parameters and Fv/Fm, the effect of the NaCl (fixed-factor), time (fixed-factor) and NaCl through time (fixed-factor) was tested with a repeated-measures ANOVA. Since data were non-parametric, the models were fitted with restricted maximum likelihood (REML) standard least-squares personality tests as per To test for significant NaCl-effects on 2 values of all models that were significant for each trait . To test that the data met the assumptions of each separate linear model, Brown-Forsythe tests were performed on the variance of the model’s residual values above and below the median predicated values to determine homoscedasticity; and normality was tested by Shapiro-Wilk W Tests on the distribution of the model’s residual data. To determine differences in dry inflorescence biomass, the effect of NaCl on dry inflorescence biomass was also tested with a one-way ANOVA, followed by Tukey’s-HSD (honestly significant difference) pairwise comparisons of NaCl treatments if significance was detected in the model.The effect of NaCl on dry inflorescence biomass (yield), cannabinoid concentrations (potency), and nutrient removal at 35 DFS were tested with linear regressions. Linear models were determined to be best fit models by comparing adjusted Rgs) and dry inflorescence biomass.To determine the mechanisms responsible for observed yield responses to NaCl, bivariate analyses were conducted determining the coefficients of Pearson correlations between physiological parameters , sg , and Fv/Fm .NaCl concentration affected the growth and physiology of -1), in plants grown in hydroponic but not in aquaponic solution . Plants grown in hydroponic solution had the highest dry inflorescence biomass in the 1 mM NaCl treatment with 19.8 ± 1.38 g·plant-1 (mean ± standard error of mean), and the lowest dry inflorescence biomass in 40 mM NaCl treatment, with 7.9 ± 1.24 g·plant-1. Plants in the 40 mM NaCl treatment had 150, 136, and 104% lower dry inflorescence biomass than plants in the 1, 5, and 10 mM treatments, respectively . Alternatively, there was no relationship between the dry inflorescence biomass of plants grown in aquaponic solution to NaCl concentration. Plants grown in aquaponic solution had a mean dry inflorescence biomass of 18.3 ± 6.56 g·plant-1.The response of dry inflorescence biomass was concentration-dependent, decreasing linearly with increasing NaCl concentrations , total CBD equivalent (CBD + CBDA x 0.877), Δ9-THCA, CBDA, CBGA and CBCA decreased linearly in response to increasing NaCl concentrations in plants grown in aquaponic and hydroponic solutions  and hydroponic solution (% = 9.9 - 0.09 x NaCl (mM); R2 = 0.539; P = 0.0018). The rate of decrease in cannabinoid concentration for increase in NaCl concentration was higher for plants grown in hydroponic solution  compared to plants grown in aquaponic solutions , for all cannabinoid relationships. Interestingly, Δ9-THC and CBD concentrations increased with increasing NaCl concentrations in plants grown in hydroponic solution, while no correlations to these cannabinoids were found in plants grown in aquaponic solution . When Δ9-THC and CBD concentrations in hydroponic plants were expressed as content however, they did not increase with increasing NaCl concentration. For example, hydroponic plants in 1 mM NaCl solution had an average CBD and Δ9-THC content of 1.39 and 2.71 g·plant-1, respectively; while plants in 40 mM NaCl solution had 0.77 and 1.64 g·plant-1, respectively.Unlike growth, physiology, and biomass responses, the production of secondary metabolites was uniformly concentration-dependent in both solution types. -1 of 0.20 ± 0.015 and 0.18 ± 0.013, respectively. Between 30 and 63 DFS aquaponic and hydroponic solutions had a decreasing pH·day-1 of 0.08 ± 0.001 and of 0.06 ± 0.010 mS·cm-1, respectively. Both aquaponic and hydroponic solution had an increasing EC per day of 0.12 ± 0.008 and 0.13 ± 0.008 mS·cm-1, respectively, over the duration of the experiment. Plants grown in hydroponic solution had decreasing solution removal·day-1 with increasing NaCl concentrations when data was pooled over time (L·day-1 = 0.45 – 0.0044 x NaCl (mM); R2 = 0.350; P = 0.0007). Plants grown in aquaponic solution removed approximately 0.39 ± 0.010 L of solution per day. Water use efficiency had a negative correlation with NaCl concentration in both aquaponic  and hydroponic  solutions.Between 14 and 28 DFS, both aquaponic and hydroponic solutions had an increasing pH·day4 removal (NH4 (mg) = 44.1 – 0.68 x NaCl (mM); R2 = 0.518; P = 0.0037); Mg removal ; Zn removal  and Si removal . There were no other significant differences calculated for absolute nutrient removal for any of the nutrients across solution and NaCl combinations at 35 DFS.At 35 DFS, after three days of exposure to plant roots, increasing NaCl concentrations in aquaponic solution had a negative linear relationship with NHC. sativa were described. In hydroponics, NaCl