Homeostatic synaptic plasticity, which induces compensatory modulation of synapses, plays a critical role in maintaining neuronal circuit function in response to changing activity patterns. Activity in the anterior piriform cortex (APC) is largely driven by ipsilateral neural activity from the olfactory bulb and is a suitable system for examining the effects of sensory experience on cortical circuits. Pro-inflammatory cytokine tumor necrosis factor-α (TNF-α) can modulate excitatory and inhibitory synapses, but its role in APC is unexplored. Here we examined the role of TNF-α in adjusting synapses in the mouse APC after experience deprivation via unilateral naris occlusion. Immunofluorescent staining revealed that activity deprivation increased excitatory, and decreased inhibitory, synaptic density in wild-type mice, consistent with homeostatic regulation. Quantitative RT-PCR showed that naris occlusion increased the expression of Tnf mRNA in APC. Critically, occlusion-induced plasticity of excitatory and inhibitory synapses was completely blocked in the Tnf knockout mouse. Together, these results show that TNF-α is an important orchestrator of experience-dependent plasticity in the APC. For all sensory systems, a key function is to have faithful representation of the stimulus while maintaining the flexibility of adding associative information to it. The anterior piriform cortex (APC) is a primary sensory cortex one synapse downstream of the olfactory bulb (OB), and yet has extensive recurrent connections that resemble properties in an association cortex . InformaNeurons in APC can perform context-dependent retrieval and storage of memory . The extAR expression, respectively, on the cell surface after exogenous TNF-α treatment in cultured neurons  and strain-matched wild-type mice  were used in this study. Tnf–/– mice were bred homozygously as described previously  was performed by using an electrocauterizer (WPI) on young adult mice  with anesthesia 7 days before experiment. The closed nostril was checked for its complete closure by visual inspection. After confirmation of occlusion of 7 days, mice were used for microscopy or RNA isolation.Ct–ΔΔ and the relative amount in occluded side compared with open side was deducted.To prepare RNA samples from the anterior piriform cortex (APC), total RNA was extracted and isolated by TaKaRa MiniBEST Universal RNA Extraction Kit (Cat. #9767) according to manufacturer’s instructions. For detection and quantification of mRNA, 1 μg of total RNA was used for cDNA synthesis with TaKaRa PrimeScript RT Master Mix , and quantitative RT-PCR was performed with Fast SYBR-Green Master Mix (Applied Biosystems) detected by StepOne Plus RealTime PCR system . The sequences of qPCR primers used for mRNA quantification in this study were obtained from the Primer Bank, and were as follows: mouse Gad1 (gene encoding GAD67 protein) forward, CTTCTTCAGGCTCTCCCGTG; mouse Gad1 reverse, GTATTAGGATCCGCTCCCGC; mouse vglut1 forward, CAGCCCGCCTACTTTGAAGA; mouse vglut1 reverse, GTGACGACTGCGCAAAAAGT; mouse pvalb forward, ATCAAGAAGGCGATAGGAGCC; mouse pvalb reverse, GGCC AGAAGCGTCTTTGTT; mouse Tnf forward, TAGCTCCCAG AAAAGCAAGCA; mouse Tnf reverse, CCATCTTTTGGGGGA GTGCC; mouse Tnfr1 forward, GCTGTTGCCCCTGGT TATCT; mouse Tnfr1 reverse, ATGGAGTAGACTTCGGGCCT; mouse gfap forward, CAGATCCGAGAAACCAGCCT; mouse gfap reverse, ACACCTCACATCACCACGTC; mouse gapdh forward, AGGTCGGTGTGAACGGATTTG; mouse gapdh reverse, TGTAGACCATGTAGTTGAGGTCA. After 40 cycles, the Ct values were determined. To normalize the samples, ΔCt between target genes and gapdh was calculated. The fold difference in expression between open and occluded side was then determined by subtraction of the ΔCt values and termed ΔΔCt. Finally, the total change was calculated as 2Mice were deeply anesthetized with ketamine/xylazine mix (100 mg/kg ketamine and 19 mg/kg xylazine) prior to fixation with 4% paraformaldehyde (PFA) in a 0.1 M phosphate buffer solution  following an initial flush with ice-cold 0.1 M PBS. Brains were post-fixed overnight in PFA and cut into 50 μm thick coronal sections in 0.1 M PBS using vibratome (Leica VT1000S). Sections were incubated for 1 h with blocking solution  before incubation overnight at 4°C with primary antibodies in blocking solution . Sections were washed 3 × 10 min with PBS at RT and incubated for 1 h at room temperature in secondary antibodies ]. Sections were washed 3 × 10 min with PBS at RT. The stained sections were mounted on slides in VectorShield mounting medium (Vector Labs) and imaged using Zeiss LSM 880 Confocal Microscope with Zeiss imaging software. Images of anterior piriform cortex were acquired with a 10×, 20× or 40× objective.+ cell density in sublayers of anterior piriform cortex was determined from confocal-acquired images (40×) using the FIJI-ImageJ particle analyzing tool  with synapses or cells on edge excluded after filtering background noise (Image\Adjust\Threshold). Threshold was chosen to exclude background and include structure that appears punctate after measuring gray value of background and target puncta. Maximal threshold was left at a maximum (255 for 8-bit images). Threshold was determined for each protein individually. PV+ neuron density in APC were quantified from confocal-acquired images (10×) using the FIJI-ImageJ particle analyzing tool, with neurons on edge excluded. Astrocyte (GFAP+) and microglia (Iba1+) densities in APC were quantified from confocal-acquired images (20×) by manual counting. Only cells co-localized with DAPI+ nuclei would be considered as astrocytes or microglia. GFAP or Iba1 expression covered area percentage and mean intensity level were quantified using FIJI-ImageJ analyzing tool  after filtering background noise (Image\Adjust\ Threshold).Synapse density and DAPIUnless noted otherwise, data were analyzed with paired t-test  or Mann-Whitney U-test and presented as mean ± SEM. Significance was indicated if P < 0.05. Power analysis: for a t-distribution with mean of μ and standard deviation of 10% of μ, to detect a 10% change in mean with 0.8 power (1-β) we will need an n of 10 animals for each condition. Images for both conditions (open vs. occluded APC) were taken from the same animal. Two (2) – 4 sections were stained for each condition and protein. Two (2) images were taken for each section. Mean values were calculated for one animal. The N numbers reported in the results and figure legends represent the number of animals.3 mm–2, p = 0.04, N = 8; 3 mm–2, p = 0.03, N = 5; L2: (11.4 ± 4.8 to 23.8 ± 5.7) × 103 mm–2, p = 0.02, N = 5; 3 mm–2, p = 0.84, N = 8, L2: (49.5 ± 13.4 to 38.7 ± 6.8) × 103 mm–2, p = 0.22, N = 7, L3: (29.0 ± 2.0 to 29.6 ± 2.6) × 103 mm–2, p = 0.87, N = 6; Homer1, L1: (31.9 ± 4.1 to 24.3 ± 6.8) × 103 mm–2, p = 0.23, N = 6]  in young adult mice (∼P21), waited for 7 days for plasticity to occur, and fixed their brains to examine the neurons and synapses . The ope, N = 6] . These r3.mm–2, p = 0.002, N = 8, 3.mm–2, p = 0.93, N = 6; –2, p = 0.008, N = 6; Parvalbumin and SST interneuronal cell bodies reside mostly in L2/3 and are two non-overlapping subtypes of interneurons with sizeable population in APC . As PV aOur previous results showed that NO altered excitatory and inhibitory synapses and neurons in a manner that is consistent with homeostatic plasticity. It remains unknown, however, whether these are independent events or that there is an external signal that coordinates this regulation.–2, p = 0.037, N = 7; intensity: 0.41 ± 0.04 to 0.47 ± 0.02, p = 0.031, N = 7; –2, p = 0.599, N = 6; intensity: 0.32 ± 0.04 to 0.32 ± 0.03, p = 0.676, N = 6; In the central nervous system, immune molecules are not only responsible for immune response under pathological condition but also important in regulating normal synaptic function . CytokinA receptors in hippocampal neurons in culture  × 103 mm–2, p = 0.148, N = 6; VGluT1, L1: (29.6 ± 6.5 to 32.8 ± 8.1) × 103 mm–2, p = 0.661, N = 6, L2: (14.5 ± 3.1 to 16.0 ± 4.4) × 103 mm–2, p = 0.703, N = 6; PV puncta, L2: (13.9 ± 4.7 to 6.9 ± 1.7) × 103 mm–2, p = 0.156, N = 7; PV soma: 34.2 ± 3.2 to 35.6 ± 7.1 mm–2, p = 0.885, N = 4; –/– mice was similar in the open side (WT open side GAD67 L2: (20.1 ± 3.5) × 103.mm–2, n = 8; Tnf–/– open side GAD67 L2: (14.3 ± 3.9) × 103 mm–2, n = 6; p = 0.287). Additionally, in the Tnf–/– mouse, we did not observe alterations in the expression of VGAT, Homer1, or SST, in a similar manner as in the WT mice  × 103 mm–2, p = 0.15, N = 5, L2: (15.9 ± 5.5 to 23.0 ± 6.4) × 103 mm–2, p = 0.5, N = 5, L3: (22.2 ± 10.9 to 19.2 ± 6.3) × 103 mm–2, p = 0.81, N = 4; Homer1, L1: (44.4 ± 14.7 to 35.2 ± 17.0) × 103 mm–2, p = 0.41, N = 5; SST, L3: (23.1 ± 6.5 to 24.9 ± 11.4) × 103 mm–2, p = 0.89, N = 5; Although TNF-α is known to regulate both AMPA and GABA culture , its rolCytokines secreted by glial cells are increasingly recognized to be important mediators of neuroglial interactions, particularly during experience-dependent plasticity. Here we show that activity deprivation in the form of NO induces structural plasticity of certain excitatory and inhibitory markers in an opposite manner: NO increases VGluT1 expression and decreases GAD67 and PV expression. Importantly, genetic deletion of Tnf entirely prevented these plastic regulations. Overall, our findings indicate that TNF-α is a critical orchestrator in modulating sensory deprivation-induced plasticity.+ synapses, are decreased after NO. Possible reasons for this discrepancy between anatomy and physiology could be: First, not all structural synapses are functional (or connected) in synaptic transmission; Second, synaptic conductance can be influenced by properties such as release probability  of TTX , while Tin vitro studies, other reports have revealed the role of TNF-dependent plasticity in sensory systems of intact animals. In layer 5 of the barrel cortex, a slower potentiation in response to unilateral whisker trimming deprivation in principal neurons is abolished in Tnf–/– mice  and L3 (glutamatergic and GABAergic)1. Hence, it is possible that both glutamatergic and GABAergic neurons are receptive to the effects of secreted TNF-α. A parsimonious explanation is that TNF-α is a key astrocytic molecule induced during activity deprivation that orchestrates the various plastic events occurring in the presynaptic terminals of glutamatergic and GABAergic neurons. Our study provides an insight into the role of TNF-α in regulating plasticity in olfactory cortex. The signaling mechanism, however, by which TNF-α exerts control on both neuron types remains to be deciphered.In addition to –/– mice . In the –/– mice . In deve–/– mice . While t–/– mice . Moreoveproteins . How canproteins . A survevia the TNF-α system. We believe that any new scheme for modeling APC circuit function and computation should include TNF-α-mediated deprivation-induced plasticity.Taken together, we propose a model where deprivation of olfactory sensory activity in the APC induces homeostatic plasticity of excitatory and inhibitory synapses and neurons such that the overall E-I synaptic density ratio is increased in the network . To the The original contributions presented in the study are included in the article/The animal study was reviewed and approved by Animal Research Ethics Sub-Committee of City University of Hong Kong and Department of Health of Hong Kong SAR government.AG performed all experiments including naris occlusion, immunofluorescent staining, quantitative PCR, and analyzed data. AG and CGL designed the research, wrote and edited the manuscript. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Crystals of the title salt are twinned by pseudo-merohedry and the structure shows extensive disorder. A strong N—H⋯O hydrogen bond links the cation and the anion and the anions are linked into [010] chains by O—H⋯O hydrogen bonds. tert-butylphenyl)-4-oxobutyl]-4-(diphenylmethoxy)piperidin-1-ium (E)-3-carboxy-1-hydroxyprop-2-en-1-olate}, C32H40NO2+·C4H3O4−, a 1:1 salt formed in the reaction between ebastine and fumaric acid is presented. All examined crystals were found to be twinned by pseudo-merohedry. The structure is extensively disordered, with over half (20 out of 35) its non-hydrogen atoms modelled as lying over two sets of sites. In the crystal, cation–anion pairs are linked by a strong N—H⋯O hydrogen bond [N⋯O = 2.697 (11) Å]. These units interact via weaker C—H⋯O and C—H⋯π contacts to form layers lying parallel to the bc plane. The hydrogen fumarate anions are linked by a very short O—H⋯O hydrogen bond [O⋯O = 2.5402 (17) Å], augmented by weak C—H⋯O contacts into pairs of R22(6) ring motifs to form chains that extend parallel to the b-axis direction. Comparisons to similar crystal structures are presented.The structure of ebastinium hydrogen fumarate {systematic name: 1-[4-(4- The asymmetric unit of I °, minor]. Additional details concerning the disorder are given in section 6 .The ebastinium cation is protonated at N1 Fig. 1, which i al. 2017, who alsThe hydrogen fumarate anion deviates substantially from planarity, as indicated by the dihedral angle between its carboxylate and carboxylic acid groups of 23.51 (14)°. As expected, the C—O bond lengths in the deprotonated carboxylate group  are the same within the accuracy limitations of the spherical-atom scattering-factor approximation  Å, Table 1O⋯O3iii hydrogen bond between hydrogen fumarate anions (vide infra). Much weaker C—H⋯O hydrogen bonds connect the ebastinium cations along the b-axis direction (C7—H7A⋯O2i), ebastinium and hydrogen fumarate ions via the c-glide (C8—H8B⋯O6ii) and hydrogen fumarate anions into chains parallel to the b-axis direction (C34—H34⋯O5i and C35—H35⋯O3iii). The symmetry operations are those defined in the footnote to Table 1For the sake of clarity, the following description is restricted to the major component of disorder except where stated otherwise. The packing in I is the cation–anion pair  is not disordered, but HECMIO has a relatively simple two-component disorder of the benzene ring of its 4-t-butylphenyl substituent. Of particular note 2CHO group relative to the piperidine/piperidinium ring, which is equatorial in ebastine, but axial in the ebastinium salt. The (C6H5)2CHO substituent in both disorder components of the hydrogen fumarate salt presented here is axial, as in HECMIO. The conformation of the C4H6O-4-t-butylphenyl fragment in I, however, is more similar to that in the neutral molecule (QATJIF and QATJIF01). An overlay of the major and minor disorder components of I with QATJIF01 and HECMIO highlights these conformational differences  with the 3,5-dinitrobenzoate salt (HECMIO) was made by Shaibah  al. 2017. The frees Fig. 7.5.via slow evaporation to give plate-shaped crystals in about a week (m.p. 468–470 K). All crystals observed were twinned by pseudo-merohedry, but those grown from acetonitrile were the largest and gave the best diffraction patterns [see section 6  for further details].A sample of ebastine was obtained as a gift from R. L. Fine Chemicals, Bengaluru, India. Ebastine  and fumaric acid  were dissolved in hot ethyl acetate and DMF and stirred over a heating magnetic stirrer for 30 minutes at 333 K. The resulting solution was allowed to cool slowly to room temperature with slow evaporation. Crystallization was carried out using several solvents  1H NMR: DMSO-d6 : 1.294 ; 1.615–1.592 ; 1.871–1.818 ; 2.400 ; 2.576–2.541 ; 2.870 ; 3.024–2.989 ; 3.423–3.406 , 5.63 ; 6.557 ; 7.250–7.207 ; 7.372–7.297 ; 7.533–7.512 ; 7.891–7.870 ; 11.6–14.2 . 13C NMR: DMSO-d6 : 20.04, 29.53, 30.78, 34.76, 35.25, 49.52, 55.93, 79.06, 125.39, 126.57, 127.13, 127.80, 128.25, 134.15, 134.57, 142.96, 156.03, 167.03, 198.80.NMR spectra were recorded on an SA-AGILENT 400MHz NMR spectrometer: 6.Synthesis, crystallization and spectroscopic details)] were thin plates that indexed to give essentially the same unit-cell dimensions. All specimens were pseudo-merohedral twins by virtue of the β angle being close to 90° and had roughly equal component volume fractions, as determined by the refined BASF parameter in SHELXL %, its standard uncertainty is only about one tenth as large, and is therefore of statistical significance. Even with such a tiny residual minor individual fraction, refinement statistics were marginally better with TWIN/BASF (in SHELXL) included. For a concise description of the various types of twinning that commonly affect molecular crystals, particularly twinning by pseudo-merohedry and the attendant twin operations that constitute the twin law, see Parkin 2CHO group of the cation followed by a relaxation into the available space, which in turn places the whole of the (C6H5)2CHO group in two distinct orientations [see section 2 ]. This of necessity must also cause minor site splitting of the piperidinium ring, albeit not discernible in electron-density maps calculated to 0.77 Å resolution. The two largest difference map peaks are only about 0.5 and 0.4 electrons, but are in positions that suggest a third, much smaller, disorder component. Such an additional disorder component, however, was not modelled due to its necessarily minuscule occupancy fraction. To ensure satisfactory refinement for disordered atom sequences in the structure, a combination of restraints were employed. The SHELXL commands SAME and SADI were used to maintain the chemical integrity and similarity of the disordered groups, while RIGU and SIMU were used to ensure physically reasonable displacement parameters for closely proximate disordered atom pairs.In addition to the twinning, the structure is extensively disordered. This disorder consists of a rotation of the (CR2Csp2H), 0.98 Å (RCH3), 0.99 Å (R2CH2) and 1.00 Å (R3CH). The N—H hydrogen atom was included using a riding model that allowed the N—H distance to refine, while that of the minor component was constrained. The O—H hydrogen atom coordinates of the hydrogen fumarate anion were refined freely. Uiso(H) parameters were set to values of either 1.2Ueq  or 1.5Ueq  of the attached atom. The final structure was checked using validation tools in PLATON  global, I. DOI: 10.1107/S2056989022008118/hb8035Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989022008118/hb8035Isup3.cmlSupporting information file. DOI: 2201292CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Background: Malaria elimination and eradication efforts can be advanced by including transmission-blocking or reducing vaccines (TBVs) alongside existing interventions. Key transmission-blocking vaccine candidates, such as Pfs230 domain one and Pfs48/45 domain 3, should be genetically stable to avoid developing ineffective vaccines due to antigenic polymorphisms. We evaluated genetic polymorphism and temporal stability of Pfs230 domain one and Pfs48/45 domain three in Plasmodium falciparum parasites from western Kenya.Methods: Dry blood spots on filter paper were collected from febrile malaria patients reporting to community health facilities in endemic areas of Homa Bay and Kisumu Counties and an epidemic-prone area of Kisii County in 2018 and 2019. Plasmodium speciation was performed using eluted DNA and real-time PCR. Amplification of the target domains of the two Pfs genes was performed on P. falciparum positive samples. We sequenced Pfs230 domain one on 156 clinical isolates and Pfs48/45 domain three on 118 clinical isolates to infer the levels of genetic variability, signatures of selection, genetic diversity indices and perform other evolutionary analyses.Results:Pfs230 domain one had low nucleotide diversity (π = 0.15 × 10–2) with slight variation per study site. Six polymorphic sites with nonsynonymous mutations and eight haplotypes were discovered. I539T was a novel variant, whereas G605S was nearing fixation. Pfs48/45 domain three had a low π (0.063 × 10–2), high conservation index, and three segregating sites, resulting in nonsynonymous mutation and four haplotypes. Some loci of Pfs230 D1 were in positive or negative linkage disequilibrium, had negative or positive selection signatures, and others  and  had a history of recombination. Mutated loci pairs in Pfs48/45 domain three had negative linkage disequilibrium, and some had negative and positive Tajima’s D values with no history of recombination events.Conclusion: The two transmission blocking vaccine candidates have low nucleotide diversity, a small number of zone-specific variants, high nucleotide conservation index, and high frequency of rare alleles. With the near fixation a polymorphic site and the proximity of mutated codons to antibody binding epitopes, it will be necessary to continue monitoring sequence modifications of these domains when designing TBVs that include Pfs230 and Pfs48/45 antigens. Plasmodium falciparum antigens has hampered efforts to develop an effective vaccine that is protective against pre-erythrocytic and asexual blood-stage parasites  candidates, P. falciparum surface protein 230 (Pfs230)    and P. ffs48/45)  have beeP. falciparum infection in mosquitoes  were collected on filter paper from febrile malaria patients at health clinics in Homa Bay County (Kochia), Kisumu County (Chulaimbo), and Kisii County (Eramba) in 2018 and 2019 . The stuPlasmodium species-specific real-time PCR targeting 18S ribosomal RNA gene was used to confirm P. falciparum positive DNA samples before amplification of specific target fragments of each gene  and were used to amplify Pfs230 D1 and Pfs48/45 D3 in a T100™ Thermal Cycler . Briefly, 3 µL of sample DNA was added to a mixture of 11.5 µL of DreamTaq Green PCR Master Mix (2X), 0.5 µL of Pfs230 D1 forward (5′-TGGTGAAGCTGTCGAAGATG-3′) and reverse primers (5′-GTGTACCACAGGGGGAAGAG-3′) targeting 514 base pairs and 7.5 µL of double-distilled water. The thermal profile was set as follows 95 °C for 3 min, 34 cycles  and final extension at 72 °C for 6 min. For Pfs48/45 D3, similar reaction volume was prepared using forward (5′-TTTTCAAGAAGGAAAAGAAAAAGC-3′) and reverse primers (5′-GCCAAAAATCCATAATATGCTGA-3′) targeting 600bp. The PCR conditions were set as follows 95°C for 3 min, 34 cycles  final extension at 72 °C for 6 min. All the amplicons were assessed by gel electrophoresis in 1.5% w/v agarose gel before sequencing. For Pfs230 D1, 82, 39, and 35 samples from Homa Bay, Kisumu, and Kisii, respectively, were amplified. For Pfs48/45 D3, 36, 44, and 38 samples from Homa Bay, Kisumu, and Kisii, respectively, were amplified. All the PCR amplicons, together with positive controls, were purified using Exonuclease I and Shrimp Alkaline Phosphatase (ExoSAP-IT) and bi-directionally sequenced using 3730 BigDye® Terminator v3.1 Sequencing Standard kit on ABI PRISM® 3700 DNA Analyzer .Genomic DNA was extracted from filter paper using the modified Chelex resin (Chelex -100) method and stored at -20°C. As a positive control, DNA from the cultured laboratory strain NF54 was extracted and stored. ach gene . Primer on Suite . Among tPfs230 and PF3D7_1346700 for Pfs48/45). DnaSP Version 6.12.03 (π), haplotype diversity (Hd), number of haplotypes (h), number of segregating sites (S) and mean number of pairwise difference (k). Population Analysis with Reticulate Trees (Popart) version 1.7 software  and linkage disequilibrium (LD) were computed in DnaSP Version 6.12.03 and Arlequin version 3.5.2. Generated Tajima’s D values were plotted using GraphPad version 8.3.0. Both antigen structural delineation was done using Protein Homology/analogY Recognition Engine (PHYRE2) version 2.0 and generated models visualized and edited in UCSF Chimera version 1.15  as well as 3D7  pleated sheets connected by a loop containing mutated codons E655V and T656N , followed by progressively lower frequencies of K661N, T652R, and I539T. E655R and T656N were each