Sargassum fusiforme, containing the preferential LXRβ-agonist 24(S)-saringosterol, prevented memory decline and reduced amyloid-β (Aβ) deposition in an Alzheimer’s disease (AD) mouse model without inducing hepatic steatosis. Here, we examined the effects of 24(S)-saringosterol as a food additive on cognition and neuropathology in AD mice. Six-month-old male APPswePS1ΔE9 mice and wildtype C57BL/6J littermates received 24(S)-saringosterol (0.5 mg/25 g body weight/day)  or vehicle  for 10 weeks. Cognition was assessed using object recognition and object location tasks. Sterols were analyzed by gas chromatography/mass spectrometry, Aβ and inflammatory markers by immunohistochemistry, and gene expression by quantitative real-time PCR. Hepatic lipids were quantified after Oil-Red-O staining. Administration of 24(S)-saringosterol prevented cognitive decline in APPswePS1ΔE9 mice without affecting the Aβ plaque load. Moreover, 24(S)-saringosterol prevented the increase in the inflammatory marker Iba1 in the cortex of APPswePS1ΔE9 mice (p < 0.001). Furthermore, 24(S)-saringosterol did not affect the expression of lipid metabolism-related LXR-response genes in the hippocampus nor the hepatic neutral lipid content. Thus, administration of 24(S)-saringosterol prevented cognitive decline in APPswePS1ΔE9 mice independent of effects on Aβ load and without adverse effects on liver fat content. The anti-inflammatory effects of 24(S)-saringosterol may contribute to the prevention of cognitive decline.We recently found that dietary supplementation with the seaweed  APOE gene. APOE encodes the cholesterol carrier apolipoprotein (Apo) E, the main extracellular lipid transporter in the central nervous system [Alzheimer’s disease (AD) is the most prevalent form of dementia in the elderly. This neurodegenerative disorder is characterized by a progressive cognitive decline, accumulation of amyloid-β (Aβ), formation of neurofibrillary tangles, neuroinflammation, and loss of neurons and synapses . Althougs system ,9. Moreos system .Liver X receptors (LXRα and -β) are oxysterol-activated nuclear receptors that not only act as master regulators of cellular cholesterol and lipid metabolism but also control inflammatory processes ,12, makiSargassum fusiforme polysaccharide 65 [Sargassum fusiforme, as well as purified 24(S)-saringosterol, preferentially activate LXRβ [Sargassum fusiforme, as well as its lipid extracts, can prevent cognitive impairment and reduce Aβ plaque load in an AD mouse model without causing the adverse effects induced by pan synthetic LXR agonists [Sargassum fusiforme on cognition and neuropathology in APPswePS1ΔE9 mice. We show that the phytosterol 24(S)-saringosterol prevented cognitive decline in APPswePS1ΔE9 mice, despite having no effect on Aβ plaque load. Our data point to the immunomodulatory effects of 24(S)-saringosterol treatment contributing to the observed neuroprotective properties.By virtue of their distinct nutritional composition, various health benefits have been attributed to seaweeds ,23. Theyaride 65 , fucostearide 65 ,29,30, faride 65 ,33,34,35aride 65 . We prevate LXRβ  and therate LXRβ . We demoagonists . Moreoveagonists . Here wep = 0.787 and p = 0.807 for OLT and ORT, respectively), while memory was intact in APPswePS1ΔE9 mice that received 24(S)-saringosterol (p < 0.01 and p < 0.01) as well as in WT mice that received the vehicle control (p < 0.001 and p < 0.001) or 24(S)-saringosterol (p < 0.01 and p < 0.05) (To test the effect of 10-week administration of 24(S)-saringosterol on cognition of WT and APPswePS1ΔE9 mice, the mice were subjected to an object location task (OLT) and an object recognition task (ORT). Cognitive decline in APPswePS1ΔE9 mice was significantly prevented by daily administration of 24(S)-saringosterol, as shown by enhanced spatial and object memory as assessed with OLT and ORT a,b. Vehi < 0.05) a,b.p < 0.001) and in the cerebellum from 0.3 ± 0.04 to 4.0 ± 0.9 ng/mg dry weight  = 576.110, p < 0.001) (Administration of 24(S)-saringosterol increased its concentrations in serum from 11.4 ± 0.5 to 38.5 ± 12.0 µg/dL  = 176.418, < 0.001) a,c. The p < 0.001) and in the cerebellum  = 4.851, p < 0.05) (p = 0.001), campesterol  = 19.236, p < 0.001), stigmasterol  = 17.951, p < 0.001), and brassicasterol  = 15.026, p < 0.001) also decreased (p = 0.050), but not in WT mice -saringosterol treatment resulted in decreased concentrations of fucosterol in the circulation  = 19.535,  < 0.05) b,d. Seruecreased b. With eecreased d. The co WT mice e–j.p = 0.963) and in the hippocampus (p = 0.450) of APPswePS1ΔE9 mice   d–g.p = 0.001 and F = 12.626, p < 0.01, respectively), and decreased upon 24(S)-saringosterol administration  (p < 0.05) (p < 0.05) disappeared upon 24(S)-saringosterol administration (p = 0.718). There were no differences in CD68 levels in the cortex of WT and APPswePS1ΔE9 mice  = 0.969, p = 0.339), and no effects of 24(S)-saringosterol treatment  = 0.109, p = 0.746) -saringosterol, we investigated the impact of 24(S)-saringosterol administration on the microglia/macrophage markers ionized calcium-binding adapter molecule 1 (Iba1) and cluster of differentiation 68 (CD68). Compared to WT mice, Iba1 levels, as determined by the surface area%, were higher in the cortex and hippocampus of APPswePS1ΔE9 mice  = 17.777, ctively) a,b. In l < 0.05) c. The di= 0.746) d.ABCA1 -saringosterol than by T0901317. However, no effect of 24(S)-saringosterol administration could be detected on the expression of Abca1, Abcg1, Apoe, Scd1, or Srebf1 in the hippocampus of WT or APPswePS1ΔE9 mice (p > 0.05) (We assessed the effect of 24(S)-saringosterol on the expression of lipid metabolism-related LXR target genes in CCF-STTG1 cells  and in tABCA1 a, ABCG1 1  .Abcg8 in the liver  = 8.533, p < 0.01)  = 4.234, p < 0.05)—, Abca1, and Apoe was not significantly increased -saringosterol increased the expression of  < 0.01) a, while ncreased b–d. The affected e–h. Admiaffected i,j. Becap = 0.683 and p = 0.562, respectively) -saringosterol administration, although the expression of Fasn did increase (t(8) = −2.64, p < 0.05)  . The exp < 0.05) .In this study, we examined whether purified 24(S)-saringosterol can preserve cognition and prevent the development of neuropathology in a mouse model for AD. Our data show that 10 weeks of administration with the semi-synthetic preferential LXRβ activating phytosterol 24(S)-saringosterol prevented cognitive decline in APPswePS1ΔE9 mice, without affecting the Aβ plaque load. Concentrations of 24(S)-saringosterol in the brain were significantly increased upon 24(S)-saringosterol administration, and microglial activation in the brain of APPswePS1ΔE9 mice was found to be reduced.Sargassum fusiforme or its lipid extract [42 release and promotes microglial Aβ clearance in vitro [Sargassum fusiforme, either in crude form or as a lipid extract, did decrease the Aβ plaque load in APPswePS1ΔE9 mice [Sargassum fusiforme including the phytosterols β-sitosterol [The prevention of the cognitive decline in APPswePS1ΔE9 mice upon 24(S)-saringosterol administration is in accordance with our previously reported data demonstrating neuroprotective effects of T0901317 and 24(S)-saringosterol-containing  extract ,21. Althin vitro . Howevertosterol ,38 and stosterol ,40, the tosterol ,31,32, aABCA1, ABCG1, and APOE. Chen et al. (2014) obtained similar results after saringosterol administration to HEK293T, HepG2, THP-1 monocytes, and RAW264.7 cells [in vivo may be caused by the chronic nature of the 24(S)-saringosterol administration leading to a new balance in gene expression levels or the difference in 24(S)-saringosterol concentration in vivo (9 × 10−4 mM in serum) compared to in vitro (1.25–7.5 mM). Previously, we observed a limited increase in