at a concentration of 40 mM resulted in phytotoxicity, evident through reticent growth and physiology. The negative effects of NaCl on reproductive parameters was evident at a much lower concentration, as yield and potency began declining at NaCl concentrations of 5 mM. Significant associations were found between leaf-level physiological performance and biomass production, suggesting impaired photosynthesis is one mechanism responsible, at least in part, for yield declines in high salinity. Notably, plants in the aquaponic solution had a certain NaCl tolerance, whereby the only effects of NaCl were the reduction of cannabinoid concentrations.For the first time concentration-dependent ecophysiological responses to NaCl in a drug-type cultivar of Rosa × hybrida L. (rose) (Lycopersicon esculentum (tomato)  (Cucumis melo L. (melon) (Solanum melongena L. (eggplant)   and Lyco(tomato) ; but nevt 0.7 mM  and in seatment) . The red (melon)  and the ggplant) . Furtherggplant) .-1  caused only a 1.1% reduction in the Fv/Fm in roses . In the present study, Fv/Fm was reticent: values found to be lower than 0.75 in the 20 and 40 mM NaCl treatments [i.e., significantly lower than a plant considered healthy (0.83) according to in roses  and soluin roses . Substanin roses . Fv/Fm mn 150 mM . Previoun 150 mM . Given tultivars .+ accumulation in the leaves and nutrient antagonisms in the rootzone environment, particularly with K+. Glycophytes are unable to exclude salt at the roots, this allows ions to enter the plants and potentially accumulate in the leaves through transpiration forces. For example, + and an increase in leaf Na+ in eggplant. NaCl has also been shown to reduce leaf Mg2+  supplemented with K+ can alleviate some of the effects of NaCl  was observed in the dry inflorescence biomass of plants grown in hydroponic solution with increasing NaCl. The lack of natural cooling caused by osmotic pressure in the leaf, paired with the high radiation provided by the lights, may have mimicked and accelerate “drying effect”, causing an increase in decarboxylation of THCA to THC and CBDA to CBD, particularly in the 40 mM treatment where plants had necrotic inflorescence tissue , but none in the aquaponic solution. Furthermore, aquaponic plants in the 4 mM NaCl treatment had a higher dry inflorescence biomass than hydroponic plants in the 1 mM NaCl treatment. This indicates that aquaponics can be competitive with hydroponic production when supplemented with nutrients. Despite Nauaponics . It had uaponics ; however4-2 and Mg2+. Recent research has identified Si as an important molecule in tolerating salt stress by regulating osmoticum in bast-fibers  comprising different aqueous, solid, and gaseous fractions to better understand the effects of NaCl. Future C. sativa NaCl experiments should also investigate multiple drug-type C. sativa cultivars given that NaCl effects have been shown to vary across food crop cultivars  and Green Relief Inc.The authors declare that this study received funding from Green Relief Inc. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication.


Amphicarpaea bracteata (Hog Peanut) or Desmodium canadense (Showy Tick Trefoil) growing in Canada, were previously characterized and placed in a novel lineage within the genus Bradyrhizobium. The taxonomic status of the novel strains was verified by genomic and phenotypic analyses. Phylogenetic analyses of individual and concatenated housekeeping gene sequences  placed all novel strains in a highly supported lineage distinct from named Bradyrhizobium species. Data for sequence similarities of concatenated housekeeping genes of novel strains relative to type strains of named species were consistent with the phylogenetic data. Average nucleotide identity values of genome sequences (84.5–93.7 %) were below the threshold value of 95–96 % for bacterial species circumscription. Close relatives to the novel strains are Bradyrhizobium amphicarpaeae, Bradyrhizobium ottawaense and Bradyrhizobium shewense. The complete genomes of strains 85S1MBT and 65S1MB consist of single chromosomes of size 7.04 and 7.13 Mbp, respectively. The genomes of both strains have a G+C content of 64.3 mol%. These strains lack a symbiosis island as well as key nodulation, nitrogen-fixation and photosystem genes. Data from various phenotypic tests including growth characteristics and carbon source utilization supported the sequence-based analyses. Based on the data presented here, the four strains represent a novel species for which the name Bradyrhizobiumsymbiodeficiens sp. nov., is proposed, with 85S1MBT (=LMG 29937T=HAMBI 3684T) as the type strain.Four bacterial strains isolated from root nodules of soybean plants that had been inoculated with root-zone soil of either  Amphicarpaea bracteata (Hog Peanut) and Desmodium canadense (Showy tick trefoil) form symbiotic associations with diverse species of soil bacteria belonging to the genus