observed at a frequency of <1%. The prevalence of various alleles was almost similar across the various study sites. For example, as shown in P. falciparum isolates from Kisumu and Homa Bay County lacked this mutation.Six loci  in utations . The mutnd T656N . G605S wPfs230 D1, Pfs48/45 D3 had three segregating sites  and parsimony-informative sites 757 and 762 (K253E and N254K) (In contrast to the six nonsynonomous mutated sites observed in ng sites . Singletd sheets . Codon cd sheets . The abid N254K) . Except d N254K) .Pfs230 D1 from the three sites had π of 0.15 × 10–2, k; 0.68 and haplotype diversity (Hd) of 0.57 (π (0.18 × 10–2) followed by Kisii (0.15 × 10–2) and Homa Bay (0.12 × 10–2). The site also had the most haplotypes 6) and the highest Hd (0.63) when compared to Kisii and Homa Bay, which had four haplotypes each and Hd of 0.60 and 0.52, respectively ( of 0.57 . The dom of 0.57 . Kisumu ectively . Haplotyectively . The remectively .Pfs48/45 D3 from western Kenya had low π (0.06 × 10–2) and Hd (0.25) (π (0.10 × 10–2) followed by Homa Bay (0.05 × 10–2) and Kisumu (0.03 × 10–2) (The d (0.25) . The domd (0.25) . Kisii p × 10–2) . Kisii a × 10–2) . The maj × 10–2) .Pfs230 D1 from P. falciparum isolates deviated from a standard neutral model. Tajima’s D (-0.8), FLD* (-0.9) and FLF* (-1.0) tests were all negative and non-significant (p > 0.05) (S result (-3.3), was significant (p = 0.023). Tajima’s D test results were also non-significant (p > 0.05) in each site (Pfs230 D1. Locus 1983 (codon change K661N) had a significant (p < 0.05) positive (1.9) Tajima’s D result, whereas the rest had negative results (p < 0.001) χ 2 values of 19.7 and 12.7 among Homa Bay and Kisumu sequences, respectively . Other loci with positive D′ results, low r2 values and non-significant (p > 0.05) χ2 values in Homa Bay and Kisumu parasites include loci pairs   and . Some loci pairs on Kisumu and Kisii sequences had negative D′ results . On Pfs230 D1 from parasites in Homa Bay and Kisumu, recombination events were detected across loci pairs  and  ( > 0.05) . Howeverach site . Despite results . Apart fectively . Among K results . On Pfs3, 1983) .Pfs48/45 D3 sequences had non-significant (p > 0.05) negative Tajima’s D -1.9, FLD*: 2.1 and FLF*: 2.0 results. The Fu’s FS (-1.9) result was, however, significant (p = 0.096). Locus 940 (L314I) among Kisii sequences had a significant (p > 0.05) positive Tajima’s D (0.8) results  and African  countries  or Homa Bay (endemic site with declining parasite population size) compared to Kisumu (Chulaimbo)  than in other geographical regions, as described by  AF = 0., AF = 0 (AF = 0.ulaimbo) . In contulaimbo)  as opposulaimbo) . This coulaimbo) . With neulaimbo) . Other p4F12 mAb . When th4F12 mAb . PlasmodP. falciparum populations in other malaria endemic regions  on the 85RF45.1 mAb epitope . Other p regions . However regions . Though  regions . The thrPfs230 D1 and Pfs48/45 D3. The presence of linkage disequilibrium confirmed the history of selection pressure and inbreeding across various loci in Pfs230 D1 and Pfs48/45 D3 (Inbreeding, recombination, and natural selection were identified as major drivers of the observed mutations in 48/45 D3 . Some po48/45 D3 . The neg48/45 D3 .D results revealed that purifying selection was affecting the majority of loci within Pfs230 D1 and Pfs48/45 D3, reducing genetic diversity (D values in both antigens were not significant. Individual Tajima’s D results for each codon revealed all other dimorphic codons to be under purifying selection, with the exception of K661N on Pfs230 D1 from all study sites and V304D on Pfs48/45 D3 from Kisii, which are under strong and weak balancing selection, respectively. The two mutated loci under balancing selection may play an important role within the Pfs230 D1 and Pfs48/45 D3 fusion proteins, which may explain why they are maintained in the P. falciparum population from western Kenya (The presence of natural selection was confirmed by the Tajima’s D values. Overall negative Tajima’s iversity . The aforn Kenya . These frn Kenya  that selThe presence of weak purifying selection acting on dimorphic sites may impact not only host mAb binding and functional activity but also be affected by selective pressure in the mosquito vector . This prPfs230 D1 and Pfs48/45 D3 in P. falciparum from western Kenya have low nucleotide diversity and a high conservation index with high frequency of rare alleles. Among the observed polymorphisms in Pfs230 D1, G605S is nearly fixed in the population. Natural selection, inbreeding, and, to some extent, recombination are important driving forces in shaping these alleles in the two antigens. With the discovery of novel polymorphic sites, the two domains of the Pfs230 and Pfs48/45 from different malaria-prone regions, including areas where clinical trials have been conducted, should be monitored indefinitely. This will help track the genetic stability of the two TBV candidates.The


Primary pulmonary lympho-epithelioma-like carcinoma (PPLELC) is a rare subtype of primary non-small cell lung cancer (NSCLC). Currently, there is still lack of research data on anti-angiogenic therapy of advanced PPLELC. The purpose of this study was to investigate the efficacy and safety of anti-angiogenic therapy combined with chemotherapy compared with traditional chemotherapy for these patients.Advanced PPLELC patients admitted to six grade A hospitals from January 2013 to January 2021 were selected. The patients received anti-angiogenic therapy combined with chemotherapy (AT group) or chemotherapy (CT group) alone.P = 0.008]. The 1-year PFS rates were 41.9% and 17.6%, respectively. The overall response rates (ORR) of two groups were 45.2% , 38.2% , (P = 0.571). The disease control rates (DCR) of two groups were 93.5% , 88.2% , (P = 0.756).A total of 65 patients were included in this study, including 31 patients in the AT group treated with anti-angiogenic therapy combined with chemotherapy and 34 patients in the CT group treated with chemotherapy alone. As of October 1, 2021, the median progression-free survival (PFS) in the AT group was 11.2 months . The median PFS in the CT group was 7.0 months  [Hazard Ratio (HR), 0.49; 95%CI, 0.29–0.83; Among patients with advanced PPLELC, the PFS of patients with anti-angiogenic therapy combined with chemotherapy is better than that of patients with chemotherapy alone. Anti-angiogenic therapy combined with chemotherapy is an optional treatment scheme.The online version contains supplementary material available at 10.1007/s00432-022-03935-0. Lympho-epitheliomatoid carcinoma is a rare epithelial neoplasm originating mostly in the nasopharynx and also in the foregut , version 1.1 has at least one measurable lesion. This retrospective study collected data of PPLELC patients from Nanfang Hospital affiliated to Southern Medical University, Zhujiang Hospital affiliated to Southern Medical University, The First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine, Cancer Hospital of Sun Yat-sen University, Cancer Hospital affiliated to Guangzhou Medical University, Three Gorges Hospital affiliated to Chongqing University from January 2013 to January 2021. 65 patients with advanced PPLELC were eventually included in the study , endu (n = 6) or anlotinib (n = 3), the chemotherapy regimen included paclitaxel (n = 14), pemetrexed (n = 10) or gemcitabine (n = 7) combined with platinum or mono-therapy. The chemotherapy regimen in CT group included paclitaxel (n = 19), pemetrexed (n = 10) or gemcitabine (n = 5) combined with platinum or mono-therapy.In this real-world retrospective study, patients were divided into AT group treated with anti-angiogenic therapy combined with chemotherapy and CT group treated with chemotherapy. AT group received anti-angiogenic therapy, including bevacizumab (The tumor response assessment program is conducted every two to four cycles. Tumor response was assessed as complete response (CR), partial response (PR), disease stability (SD), or disease progression (PD) according to RECIST version 1.1. Adverse events and laboratory abnormalities were graded according to the National Cancer Institute Standard for Common Terminology for Adverse Events, Version 4.0.T test was used for measurement data (age) and Chi-square test (Cartesian test) was used for counting data. Univariate Cox regression was used to screen individual risk factors. The independent prognostic covariate of PPLELC patients was determined by multivariate Cox regression model and forward procedure. Cox proportional risk regression was used for univariate and multivariate survival analyses. All statistical analyses were performed in IBM SPSS Statistics . When P value was less than 0.05, the difference was considered statistically significant.Efficacy was assessed in the enrolled population and safety was assessed in the treated population. Kaplan–Meier methods were used to estimate progression-free survival. P > 0.05). . There were 38 female patients (58.5%) and 27 male patients 41.5%). ECOG score was 0 in 24 patients (36.9%) and 1 in 41 patients (63.1%). 18 patients (27.7%) were smokers, and 10 patients (15.4%) had a family history. There were 10 patients (15.4%) in stage IIIB and 55 patients (84.6%) in stage IV. Patients with single organ metastasis accounted for 19 (29.2%), and patients with multiple organ metastasis accounted for 46 (70.8%). The most common tumor sites were middle lobe of right lung and lower lobe of right lung, with 18 (27.7%) and 17 (26.1%) patients, respectively. There were 61 patients (95.3%) who were positive for Epstein–Barr virus, 15 patients (23.1%) had received radiotherapy, and 4 patients (6.2%) with gene mutations, including 3 gene mutations in the AT group, which were NRAS Q61L (14.5%) missense mutation, SMAD4 K110Ter (19.5%) mutation and FGFR3–TACC3 (F17:T10) fusion. MYC amplification was found in 1 case in CT group. There was no significant difference between the two groups at baseline , while chemotherapy was used in the first-line therapy.The minimum follow-up time was about 1.4 months. Median follow-up time was 18 months in the AT group  and 19 months in the CT group . AT the end of the follow-up period, 11 patients (35.5%) in the AT group and 12 patients (35.3%) in the CT group continued treatment. Anti-angiogenic therapy was administered either as up-front or salvage therapy  and 30 , P = 0.756  reached PR, 15 patients (48.3%) reached SD, and 2 patients (6.5%) did not respond to treatment. In CT group, 13 patients (38.2%) reached PR, 17 patients (50.0%) reached SD, and 4 patients (11.8%) did not respond to treatment. The ORR of AT group and CT group were 14  and 13 , % reachedP = 0.008]. The 1-year PFS rates were 41.9% and 17.6%, respectively  fusion, no smoking history, tumor located in the right lung with lymphatic, liver, and bone metastases, clinical stage T4N3M1, pathologically confirmed as PPLELC, Genetic analysis revealed FGFR3–TACC3 (F17:T10) fusion without EGFR, ALK and other conventional mutations. The patient was treated with anlotinib in combination with albumin–paclitaxel and carboplatin at the physician's recommendation, achieving disease control and PFS for 8 months  occurred more frequently in the AT group than in the CT group. In the AT group, there were 17 cases (54.8%) of grade 3 and 8 cases (25.8%) of grade 4 treatment-related adverse events. There were 13 cases (38.2%) of grade 3 and 12 cases (35.3%) of grade 4 treatment-related adverse reactions in the CT group. The most common adverse reactions in AT group were decreased appetite 7 (22.6%), anemia 8 (25.8%), leukopenia 14 (45.2%), neutropenia 16 (51.6%), and decreased platelet 8 (25.8%). Anemia 10 (29.4%), leukopenia 14 (41.2%), neutropenia 15 (44.1%), and thrombocytopenia 10 (29.4%) were most common in the CT group Below is the link to the electronic supplementary material.