expression of LXR target genes in the brains of WT and APPswePS1ΔE9 mice upon administration of Sargassum fusiforme containing a similar concentration of 24(S)-saringosterol [Sargassum fusiforme contains additional LXR agonists, including fucosterol, that may contribute to the gene expression profile resulting from Sargassum fusiforme supplementation. Therefore, it remains to be established via which biological pathways, involving LXR activation or not, 24(S)-saringosterol exerts its neuroprotective effects in AD mice.Although 24(S)-saringosterol concentrations were significantly increased in serum and in the brain of the mice after its administration, this did not affect the concentrations of cholesterol, its precursors, or metabolites. Phytosterol concentrations in serum were reduced, possibly as a result of competition for incorporation in micelles and the subsequent intestinal absorption or an inhibitory effect of 24(S)-saringosterol on intestinal sterol absorption. An alternative explanation is an enhancing effect of 24(S)-saringosterol on sterol excretion via LXR activation and upregulation of Abcg5/8 in the liver and the intestine ,42. The .7 cells . The absgosterol . Sargassin vitro and not in vivo [Activation of LXR is known to transrepress inflammatory pathways in immune cells in the central nervous system through SUMOylation . Because in vivo . TherefoSargassum fusiforme, either crude or as an extract, in the prevention and retardation of AD-related symptoms.Our observation that administration of 24(S)-saringosterol as a preferential LXRβ activator does not induce hepatic steatosis is in line with the assumption that triglyceride accumulation in the liver is predominantly driven by LXRα activation . Further42 exposure-induced cognitive decline in aging rats [An advantage to the use of crude seaweed or a seaweed extract over pure 24(S)-saringosterol could be the presence of constituents with potential additional or synergistic effects. Seaweed constituents other than 24(S)-saringosterol have been reported to display beneficial effects in AD models ,49. Howeing rats . The caring rats  and by ping rats ,31,32,33ing rats . The oliing rats . Furthering rats . Becauseing rats ,51,52. TIn conclusion, we showed that semi-synthetic purified 24(S)-saringosterol, which is bioavailable in the central nervous system, prevents cognitive decline in a well-established AD mouse model, despite having no effect on Aβ plaque load, or any detectable effects on lipid or cholesterol homeostasis. Our data point to immunomodulatory effects of 24(S)-saringosterol contributing to its neuroprotective properties, but detailed mechanisms remain to be elucidated. Moreover, 24(S)-saringosterol can be regarded as a promising agent for the prevention of deterioration of AD-related symptoms.Prior to this experiment, two routes of 24(S)-saringosterol administration were compared: oral gavage and subcutaneous injection. Six C57BL6/J mice received an oral gavage and six C57BL6/J mice received a subcutaneous injection containing 0.5 mg 24(S)-saringosterol—semi-synthesized from kelp-derived fucosterol— per 25 g body weight twice-daily for three consecutive days, whereafter sterol concentrations in serum, brain, and other tissues were determined by gas chromatography/mass spectrometry (GC/MS) as described previously ,53 Supp. Oral gan = 20, C57BL6/J littermates: n = 19) in 200 μL of vehicle (0.5% methylcellulose containing 1% ethanol and 2% Tween) per 25 g body weight or vehicle only  for 10 consecutive weeks. The body weight of the mice (41.4 ± 6.0 g) was stable over the treatment period with no effect of diet  = 0.689, p = 0.412) or genotype  = 2.373, p = 0.132). To test for potential adverse effects of saringosterol administration on lipid metabolism, a second batch of C57BL6/J mice  received a daily oral gavage containing 0.5 mg saringosterol /24(R)-saringosterol