Bradyrhizobium [Bradyrhizobium were identified. One of these novel lineages, consisted of four strains that lacked key nodulation (nod) and nitrogen-fixation (nif) genes and did not elicit root nodules on any of the legume hosts tested. In this work we used genomic and phenotypic analyses to further characterize strains of this novel lineage and based on the results, the species, Bradyrhizobium symbiodeficiens sp. nov., is proposed.Legumes native to east North America such as izobium . In a prizobium  bacteriaA. bracteata  or D. canadense (strain 141S2) plants growing in natural woodland sites in Canada [Four novel strains were isolated from root nodules of soybean plants that had been inoculated with suspensions of root zone soil from either n Canada . The typatpD, glnII, gyrB, recA and rpoB housekeeping genes were used for phylogenetic analyses. GenBank accession numbers of nucleotide sequences are shown in Table S1. Sequences were aligned as described previously [Sequences of the 16S rRNA, eviously . Best fieviously . Bayesiaeviously . Maximumeviously  were careviously . The topBradyrhizobium species , it was necessary to trim the aligned length of sequences to 1238 bp. The Bayesian tree of 16S rRNA gene sequences  showed that type strains of all Bradyrhizobium species were placed in two superclades: one represented by Bradyrhizobium japonicum and the other by Bradyrhizobium elkani. All four novel strains had identical 16S rRNA gene sequences and were placed in the superclade represented by B. japonicum. It should be noted, however, that the 16S rRNA gene is highly conserved and its usefulness as a taxonomic marker for species differentiation in the genus Bradyrhizobium is limited [Bradyrhizobium species, computed using software implemented in EzBioCloud [In order to reconstruct a 16S rRNA gene tree of type strains of all named BioCloud , are in Bradyrhizobium [Bradyrhizobium species. The closest relatives of novel strains are B. amphicarpaeae 39S1MBT, B. ottawaense OO99T and B. shewense ERR11T.Phylogenetic analysis based on MLSA of at least five protein encoding housekeeping genes is a reliable and powerful method for the delineation of species within the genus izobium . Corroboizobium , the BayBradyrhizobium species are not available in public databases, we reconstructed a supplementary phylogenetic tree using the two gene sequences (recA and glnII) that are available for all named species. In order to include type strains of all these species in the analysis, it was necessary to trim aligned sequence lengths to 375 and 479 bp for the recA and glnII genes, respectively. The Bayesian tree of recA-glnII concatenated gene sequences confirmed the placement of novel strains in a lineage that is distinct from named species of the genus Bradyrhizobium .As one or more protein encoding housekeeping gene sequences for type strains of several T in a novel lineage within the genus Bradyrhizobium.It is noteworthy that recent phylogenomic studies  using muT with reference taxa for two and five concatenated housekeeping gene sequences were calculated using the method of Stothard [T, novel strains 65S1MB, 101S1MB and 141S2 had sequence similarities for the five concatenated housekeeping genes that were >99 % whereas in comparisons with type strains of named species similarity values were at or below the 97 % cut-off value proposed for species delineation in the genus Bradyrhizobium [Sequence similarities for pair-wise comparisons of novel strain 85S1MBStothard . Table 1izobium ; similarEach of the four novel strains are genetically distinct and were assigned to a unique multilocus sequence genotype based on MLSA of concatenated housekeeping genes .Genetic differences between novel strains were further verified by generating random amplified polymorphic DNA (RAPD) fingerprints using four random primers  and the amplification methods described previously . An examA dendrogram based on the combined character matrix of fingerprint profiles generated by the four primers was reconstructed using UPGMA and the Dice coefficient implemented in GelCompare II software version 5.10 (Applied Maths). The novel strains were readily distinguished and were placed in a cluster distinct from reference taxa .T and 65S1MB. Sequencing was done at Genome Quebec Innovation Centre, Montreal, Canada, using the Pacific Biosciences (PacBio) RS II single-molecule real-time (smrt) platform [T whereas two smrt cells were used for strain 65S1MB.For further characterization, we sequenced the complete genomes of novel strains 85S1MBplatform  as descrplatform . One smrT and 65S1MB in pair-wise comparisons with genome sequences of type strains of named Bradyrhizobium species obtained from public databases using the MUMmer (ANIm) algorithm implemented in J-species Web Server version 3.0.20 [T and 65S1MB, the ANI values of named species varied between 84.5 % (B. icense) and 93.7 % (B. amphicarpaeae), which is below