The developedmethod gave a linear response within the F– concentrationrange of 0.11–1.32 mM with the LOD and LOQ values of 0.026mM (0.5 mg L–1) and 0.079 mM (1.5 mg L–1), respectively. The LOD value for fluoride was below the WHO-permissiblelimit (1.5 mg L–1) and the US-EPA-enforceable limit(4 mg L–1) in water. The possible interference effectsof common anions  and cations  were investigated;the observed interferences from Fe2+, Fe3+,and PO43– were easily eliminated by maskingiron with the necessary amount of Na2EDTA without affectingthe blank absorbance of the Al(III)-flavonoid complex, precipitatingphosphate with Ag(I) salt, and partly neutralizing alkaline watersamples to pH 4 with acetic acid. The developed method was appliedto real water samples and also validated against a reference spectroscopicmethod at the 95% confidence level.In this study, we applied an innovative approach of greenanalyticalchemistry to develop a novel and eco-friendly chromogenic agent forfluoride determination by making use of the nontoxic Al(III)-flavonoidcomplex in a natural extract from St. John’s wort plant. Theinitial intensely yellow-colored Al(III)-flavonoid complex formedin the plant extract was converted to a colorless AlF One of the importantparameters of water quality is the amount of fluoride. Fluorine isthe 13th most abundant element in the world and cannot be freely foundin the environment unless it combines with other substances to formfluoride. Fluorides can be classified such as ionizable/non-ionizableand organic/inorganic. Organic fluorides do not dissolve as rapidlyin water as inorganic fluoride ions not having a chemical reactionwith the solvent.9 gas chromatography (GC),10 ICP-MS,11 AAS ,12 and ion-selective electrode-based potentiometry(ISE).14 Among these methods of F– determination, ISE and spectrophotometry stand out to give satisfactoryresults.15 The disadvantages of the ISEmethod were reported as limited selectivity, poor precision, longequilibration time, electrode drifting, and low solubility of thelanthanum fluoride membrane crystal19 when determining fluoride atlow concentrations. In addition, the ion activity, presence of interferingions and colloidal particles, and color and temperature of solutionmedium may cause problems on the ISE method.20 Chromatographic methods for the determination of fluoride are expensiveand time-consuming and require skilled specialists.21 Spectrophotometry is a versatile and good alternative techniquefor determining the concentration of inorganic ions in water samples,having many advantages such as low cost, ease of applicability, fastanalysis, reliability, high sensitivity at low concentrations, andwide analytical working range.22 In spectrophotometricmethods, dyestuff-metal complexes,26 nanoparticles,28 or synthesized organic molecules30 have beenstudied as the three kinds of analytical probes. However, it is knownthat lots of dyestuffs are both toxic and carcinogenic.31 Most dyes, especially synthetic ones, are nondegradabledue to their stability to light and oxidants.32 On the other hand, considering the nanoparticle-based syntheses,some of the reducing agents used to synthesize nanoparticles may betoxic, high-priced, of low reducing capability, and may bear a contaminationrisk (as they can bring other impurities to the system).33 The synthesis of organic molecules as a fluoridereceptor requires both a long time and organic solvents, limitingthe use of these analysis systems in aqueous solutions.34 Organic solvents cause formation of hazardouswastes and also constitute a major source of volatile organic compound(VOC) emission, threatening human health.36 The US-EPA declared that to overcome the problem of disposal ofhazardous wastes used in academic studies, attention should be givento the reduction of hazardous wastes.37 The usage of naturally sourced reagents like plant compounds insteadof synthetic toxic chemicals is one of the ways to apply sustainabledevelopment principles in analytical laboratories.38 Lately, it has become popular to approach green analyticalchemistry by enhancing currently applied methods and/or developingnew methods using eco-friendly materials.39 Flavonoids are plant-derived polyphenolic compounds having manyfavorable biochemical properties.40 St.John’s wort (Hypericum perforatum L.) is one of the flavonoid-rich plants41 and is conventionally consumed as a herbal tea and nutritional supplementdue to its remarkable bioactive properties.42 Flavonoids have the ability to form colored complexes with metalions due to their carbonyl and hydroxyl groups arranged in a special geometry. Metal ion coordination by flavonoidscauses significant differences in certain properties, e.g., color,fluorescence, oxidation state, catalytic ability, stability, and toxicity,explaining the wide use of flavonoids in analytical chemistry, photochemistry,medicinal chemistry, and textile dyeing.43 In addition, a method based on complexing flavonoids with aluminum(III)is used to find the total flavonoid content of some species.44 Pękal and Pyrzynska45 studied and compared two common spectrophotometric procedures,named procedure 1 and procedure 2, to determine the “totalflavonoid content” of food and medicinal plant samples. Method1 involved the measurement of flavonols and flavone luteolin at 410–430nm after addition of (only) AlCl3 solution, whereas method2 investigated the same Al(III)-complexation procedure in the presenceof NaNO2 in alkaline medium and was found specific forrutin, luteolin, and catechins but also phenolic acids at an analyticalwavelength of 510 nm. Although these two procedures yielded a differentorder of flavonoid content for the studied plant extracts , St. John’s wort had thehighest total flavonoid content with respect to (wrt) procedure 1and one of the three high ones wrt procedure 2.45 In addition, rutin was found as the main flavonoid constituentof aqueous and different solvent extracts of St. John’s wort.46 It is also known that compared to the highlycolored Al-flavonoid complex, Al(III) easily forms a colorless AlF63– complex with fluoride.23 With this background, we devised a readily available, nontoxic,economical, and flavonoid-rich aqueous extract (organic solvent-free)from St. John’s wort as a natural alternative chromogenic reagentfor fluoride when complexed with Al(III), because the color of theAl(III)-flavonoid complex could be bleached by fluoride  complex) in a concentration-dependentmanner, thereby enabling a selective, eco-friendly, and accurate determinationof fluoride.The traditional analyticalmethods for the determination of fluorideions in water are ion chromatography (IC),22.13)3·9H2O) and ethylenediaminetetraacetic acid disodium salt dihydrate(Na2EDTA) were purchased from Sigma-Aldrich. Methyl salicylateC6H4CO2CH3) was obtained from JQC (Huayin) Pharmaceutical Co., Ltd.,and sodium acetate (CH3COONa), acetic acid (CH3COOH), ethanol, iron(III) nitrate nonahydrate (Fe(NO3)3·9H2O), iron(II) chloride tetrahydrate (FeCl2·4H2O), manganese(II) nitrate tetrahydrate(Mn(NO3)2·4H2O), ammonium chloride(NH4Cl), potassium nitrate (KNO3), calcium nitratetetrahydrate (Ca(NO3)2·4H2O),magnesium nitrate hexahydrate (Mg(NO3)2·6H2O), sodium nitrate (NaNO3), sodium chloride (NaCl),sodium bromide (NaBr), sodium sulfate (Na2SO4), and boric acid (H3BO3) were purchased fromMerck. Sodium phosphate monobasic (NaH2PO4)was obtained from Riedel-de Haen.All reagents were of analyticalreagent grade. Dried St. John’s wort was obtained from a localmarket. Sodium fluoride (NaF) was purchased from La Chema, Czech Republic.Aluminum nitrate nonahydrate were used for all absorbance measurements. A Precisa XB 220A AnalyticalBalance was used to weigh all the chemicals, and a Glassco 710 DNAGhot plate with a magnetic stirrer was used to boil the ultrapure waterrequired for the aqueous extract of St. John’s wort. A Wisetherm-fuzzycontrol, wısd HB-48 dry bath was used to find the optimal temperaturefor the recommended method.2.33+ and F– were prepared at 1000.0mg L–1 in ultrapure water and stored at +4 °C.Working solutions of different initial concentrations of F– (12.5–150.0 mg L–1) were freshly prepared.The initial concentration of Al(III) solution at 250.0 mg L–1 was prepared by diluting 1000.0 mg L–1 stock solutionfor the proposed method. Acetic acid-sodium acetate buffer (pH 4.0)was prepared by mixing 0.1 M CH3COOH and 0.1 M CH3COONa solutions at an appropriate ratio.Stock solutionsof Al–, Br–, I–, NO3–, HCO3–, SO42–, andPO43–) and cations  were preparedseparately at initial 75.0 g L–1 concentrationsexcept HCO3– and then mixed with F– to be at several-fold  of analyte in ultrapure water. Only bicarbonate (HCO3–) was prepared at an initial concentrationof 50 g L–1 due to its limited solubility.The stock solutionsof common water anions 3·9H2O was dissolvedin the mixture of 1.75 mL of ultrapure water and 1.25 mL of nitricacid (1 M), the total volume was completed to 25.0 mL with ultrapurewater for preparing 2 × 10–2 M Fe3+ solution. To prepare a 1.0% methyl salicylate solution, 0.5 g ofmethyl salicylate was weighed in a 50.0 mL volumetric flask and thetotal volume was completed with pure ethanol. The final concentrationsof the working solutions of F– to generate the calibrationcurve in the UV–Vis reference method were set by adding differentvolumes  of 625.0 mg L–1 stock solution to be 5.0, 10.0, 15.0, 20.0, 25.0, and 30.0 mg L–1.To apply the UV–Vis reference method, 2 × 102.4A volume of 100.0 mL of freshly boiled ultrapurewater was added to carefully weigh 2.0 g of dried St. John’swort in a glass beaker. It was filtered into a 100.0 mL volumetricflask through a filter paper after waiting for 10.0 min. The finalfiltrate volume was completed to 100.0 mL with ultrapure water toprevent volume loss. The preparation of St. John’s wort aqueousextract as a natural and green chromogenic agent followed by Al(III)addition and the recommended method for fluoride detection are shownin –1  Al3+ solution,and 0.5 mL of F– solution at initially differentconcentrations (12.5–150.0 mg L–1) were addedto the test tube in this order. For the blank solution, 0.5 mL ofultrapure water was added instead of fluoride. After waiting for 10.0min at room temperature , the absorbance valuesof the solutions were recorded at 420 nm wavelength against water.A volume of 1.0 mL of St. John’s wortextract, 1.0 mL ofpH 4.0 acetic acid-acetate buffer solution (0.1 M), 0.5 mL of 250.0mg L–1 Al3+ solution + 0.5 mL of F– at initiallydifferent concentrations (12.5–150.0 mg L–1), wait for 10.0 min at RT, and measure the absorbance at λ= 420 nm (A420 nm) against water.Summarized procedure: for sample solutions, add 1.0 mL of St. John’swort extract + 1.0 mL of pH 4.0 acetic acid-acetate buffer solution(0.1 M) + 0.5 mL of 250.0 mg L–1  Al3+ solution + 0.5 mL of ultrapure water, wait for 10.0 min at RT, andmeasure A420 nm against water .For blank solution, add 1.0 mL of St. John’s wort extract+ 1.0 mL of pH 4.0 acetic acid-acetate buffer solution (0.1 M) + 0.5mL of 250.0 mg L2.5–, I–, NO3–, HCO3–, SO42–, and PO43–) and cations .The interferences of Fe2+, Fe3+, and PO43– could be easily eliminated.The recovery values of fluoride were calculatedby applying the recommended method in the presence of anions -flavonoid complex in the recommended method was determined.If an EDTA optimization was not carried out, an excess of EDTA coulddecolorize the Al(III)-flavonoid complex as the target