of 1:1)—semi-synthesized from commercially available hyodeoxycholic acid—in 200 μL vehicle (0.5% methylcellulose containing 1% ethanol and 2% Tween) per 25 g body weight (n = 7) or vehicle only (n = 7) for 3 weeks from the age of 12 weeks.Male APPswePS1ΔE9 (AD) and wildtype C57BL6/J (WT) littermate mice were obtained by backcrossing male APPswePS1ΔE9 mice  with female C57BL6/J mice . The animals were housed in a conventional animal facility at Hasselt University. Two weeks prior to the start of the behavioral experiments, 5-month old mice were housed individually. Mice were fed ad libitum and kept in an inversed 12/12 h light/dark cycle with behavioral experiments performed during the dark phase of the cycle. The cognitive performances were scored blindly. The body weight of the mice was monitored twice a week. Two series of animal experiments were conducted. The animal procedures were approved by the ethical committee for the animal experiments of Hasselt University and performed in accordance with institutional guidelines (protocol ID201849). From 6 months of age, mice received a daily oral gavage containing 0.5 mg 24(S)-saringosterol  (200 mg per kg body weight) followed by transcardial perfusion with Heparin-phosphate-buffered saline (PBS). Blood was collected via cardiac puncture and was centrifuged for 10 min at 200 g to separate serum, which was stored at −80 °C until use. Brains were isolated and divided into the forebrain (above bregma 0), the cerebellum, and the remaining two hemispheres. The cerebellum was snap-frozen and stored at −80 °C until sterol profiling. The left hemisphere was fixed in formalin and embedded in paraffin for immunohistochemistry. The cortex from the right hemisphere was snap-frozen and cryopreserved for Aβ ELISA analyses. Half of the liver was directly snap-frozen for mRNA expression analyses, the other half was stored at −80 °C in O.C.T. embedding compound  for Oil Red O staining.Sterol profiles in serum and in the cerebellum were determined by GC/MS as described previously ,53. In sTriglyceride and cholesterol concentrations in serum were determined with enzymatic reagent kits according to the manufacturer’s instructions .202/methanol for 10 min, sections were rinsed with TBS/0.3% Triton X-100 and incubated with blocking solution  for 1 h. Thereafter, sections were incubated overnight with the primary antibody in blocking solution (1:8000 Aβ antibody (clone 3D6), 1:1000 Iba1 antibody , or 1:235 CD68 antibody ) at 4 °C. After rinsing with TBS/0.3% Triton X-100, sections were incubated with appropriate biotinylated secondary antibodies for 30 min at room temperature followed by 30 min of incubation with avidin-biotin-complex  and 5 min of incubation with diaminobenzidine . Sections were counterstained with hematoxylin, dehydrated , and covered with a coverslip. Digital images of the sections were obtained using a Leica DRMB microscope  equipped with Leica Applications Suite software . The surface area of the staining was quantified using Fiji ImageJ software by defining the pixel intensity of the staining in the total cortical or hippocampal area.For quantification of Aβ, Iba1 ), and CD68 using immunohistochemistry, the left hemispheres of the brains were fixed in 10% formalin and embedded in paraffin by incubation in increasing concentrations of ethanol (70% (1 h), 80% (1 h), 95% (1 h), 100% (1.5 h)), xylene (1.5 h), and paraffin wax (2 h at 60 °C) using a Thermo Scientific Excelsior ES Tissue Processor . Embedded hemispheres were cut with an HM 340E Electronic Rotary Microtome  to obtain 4 µm sections which were mounted on glass slides, air-dried overnight, and stored at room temperature until use. For Aβ, Iba1, and CD68 staining, sections were deparaffinized (incubation in xylene (10 min) and decreasing concentrations of ethanol (100% (6 min), 96% (3 min), 70% (3 min))), rinsed with TBS/0.3% Triton