the threshold value of 95–96 % for bacterial species circumscription [T versus 65S1MB, is consistent with these strains belonging to the same species.Average nucleotide identity (ANI) of genomic sequences is recommended as a replacement for outdated DNA–DNA hybridization methods for bacterial species circumscription . We esticription . In contT and 65S1MB consist, respectively, of single circular chromosomes of 7039503 bp and 7133277 bp, which are similar in size to their close relative, B. amphicarpaeae 39S1MBT (7044517 bp) but smaller than the chromosomes of B. ottawaense OO99T (8606328 bp) and B. shewense ERR11T (9163226 bp)  were found for strain 85S1MBT whereas 6795 genes, 6507 coding sequences, 51 tRNAs, and three rRNA operons  were found for strain 65S1MB using the ncbi Prokaryotic Genome Annotation Pipeline version 4 (PGAP-4) [T and 65S1MB, respectively, were those involved in metabolism (900 and 926 genes), energy (297 and 300 genes), protein processing (234 and 233 genes), membrane transport (215 and 235 genes) and cellular processes (181 and 179 genes). Also found were genes implicated in stress response, defense and virulence (155 and 157 genes), motility and chemotaxis (82 and 82 genes), regulation and cell signalling (23 and 23 genes), and resistance to antibiotics and toxic compounds (57 and 58 genes).The complete genomes of novel strains 85S1MB3226 bp) . Novel s(PGAP-4) . Analyse(PGAP-4)  indicateT) and seven (strain 65S1MB) genomic islands were predicted based on the Island Path-DIMOB software implemented in the IslandViewer 4 platform [B. ottawaense and B. diazoefficiens, the genomes of novel strains do not contain a symbiosis island, key nodulation (nodDYABCSUIJ) or nitrogen-fixing  genes. Within the genus Bradyrhizobium, nod and nif genes have also been reported to be absent from diverse Bradyrhizobium spp. resident in North American forest soils [B. betae PL7HG1T isolated from a tumour on the roots of sugar beet [A total of five  agar medium [Bradyrhizobium. Cell morphology was investigated using a transmission electron microscope  as described previously [Bradyrhizobium [Novel strains 65S1MB, 85S1MBr medium . Producteviously . Cells aizobium .et al. [Bradyrhizobium [16 : 0 and C18 : 1ω6c/C18 : 1ω7c (summed feature 8).Analysis of fatty acids was done using the Sherlock Microbial Identification System (midi) version 6.0 and the rtsba6 database as described by Yu et al. . The novizobium  with a pT and 101S1MB could be differentiated from close relatives, B. amphicarpaeae 39S1MBT, B. ottawaense OO99T and B. shewense ERR11T as well as from B. japonicum USDA6T, B. betae PL7HG1T and B. diazoefficiense USDA110T on the basis of several of these phenotypic assays.Various phenotypic tests, including 70 carbon source utilization and 18 chemical sensitivity assays, were done using Biolog GEN III MicroPlates according to manufacturer's instructions. The results (Table S2) showed that novel strains 65S1MB, 85S1MBMacroptilium atropurpureum ‘Siratro’ or on roots of three legumes native to east Canada .Plant tests using Leonard jars  were carried out as described previously . ResultsT, 101S1MB and 141S2) represent a novel species named Bradyrhizobium symbiodeficiens sp. nov.Based on the phylogenetic, complete genome sequence and phenotypic data presented here, we propose that the four novel strains .l-fucose, l-pyroglutamic acid, l-galactonic acid lactone, d-gluconic acid, p-hydroxyphenylacetic acid, α-keto-glutaric acid, d-malic acid, l-malic acid, bromo-succinic acid, propionic acid, acetic acid and nine other carbon sources. Does not utilize d-mannose, d-mannitol, d-arabitol, l-aspartic acid, l-glutamic acid, mucic acid, quinic acid, d-saccharic acid, citric acid, Tween 40, γ-amino-butryric acid, α-hydroxybutyric acid, acetoacetic acid and 38 other carbon sources. Variable result with d-galactose. Resistant to troleandomycin, rifamycin SV, minocycline, vancomycin, tetrazolium violet, tetrazolium blue, potassium tellurite, aztreonam and three other chemical compounds. Susceptible to sodium butyrate and five other chemical compounds. Variable result with fusidic acid.Cells are Gram-stain-negative, aerobic, non-spore-forming rods (approx. 1.75 µm long x0.83 µm wide) with sub-polar flagella. Colonies on YEM agar medium are circular, convex, beige, translucent and <1 mm in diameter after 7 days at 28 °C. Growth occurs at pH 5 and pH 10 . Produces an alkaline reaction on YEM agar. The type strain grows at 10 °C, optimal at 28 °C, but no growth occurs at 37 °C. Grows in the presence of 1 % (w/v) NaCl. Utilizes 16 : 0 and C18 : 1ω6c/C18 : 1ω7c (summed feature 8). Does not elicit root nodules on Glycine max, Macroptilium atropurpureum, Amphicarpaea bracteata, Desmodium canadense and Desmodium glutinosum.Predominant fatty acids are CT (=LMG 29937T=HAMBI 3684T), was isolated from a root nodule of a soybean plant that was inoculated with root-zone soil of Amphicarpaea bracteata (Hog peanut) growing in Canada. The type strain does not contain photosystem genes, type III secretion system genes or key nodulation and nitrogen-fixation genes. The DNA G+C content of the type strain is 64.3 mol% and the genome size is 7.04 Mbp. GenBank/EMBL/DDBJ accession numbers for the complete genome and the 16S rRNA, atpD, glnII, recA, gyrB and rpoB gene sequences of the type strain are CP029427, KP768783, KP768551, KP768609, KF615036, KP768725 and KP768667, respectively.The type strain, 85S1MBClick here for additional data file.