probe for fluorideattack. For this purpose, Na2EDTA solutions were preparedat initial concentrations of 100, 200, 300, and 400 mg L–1. Each Na2EDTA solution was added instead of fluoridein the proposed method, and ultrapure water was added instead of fluoridefor the blank solution. Then, different mass ratios of Na2EDTA were tested along with iron ions in 1:1 iron:fluoride solutions to remove the interference due to Fe2+ and Fe3+ ions by masking.First,the maximum amount of Na+ ions as aprecipitation agent in acidic medium. For thispurpose, 0.5 mL of 1000 mg L–1 F–, 0.5 mL of 1000 mg L–1 PO43–, and 0.5 mL of 1000 mg L–1 Ag+ weremixed. After the volume of the mixture was made up to 4.0 mL withultrapure water, 0.2 mL of 0.1 mol L–1 HCl was addedand the solution became cloudy; ultrapure water was added for dilutionto a final volume of 5.0 mL, followed by keeping of the mixture ina centrifuge device at 10,000 rpm for 10 min and filtration. NaOHsolution (0.1 M) was added dropwise until the pH value of the supernatantphase was 3.2 to 4.0; the supernate was diluted to 10.0 mL with ultrapurewater.The interferenceeffect of phosphate was eliminated using Ag2.6–1 to each 5.0 mL volumetric flaskcontaining 4.0 mL of mineral water at pH set to 4.0 . If necessary, the total volume was completedto 5 mL with ultrapure water.The recommended method was applied to mineralwater and artificial wastewater samples. Then, the recovery (%) andRSD (%) values of fluoride were recorded. For the application of therecommended method to real water samples, the necessary preprocessesfor the determination of fluoride in mineral water were carried outas follows: Initially, the pH was adjusted to 4.0 by adding 78 μLof concentrated acetic acid (17.4 M) to 20.0 mL of mineral water.Solutions were prepared in a total volume of 5.0 mL by adding certainvolumes (0–1.0 mL) of fluoride at an initial concentrationof 625.0 mg L–1 F– and 16.7 mg L–1 B (H3BO3 was used as a boron source). Later, solutionswere prepared with a total volume of 4.0 mL by adding certain volumes(0.1–0.5 mL) of fluoride at an initial concentration of 625.0mg L–1 to each 2.0 mL of artificial wastewater.Total volumes were completed to 4 mL with ultrapure water .The prepared artificial wastewaterwas diluted to have the initial concentrations of 50.0 mg L2.747 The referenceand recommended methods were compared at the desired confidence levelusing the t- and F-statistical tests.The preparation of solution A and solution B and the application ofthe proposed method are briefly described below:–1 stock solutionof fluoride.Solution A: 625.0 mg L–2 M) of Fe3+ + 35.0 mL of 1.0% methylsalicylate + 30.0mL of EtOH + 15.0 mL of ultrapure water.Solution B: 20.0 mL .For samples, 1.0 mL of solution B + different volumes(20.0–120.0 μL) of solution A + make-up with ultrapurewater to a total volume of 2.5 mL .For blank solution, 1.0 mL of solution B + 1.5mL of ultrapurewater  for calculating the means and the standard error of themean. Results were expressed as mean ± standard deviation (SD).Validation of the recommended method for determining the fluoridecontent against the UV–Vis reference method33.1N = 3).The figures obtained for the optimization of each parameter wereformed as a result of three repetitive analyses  Al3+ solution (0.5 mL), and F– at 75.0 mg L–1 initial concentration (0.5 mL) were added to testtubes , and for the blank,0.5 mL of ultrapure water was added instead of F– solution. Then, after the blank and sample solutions were kept for10.0 min, their absorbances were recorded against water in the wavelengthrange of 370–550 nm, and the wavelength at which the absorbancedifference between the blank and the sample (ΔA) was maximum was 420 nm, as shown in To find the optimal wavelength, St. John’s wortextract (1.0 mL), pH 4.0 acetic acid-acetate buffer solution , 250.0 mg L3+ concentration,fluoride solutionsat 12.5 and 25.0 mg L–1 initial concentrations weretested separately with Al3+ solutions at different concentrations.Initial concentrations of Al3+ ranged from 100.0 to 350.0mg L–1. After adding solutions as stated below andwaiting for 10.0 min, A420nm readingswere recorded against water. St. John’s wort extract (1.0 mL)+ 1.0 mL of pH 4.0 acetic acid-acetate buffer solution (0.1 M) + 0.5mL of different initial concentrations of Al3+ solution+ 0.5 mL of F– at 12.5 or 25.0 mg L–1 initial concentration were added to test tubes . For blank solutions, 0.5 mL of ultrapurewater was added instead of F– solution. The optimalinitial Al3+ concentration was chosen as 250.0 mg L–1 due to the maximum ΔA420nm readings recorded with two different concentrations ofF– as shown in To find the optimal Al–1. After adding the solutions sequentiallyas stated below, they were kept for 10.0 min at RT, and the absorbancevalues of the blank and sample solution were read at 420 nm wavelength.To find the optimal pH value, studieswere made in the range ofpH 4.0–8.0 and also separate experiments were carried out attwo different initial concentrations of fluoride as 12.5 and 25.0mg L–1  Al3+ solution + 0.5 mL of F– at initial different concentrations (12.5 or 25.0 mg L–1) , wait for 10.0 minat RT, and measure absorbance at λ420 nm againstwater. For blank solution, 0.5 mL of ultrapure water was added insteadof F–. Acetic acid–sodium acetate buffersolution (for pH 4.0 and pH 5.0), potassium hydrogen phthalate-NaOHbuffer solution (for pH 6.0), ammonium acetate (for pH 7.0) buffersolution, and borax-HCl buffer solution (for pH 8.0) were used forthe optimization. The absorbance differences with two different fluorideconcentrations were maximum at pH 4.0, and this pH was chosen as theoptimal value as shown in For sample solutions, add 1.0 mL of St. John’s wort extract+ 1.0 mL of buffer solvents at different pH values  + 0.5 mL of 250.0 mg L–1 initial concentrationof fluoride. For this purpose, 1.0 mL of St. John’s wort extract+ 1.0 mL of pH 4.0 acetic acid-acetate buffer solution (0.1 M) + 0.5mL of 250.0 mg L–1  Al3+ solution + 0.5 mL of F– at 50.0 mg L–1 initial concentration were added to test tubes .To select the optimal time, separate experimentswere made forblank solution and 50.0 mg LA420 nm values were recordedagainst water. As seen in 63– complex, because the optimal times of the blank solution and sampleswere different. Since the absorbance readings of the blank solutionand samples were recorded against water, the optimal time was chosenas 10 min for the recommended method.For blank solution, 0.5 mL of ultrapurewater was added insteadof fluoride. After the solutions were added, the blank solutions andsamples were incubated separately for up to 25 min at 1 min intervals,and then –1 initial concentrations were studiedseparately. St. John’s wort extract (1.0 mL) + 1.0 mL of pH4.0 acetic acid-acetate buffer solution (0.1 M) + 0.5 mL of 250.0mg L–1  Al3+ solution+ 0.5 mL of F– at 25.0 or 75.0 mg L–1 initial concentration were added to test tubes . For blank solutions, 0.5 mL of ultrapurewater was added instead of fluoride. The blank and sample solutionswere left to stand for 10 min at different temperature intervals (25.0–100.0°C), and then A420 nm valueswere recorded against water.To determine the optimaltemperature, fluoride solutions at 25.0and 75.0 mg LSince it was observed that therecommended method was independentof temperature as seen in 3.248 As a result of flavonoidsforming a coordination compound with metals, their absorption spectrachange and a bathochromic (red) shift is observed in the UV–Visspectrum. According to some authors, the reason for this red shiftis a strong charge transfer transition from the flavonoids to themetal center, while others argue that the decrease in the HUMO-LUMOenergy levels of the flavonoid molecules is greater than the chargetransfer from the ligand to the center.49 For fluoride determination, the chromogenic agent was formed byproviding a red shift in the absorption band due to the formationof the Al(III)-flavonoid complex from the St. John’s wort extractsolution. Then, by adding fluoride ions to the medium, aluminum ions—capableof forming colored complexes with flavonoids—selectively forma colorless AlF63– complex. Therefore, a blue (hypsochromic) shift in the absorptionband of flavonoids is observed 420 nm) was related to the increasing fluorideconcentration when the proposed method was applied. In this recommendedmethod, as the concentration of fluoride solutions increased, thedecolorization (from dark yellow to light yellow) of the Al(III)-flavonoidcomplex was observed .Linear calibration equation for fluoride:–1), covering an order of magnitude for C–F. In addition, the molarabsorptivity (ε), limit of detection (LOD), and limit of quantification(LOQ) values were 7.67 × 102 ± 2.2 × 101 L mol–1 cm–1, 0.026 mM(0.5 mg L–1), and 0.079 mM (1.5 mg L–1), respectively. The limit of detection was found in millimole perliter units . The coefficientsof variation (CVs) of intra- and inter-assay for fluoride were 2.17and 2.59%, respectively (N = 5), showing that therecommended method has good precision. The LOD value is below thepermissible limit of fluoride in water  by WHO50 and also enforceable  and non-enforceable ) limits by EPA.52The linear concentration range wasfrom 0.11 to 1.32 mM  and cations  present in water on the proposed methodwere investigated. The interferences of Fe2+ and Fe3+ on the method were eliminated using Na2EDTA asa specific masking agent without affecting aluminum ions that complexedwith the flavonoid. PO43– interferencewas removed by the precipitation method with AgNO3. Theinterference analysis was performed using solutions of fluoride atan initial concentration of 50.0 mg L–1 with differentmass ratios of common water ions such as Cl–, Br–, I–, NO3–, HCO3–, SO42–, PO43–, K+, NH4+, Ag+, Ca2+, Mg2+, Mn2+, Fe2+, and Fe3+ . The interference effects ofanions and cations are represented in The possible interference effects of common anions  but not interacting with aluminum in the Al(III)-flavonoid complex,was investigated. For this purpose, a maximal initial concentrationof 300.0 mg L–1 Na2EDTA was added insteadof fluoride, and no change was observed in the absorbance of the blanksolution. The interference effect of Fe2+ and Fe3+ was easily eliminated by using Na2EDTA as a masking agent,which was added at different mass ratios for each valency of iron[metal–EDTA ratio: 1:3 (w/w) for Fe2+ and 1:6 (w/w)for Fe3+] to 1:1 iron:fluoride solutions before applyingthe recommended method. Since ferric ions had a higher affinity forfluoride than ferrous ions in accordance with crystal field theory,they required a higher concentration of the chelating agent (EDTA)for sufficient masking. During this procedure, the Al(III)-flavonoidcomplex was not affected because Al(III) was already bound in a stablecomplex, and Al(III) chelation with EDTA needs temperature and time has slow kinetics due to its noble gas electronic configuration).The initial concentration of Na–1 F–, 50.0 mg L–1 PO43–, and 50.0 mg L–1 Ag+ mixture obtained in the last step.Elimination of the interference effect of phosphate is based onthe principle of removing it from solution by precipitation with silverions in acidic medium  was 0.281 mg L–1.The concentrations in mg L–1  and 300.0 mg L–1 F–. Concrete sludge is an industrial waste slurry containing hydratedcement, aggregates, and water, and its wastewater may contain bothboron and fluoride.60 The proposed methodwas applied to