X-100 and incubated in 10 mM citrate buffer (pH 6.0) for 10 min at 100 °C. After blocking endogenous peroxidases by incubating with 3% Hg, 10 min). The supernatant containing the extracellular soluble Aβ was obtained and stored at −80 °C until use, the pellet was sonicated in TBS containing 2% complete protease inhibitor cocktail (Roche Diagnostics Ltd.) and centrifuged . The supernatant containing intracellular soluble Aβ was obtained and stored at −80 °C until use, the pellet was sonicated in 2% sodium dodecyl sulfate (SDS) in distilled water and centrifuged . The supernatant containing membrane-associated soluble Aβ was obtained and stored at −80 °C until use, the pellet was sonicated in 70% formic acid in distilled water and centrifuged . The supernatant containing insoluble Aβ was obtained and stored at −80 °C until use. In the obtained samples, Aβ40 and Aβ42 levels were quantified using an Aβ40 and Aβ42 ELISA  and related to the total protein content (in the extracellular soluble Aβ fraction) determined with a BCA protein assay kit  according to the manufacturer’s instructions.For quantification of Aβ using ELISA, the cortex of the right hemisphere of the brains of APPswePS1ΔE9 mice was homogenized in TBS/0.1% Triton X-100 containing 2% complete protease inhibitor cocktail  (pH 7.2) and centrifuged  were incubated with 1.25, 2.5, 5.0, or 7.5 µM 24(S)-saringosterol or a negative or positive control ) for 6, 24, or 48 h.Human Caucasian astrocytoma cells (CCF-STTG1)  cultured in DMEM/F-12 medium (Thermo Fisher Scientific) containing 10% heat-inactivated fetal calf serum (FCS)  and 1% 10,000 U penicillin/10,000 μg streptomycin/mL  at 37 °C and 5% COActb, B2m, Hprt1, and Sdha (hippocampus) or Actb (liver)), which were analyzed and selected with geNorm 3.5 and StepOnePlus. Expression levels are indicated by fold change values and compared to the WT mice on the vehicle control. Details of the primers used are shown in Hippocampus and liver tissue were homogenized using the BioSpec Mini-Beadbeater  and CCF-STTG1 cells were washed with cold PBS. RNA was prepared using Trizol  and RNA was reverse transcribed to cDNA using the QuantiTect Reverse Transcription Kit (Qiagen), according to the manufacturer’s instructions. Quantitative PCR (qPCR) was conducted, as previously described, on a CFX384 Thermal Cycler (Bio-Rad Laboratories) using SYBR Green PCR Select Master Mix  . RelativTissue Tek-embedded livers were cut with a cryostat CM3050S  to obtain 14 µm sections which were mounted on SuperFrost Plus adhesion slides , air-dried overnight, and stored at room temperature until use. For hepatic neutral lipid staining, liver sections were fixed in 4% neutral buffered formalin, washed with tap water, and rinsed with 60% isopropanol. Hepatic lipids were stained with Oil Red O  for 15 min. Next, the liver sections were rinsed with 60% isopropanol, lightly stained with hematoxylin, and covered with a coverslip. Digital images of the sections were obtained using a Leica DMLB microscope  equipped with software from the Leica Applications Suite .p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001.All statistical analyses were performed using IBM SPSS Statistics 25. The Shapiro–Wilk normality test was used to test normal distribution. Unless stated otherwise, normally distributed data are presented as mean ± SD and analyzed using two-way ANOVA (with treatment and genotype as independent variables) and the Tukey post hoc test. Not-normally distributed data are presented as median (25th–75th percentile) and analyzed using the Mann–Whitney U test. The OLT and ORT discrimination index D2 and the fold change values of CCF-STTG1 cells (compared to the DMEM-F12 medium control) were analyzed using a one-sample T-test. Animals that did not reach the minimum of 4 s of exploration in T1 or T2 were excluded from further analyses. Extreme values were excluded using Dixon’s principles of exclusion of extreme values ,57. Sign