The quantification of the analytes was achieved using the inductively coupled plasma optical emission spectrometry (ICP-OES) technique. The nanocomposite was then characterized using BET, FTIR, SEM, and EDS. Due to its high adsorption surface area, compared to traditional metal oxide-based adsorbents, MLDH nanocomposite exhibited high extraction efficiency. Several experimental parameters controlling the preconcentration of the trace metals were optimized using response surface methodology based on central composite design. Under optimum conditions, the linearity ranged from 0.1 to 500 µg L−1 and the correlation of coefficients (R2) were higher than 0.999. The limits of detection (LODs) and quantification (LOQs) were 0.11–0.22 µg L−1 and 0.35–0.73 µg L−1, respectively. The intra-day (n = 10) and inter-day precisions (n = 5 working days) expressed in the form of percent relative standard deviations (%RSDs) were below 5%. The proposed method was successfully applied for the analysis of the As(III), Cd(II), Cr(III), Co(II), Ni(II), and Pb(II) ions in different environmental water samples.A magnetic Fe This is because of their toxicity to humans and other living organisms as well as wide availability in water systems1. These metals come into contact with humans through food chain and uptake by plants4. They can accumulate in human organs such as lungs, liver, kidney, immune system, central nervous, and heart amongst others, thus leading to serious health effects5.Determination and monitoring of toxic trace metals such as lead, cadmium, mercury, thallium, and arsenic, have received more attention in recent years6, atomic fluorescence spectrometry (AFS)7, inductively coupled plasma-mass spectrometry (ICP-MS)9 electrothermal atomic absorption spectroscopy (ETAAS)10, electrochemical methods, and ICP-OES. Among the aforementioned techniques, ICP-OES is one of the most widely used detection method11. This is because ICP-OES is robust and can be applied for the qualitative and qualitative analysis of multielement in complex matrices13. Therefore, owing to the low concentration of trace metals in various samples and the complexity of the sample matrices, there is a need for separation/preconcentration methods before ICP-OES determination. This is because the preconcentration step improves the LOD/LOQs, sensitivity, accuracy, and specificity of the analytical technique14.Numerous analytical methodologies have been used for the quantification of trace elements in water systems. These include flame atomic absorption spectroscopy (FAAS)15, solid-phase extraction (SPE)17, dispersive solid-phase microextraction (DSPME), and dispersive magnetic solid-phase extraction (DMSPE)18, among others have been used for preconcentration of trace metals. Dispersive magnetic solid-phase extraction (DMSPE) is classified as a miniaturized SPE technique because smaller amounts of adsorbents are needed compared to the traditional SPE19. Other advantages of this technique include being easy to carry out, effective, and safe sample preparation method12. In this method, the adsorbent is added to the aqueous sample without conditioning step, instead, the sample with the sorbent is dispersed using external forces such as vortex, microwave, and ultrasonic bath, among others which improve extraction kinetics and adsorption process21. The adsorbent is then recovered by an external magnet and the analytes are desorbed using an acid12.Sample preparation methods such as dispersive liquid–liquid micro-extraction (DLLME)13. Among other adsorbents reported in the literature, nanomaterials have found great attention in recent years due to their high surface area and large adsorption area22. They are highly effective sorbents that can be applied for the pre-concentration of a wide range of analytes23. Nanomaterials such as layered double hydroxide (LDH) have received great attention in recent years due to their unique physical and chemical properties24. Layered double hydroxides are types of nanomaterials that are amongst a wide class of synthetic 2D anionic nano-clay materials22. The core aim of using LDH as sorbents over the other layered materials is because of its diversity and flexibility. As a result, LDH has been used as sorbents25, catalysts26, catalyst supports27, anion exchangers25, and molecular sieves28. Furthermore, LDH sorbents resemble mineral hydrotalcite (HT), a