artificial wastewater with standard additions at afinal concentration range of 4.166–16.666 mg L–1 , absorbance readings were recorded forthe recommended method and the reference method . The mean of the absorbance readings of three consecutivesamples was used for each calculation (N = 3). Theresults showed no significant difference in precision and accuracybetween the two methods. The recommended method was validated againstthe UV–Vis reference method at the 95% confidence level usingStudent’s t- and F-tests.Statistical parameters of the recommended method and the UV–Visreference method are depicted in After preparing fluoride solutionsto have an initial concentration of 75 mg L4–1) of the recommended method meets the required critical values ofWHO (1.5 mg L–1) and EPA (4 mg L–1 for the non-enforceable standard and 2 mg L–1 forthe secondary standard) for the fluoride content of water samples.The possible interferences of ionic species commonly found in waterwere investigated for establishing the selectivity of the recommendedmethod for the analyte. While Na2EDTA was used to selectivelyremove the interference effects of Fe2+ and Fe3+, the precipitation method was applied in acidic medium to eliminatethe interference of PO43– using Ag+ ions as a precipitation agent. The recommended method wasapplied to water samples  and also validated against a reference UV–Visreference method at the 95% confidence level.In this study, our mainaim was to develop a simple and inexpensivefluoride determination method based on the principles of green analyticalchemistry, free from toxic solvents. This method was eco-friendlywithout complicated steps and utilized a natural extract in a greenchromogenic method that meets today’s requirements. For thispurpose, St. John’s wort was chosen as a natural sensing reagentfor fluoride determination by utilizing the Al(III)-flavonoid metalcomplex formation due to the strong charge transfer interaction offlavonoids with metal ions and/or the decrease in HUMO-LUMO energylevels of flavonoids, which is more effective than ligand-to-metalcharge transfer. It is clear that the LOD value (0.5 mg L


Moreover, HL also showed good colorimetric selectivity to F−/CN−; a solution color change from colorless to light yellow in acetonitrile was observed by the ‘naked-eye’. The properties of HL with Al3+ and F−/CN− were studied by UV-vis absorption spectroscopy, fluorescence spectroscopy, high-resolution mass spectrometry and 1H NMR titration. Furthermore, the cell imaging experimental results indicated that the chemosensor HL could be applied for the detection of Al3+ in living cells.A novel multifunctional chemosensor HL bearing a julolidine unit and a Schiff base unit has been synthesized. As a fluorescent sensor, HL exhibited excellent selectivity and high sensitivity to Al A novel multifunctional chemosensor HL bearing a julolidine unit and a Schiff base unit has been synthesized. However, excessive intake of fluoride may affect thyroid activity and may also lead to skeletal fluorosis, depression and adverse effects on the immune system.12 The WHO has set a maximum allowable fluoride ion limit of 1.5 mg L−1 in drinking water.13 On one hand, cyanogen salts are widely used in many chemical and industrial processes such as plastic production, resin industry, organic synthesis, metallurgy and gold mining.14 Thus, CN− is present around us in different forms. It can be absorbed through the lungs, gastrointestinal track and skin, which can cause vomiting, convulsion, loss of consciousness, and even death.15 The maximum amount of CN− in drinking water is only 1.9 μM based on WHO research.16 Most importantly, it is also crucial to detect F−/CN− in chemical, biological and environmental samples.In addition, the detection of anions has also aroused much attention in recent years because it plays an important role in medical, chemical, biological and environmental processes.17,18 Compared with other test methods, colorimetric and fluorescent methods are the most attractive for the detection of these analytes because of the advantages of high sensitivity, fast response, convenience and low cost.19 Among the reported colorimetric and fluorescent sensors, Schiff base sensors are the most common candidates because of their simple synthesis; the nitrogen atom in the C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>N bonds have lone pair of electrons which can easily bond with metal ions.20 There are many Schiff base chemosensors that can detect Al3+ including diarylethene,21 naphthalimide,22 and thiazole23 derivative. In addition to the Schiff base sensors, other types of optical sensors for detecting Al3+ have also exhibited significant progress. A large number of Al3+ fluorescent sensors have been reported such as some derivatives including rhodamine derivatives,24 phenanthroline derivatives,25 tetraphenyl ethylene (TPE)–COOH26 and other rare earth metal complexes,27 and the researches of these sensors have made good results; however, the synthesis process of them was complicated, or the sensor cannot distinguish Al3+ from Ga3+.28 Moreover, there are many fluorescent or colorimetric sensors for detecting F−/CN− including benzimidazole–naphthalene conjugate molecules,29 silylated derivatives,30 and 2-phenyl-2H-1,2,3-triazole derivatives.31 Besides, some Schiff base compounds can detect F−/CN−.32,33 However, sensors that can simultaneously recognize Al3+ and F−/CN− are still relatively uncommon.34,35In recent years, many studies have focused on detecting metal ions and anions with multifarious methods.3+ and F−/CN−. Detecting multiple targets with a single receptor is more efficient and less expensive than one-to-one analysis.36 The julolidine group is an excellent fluorophore,37 and the Schiff base compounds contain a benzhydrazide unit with donor sites such as N atom and O atom for Al3+.38 Besides, benzhydrazide units contain an amide functional group, and julolidine possesses a phenolic hydroxyl group; the –NH proton and the phenolic hydroxyl group could be linked with F−/CN−via a hydrogen bond to detect F−/CN−.39,40 The synthetic route of sensor HL was shown in 3+ and F−/CN− and as a colorimetric sensor in acetonitrile to recognize F−/CN−.Therefore, we designed and synthesized a new Schiff base chemosensor bearing a julolidine unit; the sensor was easy to synthesize and could identify both Al2.2.1.2+ (its counter ion was chloride). All anion solutions were prepared from the dissolution of the corresponding potassium or sodium salts (0.1 M) in distilled water (2.0 mL). NMR spectra were recorded in a Bruker AV400 (400 MHz) spectrometer with DMSO-d6 as solvent and tetramethylsilane (TMS) as internal standard. UV-vis spectra were measured on an Agilent 8453 UV-vis spectrophotometer. Fluorescence spectra were recorded with a Hitachi F-4600 fluorescence spectrophotometer. Fluorescence quantum yield was measured with an Absolute PL Quantum Yield Spectrometer QY C11347-11. Melting points were obtained on a WRS-1B melting point apparatus. High resolution mass spectra were recorded on an AB SCIEX Triple TOF 4600 instrument. Elemental analysis was performed with a PE CHN 2400 analyzer. Fluorescent cell imaging was obtained on an Olympus FV1000 confocal laser scanning microscope.All the solvents were of analytical grade and were not purified before use. Other reagents were of spectroscopic grade. Metal ion solutions were obtained by dissolving the respective nitrates (0.1 M) in distilled water (2.0 mL) except for Hg2.2.1H NMR , δ (ppm): 11.80 , 11.63 , 8.27 , 7.84–7.83 , 7.21–7.19 , 6.72 , 3.14–3.18 , 2.61–2.49 , 1.84 . 13C NMR : 157.54, 155.27, 151.43, 145.94, 138.63, 132.15, 129.27, 128.99, 128.81, 113.14, 106.96, 106.41, 49.98, 49.52, 27.20, 22.16, 21.34, 20.86. Anal. calcd for C18H19N3O2S (%): C, 63.32; H, 5.61; N, 12.31. Found: C, 62.24; H, 5.63; N, 12.34. HRMS: m/z = 364.1096 [M + Na+]+ .  and 2-thiophenecarboxylic hydrazide  were added to ethanol (10 mL) and stirred for 10 min at room temperature. The mixture was heated to reflux for 24 h and then cooled to room temperature; the light yellow precipitate was washed three times with cold ethanol and dried under vacuum to give product HL (0.08 g). Yield 47.06%. Mp 492–493 K. 2.3.2 and 95% air at 37 °C. Two mg HL was dissolved in DMSO (40 μL) and diluted with DMEM to obtain 5 different concentrations . Different HL solutions were added to the 96-well plate in a low to high concentration sequence except for that in the control well. Cells were incubated for 24 h to test cytotoxicity. After 24 hours incubation, 20 μL  (MTT) (5 mg mL−1) was added to each well with continuous incubation for another 3.5 h. After MTT was removed, DMSO was added until the compounds were completely dissolved. The absorbance values were measured using a microplate reader. The absorbance of the solvent control cells was considered to be 100%; cell viability and the absorbance of the treated cells were used to calculate the cell viability at different concentrations of HL solution.The HeLa cells were added to a 96-well plate. About 4000 cells per well were placed in a carbon dioxide cell incubator in an atmosphere of 5% CO2.4.2 and 95% air at 37 °C. The cells were plated in a cell culture dish overnight. Before the experiments, the cells were washed with phosphate-buffered saline (PBS) buffer. Then, the cells were incubated with HL (20 μM) dissolved in DMEM medium for 30 min at 37 °C. Next, the cells were observed using an Olympus FV1000 confocal laser scanning microscope. Finally, fluorescence images were obtained after incubating the cells with Al3+ (50 μM) for another 30 min at 37 °C.The human cervical HeLa cancer cells were incubated in DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% FBS  and 1% penicillin–streptomycin in the atmosphere of 5% CO3.3.1.3+ were preliminarily tested in acetonitrile (2.0 × 10−5 mol L−1) by a UV-vis spectrometer. Upon addition of Al3+ into the solution of HL, a new absorption band appeared, and it was centered at 420 nm and 440 nm. As the concentration of Al3+ increased, the absorbance at 420 nm and 440 nm gradually increased, whereas the absorption peak at 384 nm declined slowly. The color of the solution changed from colorless to kelly green . As shown in Φ = 0.008) at 420 nm excitation due to CN isomerization and photoinduced electron transfer (PET) processes.41,42 The fluorescence emission intensity of HL at 521 nm enhanced gradually along with the increase in Al3+ concentration until the amount of Al3+ reached 5.0 equivalents with a high quantum yield (Φ = 0.464). Meanwhile, the fluorescence color of HL changed from very faint orange to strong cyan. Compared with the result of HL, the fluorescence intensity of complex HL–Al3+ was dramatically enhanced. The phenomenon of fluorescence enhancement was ascribed to the inhibition of CN isomerization and PET processes.To further research the interaction between HL and Al3+, the stoichiometry of HL to Al3+ was confirmed based on the Job's plot of the fluorescence spectrum (λem = 521 nm). When the molar fraction of (Al3+)/[(HL–Al3+)] was 0.3, the fluorescence emission intensity value reached its maximum  of HL–Al3+ was calculated to be 6.35 × 104 M−1 (R = 0.996)  was measured to be 20.5 nM according to the equation LOD = 3σ/s, where σ is the standard deviation of the blank signal, and s is the slope of the linear calibration plot  effect.50–52  Fig. S6 from theion plot . Compare− Fig. S7 confirmect.50–52 .