It is therefore of great medicinal interest to modulate the cellular responses to TNF-α. Ebosin, a novel exopolysaccharide derived from Streptomyces sp, has been demonstrated to have remarkable therapeutic actions on collagen-induced arthritis in rats, while it also suppressed the production of IL-1β, TNF-α, and IL-6 at both mRNA and protein levels in cultured fibroblast-like synoviocytes. In order to further understand the potential mechanisms involved in the anti-inflammatory effects of ebosin at molecular level, we investigated the impact of it on the activation of MAPK and NF-κB pathways following TNF-α induced in fibroblast-like synoviocytes (FLS). The results showed that the phosphorylation levels of TNF-α-induced p38, JNK1, JNK2, IKKα, IKKβ, and IκB, as well as NF-κB nuclear translocation, were reduced significantly in FLS cells in response to ebosin. Furthermore, we proved that ebosin decreased the level of NF-κB in the nucleus and blocked the DNA-binding ability of NF-κB using electrophoresis mobility gel shift assay. Besides, low levels of matrix metalloproteinases (MMP-1 and MMP-3) and chemokines (interleukin-8 and RANTES) were found in TNF-α-stimulated fibroblast-like synoviocytes treated with ebosin. These results indicate that ebosin can suppress a range of activities in both MAPK and NF-κB pathways induced by TNF-α in rat fibroblast-like synoviocytes, which provides a rationale for examining the use of ebosin as a potential therapeutic candidate for rheumatic arthritis.Tumor necrosis factor- Proinflammatory cytokines such as tumor necrosis factor (TNF) produced by fibroblast-like synoviocytes (FLS) and inflammatory cells, which are expressed at high levels in rheumatoid joint tissue, where they contribute significantly to inflammation and articular destruction. Dysfunction of TNF-uding RA , 2, whicuding RA . TNF-taruding RA  to blockα elicits its biological activities through binding to two types of cell surface receptors TNF-R1 and TNF-R2. In most of the cells, TNF-R1 was considered to be a key mediator of TNF-α signal [κB is an important downstream target of TNF-α signaling, and it has been identified to be involved in inflammatory responses [α triggers NF-κB activation by a variety of signaling molecules, including TRAF2 (TNF receptor-associated factor 2), RIP (receptor-interacting protein), and the IKK (IκB kinase) complex. Another signal pathway activated by TNF-α is mitogen-activated protein kinase pathways , which has been strongly associated with many of the processes that mediate the pathological features of RA [α also induces extracellular matrix (ECM) remodeling by regulating the expression of MMPs involved in joint damage in RA, as well as expression of multiple chemokines including IL-8, MCP-1, CCL3, CCL4, and RANTES, which contribute to chronic inflammatory [κB pathways has been well recognized [TNF-α signal . NF-κB iesponses . TNF-α tes of RA . The p38ammatory –16. Currcognized .Streptomyces sp.139 of soil samples in China, remarkably inhibits the development of CIA (collagen-induced arthritis) in rats [β (interleukin-1β), IL-6 (interleukin-6), and TNF-α at the transcriptional and posttranscriptional levels [Ebosin, a novel exopolysaccharide (EPS) extracted from  in rats , which c in rats . This anl levels .Streptomyces genus with a novel structure [α secretion was stronger than other cytokines as IL-1β and IL-6 in the CIA rat model and FLS cells, implicating that ebosin may improve rheumatoid arthritis symptoms through the TNF-α signaling pathway [α-mediated MAPK and NF-κB pathways, as well as on levels of MMPs and chemokines in rat fibroblast-like synoviocytes (FLS), which will help us to better understand the mechanism of action of ebosin.Although anti-TNF agents have been shown to improve the outcome of the management of RA, a proportion