natural magnesium–aluminium hydroxyl carbonate (Mg6Al2(OH)16CO3·4H2O) because it consists of exchangeable adsorption sites. The attributes of LDH such as excellent anion exchange capacity, high surface area, good biocompatibility, specific structural characteristics, pH-dependent solubility, and high chemical stability are some of the few admirable properties which attracted several researchers’ interest29. While many magnetic LDHs have been synthesized, there are not many studies on their application as sorbents for pre-concentration of multi-element in complex matrices such as acid mine drainage and seawater.Design and choice of adsorbents in solid-phase based sample preparation techniques plays a most important factor to get a good recovery and high selectivity3O4@Mg/Al-layered double hydroxide (MLDH) nanocomposite was used as an adsorbent for the extraction of selected trace metals. The response surface methodology (RSM) based on the central composite design (CCD) was employed to investigate the effects of important operating parameters such as sample pH, mass of adsorbent, eluent concentration, and extraction time on the preconcentration of trace metals.The main purpose of this study was to develop an ultrasound-assisted-dispersive magnetic solid-phase extraction (UA-DMSPE) technique for preconcentration of trace quantities of As(III), Cd(II), Cr(III), Co(II), Ni(II) and Pb(II) ions from complex water matrices. Magnetic Fe3·6H2O, molecular weight 270.30 g mol−1, 99%), ammonium hydroxide solution (28%), iron(II) dichloride tetrahydrate , magnesium nitrate hexahydrate (Mg(NO3)0.6H2O molecular mass 256.41 g mol−1, 99.999%), aluminium nitrate (Al(NO3)3 molecular weight 256.41 g mol−1, 99%), obtained from Sigma-Aldrich . A synthetic sample of multi-element at concentration of 50 μg L−1 was prepared from Spectrascan single element standards (1000 mg L−1) of As, Cd, Cr, Pb, Co, and Ni . A 100 mg L−1 Spectrascan multi-element standard solution  for preparation working calibration standards. Certified reference material  were obtained from Sigma. NIST SRM 1643e was purchased from the National Institute of Standards and Technology .All reagents were of analytical grade unless specified and double-distilled deionized water was used during the experiments. Iron (III) trichloride hexahydrate (FeCl3O4@Mg/Al-LDH nanocomposite. The X-ray powder diffraction (XRD) patterns recorded using a Philips X-ray generator model PW 3710/31 a diffractometer with an automatic sample changer model PW 1775. The specific surface area, pore size, and pore volume values were measured using Surface Area and Porosity Analyzer . Spectrum 100 FT-IR  spectrometer equipped with Universal Attenuated Total Reflectance (ATR) was used to investigate the functional groups on the surface of the adsorbent, X-ray photon spectroscopy (XPS) on VG ESCALAB MARK II .Inductively coupled plasma–optical emission spectrometer (ICP-OES)  equipped with a charge injection device (CID) was employed for quantification of As(III), Cd(II), Cr(III), Co(II), Ni(II) and Pb(II) ions. The ultrasound-assisted preconcentration of trace metals was performed in a Scientech ultrasonic bath system with a frequency of 50 Hz and power of 150 W . Scanning electron microscope (SEM)  was used to examine the morphology FeThe sampling of real water samples (tap and river water) was collected from south of Johannesburg, South Africa, and were kept in 1000 mL polypropylene bottles and stored in the fridge at 4 °C until analysis. Prior to analysis, the water samples were filtered using 0.25 µm acrodisc and syringe.3O4 was synthesized following the method reported previously30. Briefly, 10.1 g of FeCl3·6H2O and 5 g of FeCl2·4H2O salts were dissolved in 150 mL of deionized water. The reaction was carried out under vigorous stirring at 85 °C under a nitrogen atmosphere. Whilst continuing stirring, 25 mL aliquot of ammonium hydroxide solution was added dropwise into the solution to form a black precipitate. The mixture could stir at the same conditions for 15 min and then cooled at ambient temperature. The Fe3O4 was collected using an external magnet. The magnetic nanoparticles were rinsed with deionized water. The magnetitic nanoparticles were dried at 60 °C for 10 h in an over and then they were pulverized into fine particles using a pestle and mortar.The synthesis of Fe3O4@MgAl LDH nanocomposite was achieved following the procedure described elsewhere31. Briefly, 1.93 g of previously Fe3O4 nanoparticles were dispersed in 150 mL distilled water in a beaker for 15 min to obtain a uniform suspension. The precipitate formed was then stirred vigorously at 60 °C, whilst adding dropwise a mixture of 12.8 g Mg(NO3)0.6H2O and 9.38 g Al(NO3)0.9H2O solution. Then the pH was adjusted to 10 by adding a mixture of 6.75 g NaOH and 5.29 Na2CO3 solution. The precipitate was left to age for 8 h, then washed several times with distilled water and separated using