−5 mol L−1), and the ions included Cu2+, Zn2+, Cr3+, Al3+, Co2+, Mn2+, Pb2+, Cd2+, Ca2+, K+, Fe3+, Mg2+, Sr2+, Ba2+, Ag+ and Ni2+  were obtained in acetonitrile  at room temperature (−/CN− solution of HL resulted in the appearance of two new absorption peaks centered at 439 nm and 467 nm (− in acetonitrile (2.0 × 10−5 mol L−1) is shown in − increased from 0 to 5.0 equivalents, the absorption peaks at 439 nm and 467 nm gradually increased and reached a maximum at 5.0 equivalents of F−, whereas the absorption peak at 382 nm decreased when the color of the solution changed to light yellow. Moreover, an isosbestic point was observed at 400 nm, indicating that HL–F− complex was generated. The changes in absorption spectra could be due to intramolecular charge transfer (ICT) mechanism via the deprotonation effect. The increased negative charge density of the phenol oxygen atom accelerated ICT from O atom (Ar–OH) to aromatic rings.53,54The selective experiment of HL to anions was investigated first by UV-vis absorption spectra. Miscellaneous anions including Fperature . Upon add 467 nm . At the d 467 nm . Other a− was added to the solution of HL; the absorption spectra presented analogous changes to that observed with the addition of F−  mentioned above, various anions were also explored in acetonitrile, as depicted in − (5.0 equiv.) to HL, the fluorescence emission intensity enhanced nearly 29-folds at 540 nm (λ = 420 nm). On the contrary, there was almost no clear fluorescence signal when other anions were added , as shown in − (0–5.0 equiv.), the fluorescence emission intensity at 540 nm increased significantly and reached the maximum (Φ = 0.021) when the amount of F− reached 5.0 equivalents. Upon continuous addition of F− to the HL solution, the fluorescence spectra hardly changed. The fluorescence spectra of HL–CN− were similar to those of HL–F−  for HL–F− and HL–CN− were obtained as 1.25 × 104 M−1 (R = 0.995)   completely disappeared. The proton H2 (–N–H) shifted from 11.65 ppm to 11.70 ppm, and the peak intensity decreased  for 30 min at 37 °C; almost no fluorescence was observed (3+ (50 μM) to the above HeLa cells triggered a striking fluorescence enhancement and resulted in green fluorescence (3+.Next, to examine the biological applicability of HL to detect Alobserved . Neverthrescence . The res4.3+ and F−/CN−. HL exhibited excellent colorimetric selectivity to F−/CN− and highly specific fluorescence responses to Al3+ and F−/CN−. Upon addition of Al3+ or F−/CN−, HL showed clear fluorescence increase with different emission spectra. The results demonstrated that the compound HL could be used as a fluorescent chemosensor to detect Al3+ and F−/CN−. Besides, it could also serve as a colorimetric chemosensor to recognize F−/CN−via a clear color change from colorless to visible light yellow. Furthermore, the relatively low cytotoxicity of HL could be successfully used for cell imaging to identify Al3+, indicating its promising application prospects in living cells.In summary, we have designed and developed a new multiple-ion-responsive Schiff base chemosensor HL, which can simultaneously detect AlThere are no conflicts to declare.RA-008-C8RA05439H-s001


Schistosoma, affects over 230 million people, especially in developing countries. Despite the significant economic and public health consequences, only one drug is currently available for treatment of schistosomiasis, praziquantel. Thus, there is an urgent demand for new anthelmintic agents. Based on our continuous studies involving the chemical prospection of floristic biodiversity aiming to discover new bioactive compounds, this work reports the in vitro antiparasitic activity against Schistosoma mansoni adult worms of neolignans threo-austrobailignan-6 and verrucosin, both isolated from Saururus cernuus L. (Saururaceae). These neolignans showed a significant in vitro schistosomicidal activity, with EC50 values of 12.6–28.1 µM. Further analysis revealed a pronounced reduction in the number of S. mansoni eggs. Scanning electron microscopy analysis revealed morphological alterations when schistosomes were exposed to either threo-austrobailignan-6 or verrucosin. These relevant antischistosomal properties were accompanied by low cytotoxicity potential against the animal (Vero) and human (HaCaT) cell lines, resulting in a high selectivity index. Considering the promising chemical and biological properties of threo-austrobailignan-6 and verrucosin, this research should be of interest to those in the area of neglected diseases and in particular antischistosomal drug discovery.Schistosomiasis, a parasitic disease caused by the blood fluke of the genus  Schistosoma, is a debilitating disease with a tremendous global burden. Estimates show that over 230 million people are affected with schistosomiasis in 78 countries and approximately 10% of the world population is at risk for infection1. Schistosoma mansoni, one of the three major human species, occurs across much of Africa, the Middle East, the Caribbean, and South America. Morbidity due to schistosomiasis mansoni includes hepatosplenomegaly, liver fibrosis, and ascites; in severe cases, S. mansoni infection can be fatal2. Consequently, it is a disease of immense medical importance.Schistosomiasis, a poverty-associated parasitic disease caused by blood fluke of the genus 4. The Word Health Organization (WHO) strategy for schistosomiasis control focuses on large-scale treatment (preventive chemotherapy) of affected populations, a strategy that might select for drug-resistant parasites. In addition, numerous persistent schistosomiasis hotspots remain6, and low cure rates have been reported8. Systematic reviews and meta-analyses show that praziquantel achieves a cure rate of approximately 75% for S. mansoni infections10, demonstrating the limitations of praziquantel. With the aim to eliminate human schistosomiasis as a public health problem by 2030, in their new road map for neglected tropical diseases 2021–2030, the WHO highlights the need for new therapeutic interventions11. Thus, the pressing need to develop new anthelmintic compounds has been emphasized and the drug discovery landscape has gained momentum over the past few years14.The control of schistosomiasis is mainly dependent on the use of praziquantel, the only readily commercially available drugSaururus cernuus L.  is a type of freshwater plant widely distributed in America, including Brazil15. In folk medicine, this plant has been used as an anti-inflammatory and as a sedative16. Phytochemically, S. cernuus produces mainly lignoids but the occurrence of other compounds such as alkaloids and terpenoids was also reported19. Studies describing antiprotozoal effects of some lignoids isolated from S. cernuus were previously described, including threo,threo- and threo,erythro-manassantin A with activity against amastigote forms of Leishmania amazonensis20 and threo-austrobailignan-5, threo-austrobailignan-6, and threo-dihydroguaiaretic acid with activity against trypomastigote and amastigote forms of Trypanosoma cruzi21. Furthermore, alterations in the plasma membrane permeability, in reactive oxygen species (ROS) levels, and mitochondrial membrane potential caused by these compounds in tested parasites were observed. More recently, the molecular dereplication and the evaluation of anti-T. cruzi activity of volatile oils from inflorescences, leaves, branches, and roots of S. cernuus was reported. As described, the predominance of mono and sesquiterpenes was observed, as well as phenylpropanoids as the main compounds. Interestingly, oils from leaves, branches, inflorescences, and roots displayed activity against trypomastigotes, with reduced toxicity against NCTC cells22. This selectivity makes these compounds attractive as antiparasitic agents.25, this study reports the isolation of two neolignans from leaves of S. cernuus, of which one is dibenzylbutane—threo-austrobailignan-6—and one tetrahydrofuran—verrucosin—and their evaluation of anthelmintic properties against S. mansoni was performed, since these compounds exhibited activity against other parasites such as T. cruzi and L. infantum.As part of our continuous study aiming to discover new antischistosomal agents from plants of Brazilian biodiversitythreo-austrobailignan-6 were identical to those reported in the literature26. As observed, the 1H NMR spectrum indicated the presence of two 1,3,4-trissubtituted aromatic rings due the signals at δ 6.67 , 6.70 , 6.60 , 6.80 , and 6.57–6.53 . The presence of four double-doublets at δ 2.39 (J = 13.5 and 8.0 Hz), 2.57 (J = 13.5 and 6.5 Hz), 2.33 (J = 13.5 and 8.0 Hz), and 2.56 (J = 13.5 and 6.5 Hz), assigned to H-7a, H-7b, H-7′a and H-7′b, respectively, in association with two doublets at δ 0.86  and 0.81 , indicated the occurrence of a dibenzylbutane neolignan21. Two singlets at δ 5.93 (2H) and 3.84 (3H) indicated the presence of methylenedioxy and methoxy groups, respectively, in the aromatic rings. 13C NMR spectrum of shown signals referring to aromatic carbons from δ 107.9 to 147.3 (C-1 to C-6 and C-1′ to C-6′), two methylene carbons at δ 41.1 (C-7) and 41.0 (C-7′), two methine carbons at δ 37.9 (C-8) and 37.8 (C-8′), and two methyl groups at δ 13.9 (C-9) and 13.8 (C-9′). Furthermore, signals at δ 100.9 and 55.8 were assigned to methylenedioxy and methoxy groups, respectively. Finally, based on the chemical shifts of C-8/C-8′ and C-9/C-9′27, it was possible to infer that methyl groups at C-8 and C-8′ are positioned at trans configuration , two doublets of oxymethine hydrogens at δ 5.03  and 4.33  and two doublets of methyl groups at δ 0.98  and 0.59 , indicated the occurrence of a tetrahydrofuran neolignan29. Additional singlets at δ 5.54 (4-OH), 5.49 (4′-OH), and 3.84 (3- and 3′-OMe) inferred the presence of two ortho methoxy-phenol moieties. 13C NMR spectrum showed signals referring to aromatic carbons from δ 109.4 to 146.5 (C-1 to C-6 and C-1′ to C-6′), two oxymethine carbons at δ 87.3 (C-7) and 83.1 (C-7′), two methine carbons at δ 47.8 (C-8) and 46.0 (C-8′), and two methyl groups at δ 14.5 (C-9) and 14.9 (C-9′). Furthermore, one intense signal at δ 55.9 was attributed to methoxyl groups at C-3 and C-3′. Based on the chemical shifts of C-7/C-7′, C-8/C-8´and C-9/C-9′30, it was possible to infer the stereochemistry trans,trans,cis to the substituents in the tetrahydrofuran groups  were exposed to different concentrations for their effective concentration 50% (EC50) determination . A significant reduction in oviposition was also recorded with verrucosin at 12.5 μM (P < 0.01).The ability of tested tetrahydrofuran lignans to affect 49. Similar to these studies, threo-austrobailignan-6 and verrucosin, which also had a schistosomicidal effect, caused a reduction in S. mansoni egg production. The interference in oviposition may be associated with the separation of parasite couples or by changes in the reproductive system of S. mansoni49.Considering that egg production is an essential mechanism for both transmission and pathogenesis, the negative impact on schistosomes egg-laying is of particular interest for molecules with anthelmintic activities. Indeed, the effects of antischistosomal agents on egg output during in vitro incubation have been previously reported in several studies50, we used scanning electron microscopy to examine the surface of S. mansoni adult parasite exposed to threo-austrobailignan-6 or verrucosin. The two neolignans tested at lethal concentrations caused morphological changes in the tegumental surfaces of male and female schistosomes. As shown in Fig. threo-austrobailignan-6 at 50 µM exhibited severe tegumental alterations such as swelling, sloughing, and shortening of the tubercles . The authors reported that lignans caused serious lesions on the surface of parasites that led to death. Unlike nematodes, which are protected by a cuticle, schistosomes are covered by a living syncytium, called the tegument. Data from the present study clearly show that threo-austrobailignan-6 and verrucosin led to a pronounced change in the tegument of S. mansoni. Morphological alterations may not always result in the death of a parasite55, but the tegumental damage observed in this study could be a mechanism through which neolignans kill the schistosomes. From the point of view of an in vivo study, in addition to the direct effect on the survival of the schistosomes, tegumental alterations might result in exposure of the worm antigens to the host’s immune system56. Furthermore, considering that differential appearance of morphological changes was noted, it will be interesting to see the effect when the two drugs are combined in future experiments.Morphological alterations induced by several antischistosomal natural compounds on the tegument of threo-austrobailignan-6 and verrucosin have a significant antischistosomal effect and the morphological alterations could be a mechanism through which these neolignans kill the helminths. However, the exact mechanism is unclear and needs further investigation. In T. cruzi, Brito and coworkers21 reported that dibenzylbutane neolignans act on the plasma membrane of the parasite and induce alterations in the mitochondrial membrane potential. Moreover, because the redox system is responsible for the survival of many parasites, the authors demonstrated that neolignans cause alterations in the ROS production. Studies have reported that some antischistosomal compounds, such as licochalcone A, elicit mitochondrial and cellular membrane alterations in S. mansoni57. In addition, a wide range of natural compounds are known to generate ROS in S. mansoni, and the induction of oxidative stress has been considered an attractive treatment strategy for schistosomiasis drug discovery59. Accordingly, the possibility of threo-austrobailignan-6 and verrucosin causing disturbance in both ROS and the activities of enzymes associated with maintaining redox balance cannot be excluded.Taken together, this finding indicates that both 50). The selectivity index (SI) was calculated as the ratio between the CC50 values for the human cells and the EC50 values for S. mansoni. As shown in Table threo-austrobailignan-6 and verrucosin, respectively. In line with this study, the low cytotoxic profile of neolignans from S. cernuus had been reported21. The SI of a compound is a widely accepted criterion used to express a drug’s in vitro efficacy61. Since the WHO establishes a SI value ≥ 10 for the selection of candidate anthelminthic compounds62, this study demonstrated that threo-austrobailignan-6 and verrucosin are highly selective antischistosomal agents.Isolated neolignans were incubated with two mammalian cell lines, namely the African green monkey kidney epithelial cells (Vero) and human epithelial cells (HaCaT) to evaluate their in vitro cytotoxic concentration 50% , operating, respectively, at 300 and 75 MHz. As a solvent and internal standard were used CDCl3 (Aldrich) and TMS (Aldrich). Chemical shifts are reported in δ units (ppm) and coupling constants (J) in Hz. ESI-HRMS spectra were measured on a Bruker Daltonics MicroTOF QII spectrometer. Silica gel 60  and Sephadex LH-20 (GE) were used for column chromatographic separation procedures whereas silica gel 60 PF254 (Merck) was used for analytical (0.25 mm) and preparative (1.00 mm) thin-layer chromatography (TLC).S. cernuus leaves were obtained from a local producer of ornamental plants in the city of Suzano, São Paulo State, Brazil in August/2019 and, after collection, received a registration code A4123E4 at SISGEN . Botanical identification was performed by Dr. Fátima Otavina de Souza Buturi, from University of São Judas Tadeu, and Dr. Oriana Aparecida Fávero, from Mackenzie Presbyterian University. A voucher specimen (voucher number E. A. Ferreira—001) has been deposited at the SPF Herbarium of the Institute of Biosciences at the University of São Paulo.The use of plant parts in the present study complies with the international, national, and institutional guidelines. S. cernuus (315 g) were powdered and exhaustively extracted using MeOH at room temperature to afford 91 g of crude extract after solvent evaporation at reduced pressure. This extract was resuspended in MeOH:H2O 1:1 and partitioned using hexane to afford 18.6 g of hexane phase. Part of this material (9.0 g) was subjected to column chromatography over silica gel eluted with n-hexane containing increasing amounts of EtOAc to afford fourteen fractions (A–N). After an initial screening, fractions C and G displayed activity against S. mansoni and were selected for further purification procedures. Therefore, fraction C (671 mg) was subjected to column chromatography over silica gel eluted with n-hexane containing increasing amounts of EtOAc to afford six fractions (C1–C6). Fraction C3 (232 mg) was chromatographed over a silica gel column eluted with n-hexane:EtOAc 3:2 to afford the fraction C3-4 (25 mg) which was purified by prep. TLC . This procedure gave threo-austrobailignan-6 (13 mg)21. Part of fraction G (760 mg) was chromatographed over Sephadex LH-20 eluted with n-hexane:CH2Cl2 1:4 and CH2Cl2:acetone 3:2 to afford pure verrucosin (335 mg).Dried leaves of -austrobailignan-6. White amorphous solid. 1H NMR (CDCl3), δ /ppm 6.80 , 6.70 , 6.67 , 6.60 , 6.57–6.53 , 5.93 , 3.85 , 2.57 , 2.56 , 2.39 , 2.33 , 1.76–1.67 , 0.86 , 0.81 . 13C NMR (CDCl3), δ /ppm 147.3 (C-3), 146.3 (C-3′), 145.4 (C-4), 143.5 (C-4′), 135.5 (C-1), 133.5 (C-1′), 121.8 (C-6), 121.6 (C-6′), 114.0 (C-5′), 111.3 (C-2′), 109.3 (C-2), 107.9 (C-5), 100.7 (OCH2O), 55.8 (OCH3), 41.1 (C-7), 41.0 (C-7′), 37.9 (C-8), 37.8 (C-8′), 13.9 (C-9), 13.8 (C-9′). ESI-HRMS m/z 327.1594 [M—H]− .ThreoVerrucosin. White amorphous solid. 1H NMR (CDCl3), δ /ppm 6.97–6.76 , 5.03 , 5.54 (4-OH), 5.49 (4′-OH),4.33 , 3.84 , 3.78 , 2.20–2.11 , 1.75–1.64 , 0.98 , 0.59 . 13C NMR (CDCl3), δ /ppm 146.5 (C-3), 146.2 (C-3′), 145.2 (C-4), 144.6 (C-4′), 133.2 (C-1), 132.8 (C-1′), 119.9 (C-6′), 119.3 (C-6), 114.2 (C-5), 113.9 (C-5′), 109.7 (C-2′), 109.4 (C-2), 87.3 (C-7), 83.1 (C-7′), 55.9 (3-/3′-OMe), 47.8 (C-8), 46.0 (C-8′), 14.9 (C-9′), 14.5 (C-9). ESI-HRMS m/z 345.1798 [M + H]+ and 367.1518 [M + Na]+ .All the structural data collected are presented in the Supplementary Information Fig. .Dulbecco’s Modified Eagle Medium (DMEM) modified to contain 4 mM of L-glutamine, 4500 mg/L glucose, and 1 mM sodium pyruvate, Roswell Park Memorial Institute (RPMI) 1640 medium, trypsin/EDTA solutions, heat-inactivated fetal calf serum, and penicillin G-streptomycin solutions  were obtained from Vitrocell . HEPES buffer, thiazolyl blue tetrazolium bromide (MTT), and dimethyl sulfoxide (DMSO) were purchased from Sigma . Praziquantel was kindly provided by Ecovet Indústria Veterinária Ltda . In all in vitro experiments, compounds were solubilized in DMSO.S. mansoni (BH strain) is maintained by routine passage through Biomphalaria glabrata snails and Swiss mice at Guarulhos University . Rodents, three weeks-old, were purchased from Animais de Laboratório Criação e Comércio . Both mice and snails were kept under environmentally controlled conditions , with free access to food and water. Cercariae of S. mansoni were obtained from infected intermediate host snails in our laboratories as described previously64.The life cycle of 2. They were maintained in 25 cm2 culture flasks  and harvested using 0.25% trypsin in 0.2 g/L EDTA solution.HaCaT  and Vero (monkey kidney cells) were obtained from the Banco de Células do Rio de Janeiro  and the American Type Culture Collection , respectively. Cells were cultured in DMEM medium supplemented with 10% heat-inactivated fetal bovine serum and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin) at 37 °C in a humidified atmosphere containing 5% CO66. Briefly, S. mansoni adult parasites were obtained from infected mice at day 42 post-infection (parasite ex vivo). One adult male and one adult female worms (i.e. one worm pairs) were incubated in flat bottom 24-well plates  in the presence of threo-austrobailignan-6 and verrucosin (started at 50 µM and followed a twofold dilution series) in RPMI 1640 culture medium supplemented with 5% inactivated fetal calf serum and containing antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin). The control schistosomes were assayed in a culture medium and 0.5% DMSO (representing the highest concentration of solvent). Each concentration  was tested in five replicates, and the experiments were repeated three times. Parasites were kept for 72 h , and their viability was monitored microscopically at 2, 24, 48, and 72 h using both a Motic AE2000 inverted microscope  and a Leica EZ4E stereomicroscope . The death of adult schistosomes was defined as no movement observed for at least 1 to 2 min of examination, whereas parasites with any body movement were considered viable67. The percentage of viable adult schistosomes was calculated considering worms exposed to compounds vs control worms. For assessment of the reproductive fitness of S. mansoni, the number of eggs was counted daily using an inverted microscope (Motic) as previously described68.The antischistosomal assay was performed as previously described70. Briefly, adult worms (treated and control groups) were fixed in 2.5% glutaraldehyde, and mounted specimens were coated with gold sputter  and photographed using a high-resolution scanning electron microscope .Scanning electron microscopy studies were performed as previously described72. Briefly, cells were seeded (2 × 103/well in 96-well culture plates) and incubated with threo-austrobailignan-6 and verrucosin (started at 500 µM and followed a threefold dilution series) for 72 h at 37 °C and 5% CO2. MTT solution was added to each well and the absorbance was read on an Epoch Microplate Spectrophotometer  at 595 nm. The selectivity indices (SI) were calculated by dividing the 50% cytotoxic concentration (CC50) obtained on cells with 50% effective concentration (EC50) values determined on schistosomes73.The MTT assay was used to evaluate the cytotoxic activity as previously described50 and, CC50 values were calculated using sigmoid dose–response curves74. For S. mansoni egg production, significant differences were determined by one-way analysis of variance (ANOVA) and applying Tukey's test for multiple comparisons with a level of significance set at P < 0.05.Statistical analyses were performed using Graph Pad Prism software 8.0 . Data are presented as the mean ± standard deviation (SD) of at least three independent experiments, ECAnimal studies are reported in compliance with the ARRIVE guidelines. The protocol for experimental design was reviewed and approved by the Committee for the Ethical Use of Animals in Experimentation of the Guarulhos University  in conformity with the Brazilian law for Guidelines for Care and Use of Laboratory Animals.Supplementary Information.