a magnet. It was dried at 60 °C overnight.The synthesis of Fe32. Briefly, 50–100 mg of the magnetic LDHs nanocomposite was weighed into 100 mL plastic bottles, then an aliquot of 20 mL synthetic sample solutions (pH 3–9) was added to the sample bottles. The extraction and preconcentration of the As(III), Cd(II), Cr(III), Co(II), Ni(II), and Pb(II) ions were carried out by ultrasonication for 5–30 min. After the pre-concentration process, the sample solution and nano adsorbent were separated using an external magnetic. The analytes desorbed by adding 2.5 mL of 0.1–1.0 HNO3 to the adsorbent and sonicated for 5 min. The eluent and the nanoadsorbent were separated by magnetic decantation. The aqueous phase containing the analytes was filtered into a pre-cleaned polypropylene centrifuge tube using the PVDF syringe membrane. The filtrate was then analysed by ICP-OES. The experimental parameters affecting the preconcentration method were optimized using RSM employing a CCD. These parameters include the mass of adsorbent (MA), extraction time (ET), sample pH; and eluent concentration (EC). Under optimal conditions, the above method was repeated for the validation and application of the proposed method.The ultrasonic-assisted dispersive solid-phase extraction procedure was carried according to our previous methods®-CA713) and standard reference materials (NIST SRM 1640a and 1643e). The accuracy was expressed as percentage recovery (%R) and relative error (%RE) and they were calculated according to Eqs. , preconcentration factor (PF), enhancement factor (EF), accuracy, the limit of detection (LOD), and limit of quantification (LOQ). The accuracy and precision (intraday and interday) were investigated using certified reference materials ERM®-CA73 and sta to Eqs. . The pre3O4, and Fe3O4@MgAl LDH nanocomposite. The appearance of peaks at 2θ 11 (003), 24 (006), 35 (012), 40 (0 15), 47 (018), 62 (110), and 64 (113) in Fig. 34. These peaks are the same as those observed in Fig. 3O4 nanoparticles in the composite35. Similar peaks can be observed for pristine Fe3O4 nanoparticles. These findings proved that the prepared adsorbent was crystalline and they were similar to those reported elsewhere35.Figure 3O4@MgAl LDH was characterized by scanning electron microscope/(SEM/EDS) and transmission electron microscopy (TEM) to examine the morphological properties as well as elemental composition  which was from the carbon coating.The prepared magnetic Feion Fig. a,b. The rum Fig. c shows t3O4@MgAl LDH composite are illustrated in Fig. 0 which is a typical type IV isotherm. This implied that the prepared composite was mesoporous. The BET specific surface area pore volume and pore size  for the Fe3O4@MgAl-LDH nanocomposite and the results are presented in Fig. −1 was ascribed to O–H stretching of LDH layers and interlayer water molecules36. A sharp at 1374 cm−1 was assigned to the stretching vibration of intercalated –NH3 from the synthesis of magnetite. The vibrations at 1636 cm−1, 783 cm−1, and 641 cm−1 were ascribed to the presence of CO3− ions in the interlayer of LDHs36. The results are comparable to the literature36.The Fourier-transform infrared spectroscopy (FTIR) technique was used to assess the functional groups present on the surface of Fe3O4/MgAl LDH nanocomposite was evaluated in comparison with Fe3O4 and MgAl-LDH adsorbents. Figure 3O4/MgAl LDH nanocomposite and MgAl LDH exhibited better extraction efficacy than Fe3O4 nanoparticles. However, due to magnetism, Fe3O4/MgAl LDH nanocomposite displayed better separability compared to MgAl-LDH. Thus, the Fe3O4/MgAl LDH nanocomposite was chosen as the adsorbent for the preconcentration of trace metals.The selection of a suitable and effective adsorbent is one of the critical aspects of the extraction and preconcentration of trace metals. Therefore, in this study, the sorption and extraction ability of FeThe CCD matrix and respective recoveries  for each analyte are presented in supplementary data Table . AnalysiThe three-dimensional response surface plots of the second-order polynomial equation were used to examine interactions between independent variables and their combined effect on the analytical response. The combined effect of two variables was assessed by varying them within the determined ranges while keeping two other variables at zero level  Fig. . The res37. The optimum conditions for simultaneous preconcentration of As(III), Cd(II), Cr(III), Co(II), Ni(II), and Pb(II) ions were selected to be 6.5, 84 mg, 10 min, and 2.0 mol L−1 for sample pH, mass of adsorbent, extraction time and eluent concentration, respectively.The desirability function was employed to optimize individual variables  that affect the pre-concentration process. The desirability values of 0.0, 0.5, and 1.0 corresponded to the minimum, middle and maximum functions. The values in the desirability profile Fig.  that are−1 of As(III), Cd(II), Cr(III), Co(II), Ni(II), and Pb(II) ions was processed using the optimized preconcentration method. After equilibrium has been reached the solutions were determined using ICP-OES. The adsorption capacity was measured by using the following formula:e is the adsorption capacity, C0 and Ce are the initial and equilibrium concentrations of the analytes, m and V are the mass of adsorbent and volume of synthetic solution. The equilibrium data were fitted to nonlinear Langmuir and Freundlich equations  and for Freundlich were 0, 24.6, 22.6, 21.6, 23.5 and 25.2).The adsorption isotherms of the Fe3O4/MgAl LDH nanocomposite before and after adsorption of As(III), Cd(II), Cr(III), Co(II), Ni(II), and Pb(II)  Fig. a showed II) Fig. b–d. It cII) Fig. b showed ows Fig. d,e it is3O4@MgAl LDH nanocomposites confirm the existence of Al, Mg, Fe, O, and C atoms on the nanocomposites before and after adsorption. Noticeably, the survey spectra of Fe3O4@MgAl LDH nanocomposite after the adsorption of metal ions show an increase in the intensity signal as compared to before adsorption, thus suggesting the conceivable adsorption of metal ions in solution , preconcentration factor (PF), enhancement factor (EF), precision (intraday and interday), LODs, and LOQs were determined as discussed in “2, and the enrichment factors for each analyte are presented in Table Analytical figures of merit of the UASPME/ICP-OES method, such as linear range, correlation coefficient R, preconcThe preconcentration factor, calculated as the ratio of the sample volume (100 mL) divided by the eluent volume (2.5 mL), was found to be 40. The LODs and LOQs of the UASPME/ICP-OES method are illustrated in Table Table −1 of the target analytes the analytical results are presented in Table Additionally, the validity and applicability of the developed UASPME/ICP-OES procedure were evaluated by analysis of As(III), Cd(II), Cr(III), Co(II), Ni(II), and Pb(II) ions in complex matrices such tap and river water. The accuracy of the method was evaluated by spiking the water samples with 10 µg LThe developed UASPME/ICP-OES method was applied to the determination of trace As(III), Cd(II), Cr(III), Co(II), Ni(II), and Pb(II) ions in acid mine drainage effluents  and the results are shown in Table 43. In the addition, parameters such as liner calibration range, LOD, EF, and RSD for UASPME/ICP-OES technique were comparable to those stated in the literature44. However, the analytical performance on the developed method was relatively poor than those reported by Refs45.The comparison of analytical characteristics of UASPME/ICP-OES method for preconcentration of As(III), Cd(II), Cr(III), Co(II), Ni(II), and Pb(II) ions with previously researched preconcentration procedures specifically (solid-phase based extraction) reported in the literature are summarised in Table 3O4@MgAl LDHs composite was performed by conducting subsequent adsorption/desorption experiments. Nitric acid (2.0 mol L˗1) was used to regenerate the spent Fe3O4@MgAl LDHs composite. The amount of trace metals after every adsorption/desorption cycle was expressed in terms of percentage recovery (%R). Figure 3O4@MgAl LDHs composite retained its adsorption capabilities up to the sixth cycle. After the sixth cycle, the adsorption efficiency gradually decreased by a small margin. At the end of the eighth adsorption/desorption cycle, the %R were 85.3%, 88.1%, 83.6%, 79.9%, 88.5% and 86.7% for As, Cd, Cr, Pb, Co, and Ni, respectively. Nonetheless, it can be concluded that Fe3O4@MgAl LDHs composite had demonstrated exceptional regeneration and reusability as the percentage recoveries decrease only slightly after the sixth cycle. Furthermore, the study of the stability of the prepared magnetic Fe3O4@MgAl LDH adsorbent in the adsorption system was performed by testing for the presence of Fe, Mg, and Al to evaluate the long-term stability. There were no Fe, Mg, and Al detected in all the ten parallel cycles, indicating there was no leaching of Fe3O4@MgAl LDH. Also, the prepared adsorbent showed stable magnetic property before and after use.The reusability and regeneration of Fe3O4@MgAl LDH composite was prepared successfully via the co-precipitation method. The prepared composite was characterized through instruments such as SEM–EDS, TEM, BET surface area, and FTIR. The Fe3O4@MgAl LDH composite was applied as an adsorbent in UA-DSPME for preconcentration of trace As(III), Cd(II), Cr(III), Co(II), Ni(II), and Pb(II) ions in river water, tap water, and mine wastewater samples. Experimental parameters affecting UA-DSPME/ICP-OES method were optimized using a multivariate approach. The UA-DSPME/ICP-OES method revealed outstanding analytical characteristics such as simplicity, rapidity, low LODs, high accuracy, enrichment factors, and precision. Furthermore, the performance of the UA-DSPME/ICP-OES method was applied for the determination of trace metals in complex matrices.The FeSupplementary Information.