of patients does not respond well to anti-TNF therapy and has increased the risk of adverse events such as infections –24. Unlitructure . In a pr pathway . Howeverμg/ml streptomycin and cultured at 37°C, 5% CO2.Male Wistar rats (Certificate No.: SCXK 2005-0013) were obtained from the Institute of Experimental Animals, Chinese Academy of Medical Sciences, Beijing . All ratStreptomyces.139 was found from a soil sample in China and deposited in the China General Microbiology Culture Collection Center (No. 0405). Ebosin was purified from the supernatant of fermentation culture of Streptomyces sp.139 according to the protocol as described before [Ebosin-producing d before .6/ml) at 37°C for 24 h. Ebosin  was diluted in DMEM and added into FLS for 3 h with TNF-α (10 ng/ml). Cells were collected in 0.25% trypsin-EDTA (HyClone) and harvested by centrifugation at 500 g for 5 min. Cytoplasmic and nuclear were extracted using NE-PER Nuclear and Cytoplasmic Extraction Reagents following the manufacturer's protocol [FLS were cultivated in 6-well plates  with Image J software.The effect of Ebosin on MAPK and NF-eviously . The exp6/ml and cultivated at 37°C for 24 h and then received proper ebosin treatments  for 12 h. Cells were further induced by adding 10 ng/ml TNF-α for 24 h before supernatants were collected. The level of MMP-1, MMP-3, RANTES, and IL-8 in the medium was detected using colorimetric ELISA kits .ELISA was used to evaluate the levels of IL-8, RANTES, MMP-1, and MMP-3 in cell culture medium. FLS cells were plated in 24-well plates at 1 × 10κB binding to DNA in nuclei using the LightShift Chemiluminescent EMSA Kit (Thermo) following the instructions of the manufacturer [κ-chain binding site , was synthesized from Beyotime Biotechnology Company. Unlabeled oligonucleotide and a 50-fold excess of cold κB oligonucleotide were used as a control to confirm specific binding.EMSA was performed to detect the effect of ebosin on the ability of NF-facturer . Extract4/ml) at 37°C for 12 h. Ebosin (80 μg/ml) was added in each well followed by cultivation at 37°C for 12 h and then treated cell with TNF-α (10 ng/ml) for an additional 3 h. The cells were fixed in 5% paraformaldehyde (PFA)/PBS for 10 min at room temperature, permeabilized with PBS/0.1% Triton-X100 for 15 min, and blocked in PBS with 5% bovine serum albumin for 1 h. The cells were incubated with rabbit anti-NF-κB p65  and Alexa Fluor 488 donkey anti-rabbit IgG (Molecular Probes). The fluorescent signals were detected by fluorescence microscopy .FLS cells were cultivated in 96-well plates .Data are presented as mean ± SD values. Evaluation of significance of differences between sample groups was performed by GraphPad Prism software (Version 6.0) using Student's α [α-induced p38 MAPK activation, FLS cells were treated with ebosin at a concentration of 0.128, 0.64, 3.2, 16, 80, and 400 μg/ml for 12 h. Phosphorylated active forms of p38 MAPK were detected by western blot in FLS cells following TNF-α stimulation. As shown in P < 0.01), 38.94% (P < 0.001), 25.21% (P < 0.05), 16.51% (P < 0.01), 10.16% (P < 0.05), and 3.62%, respectively, in a dose-dependent manner, while the levels of nonphosphorylated p38 in FLS did not change , 34.84% (P < 0.001), 27.60% (P < 0.01), 13.51% (P < 0.01), 8.95%, and 1.19%, respectively (P < 0.01), 50.07% (P < 0.01), 43.00% (P < 0.01), 21.96% (P < 0.05), 13.29%, and 8.30%, respectively  p42/44 protein level in the supernatant of lysed cells was determined using western blot. The results found that ebosin did not exert any action on both phosphorylated and nonphosphorylated forms of p42/44 MAPK , 19.90% (P < 0.05), and 0.85% separately and IKKβ by 71.56% (P < 0.01), 58.11% (P < 0.05), and 40.28% (P < 0.05), respectively , 14.99% (P < 0.05), and 6.93%, respectively, at dosages 80, 16, and 3.2 μg/ml, but the levels of nonphosphorylated IκBα were enhanced by 55.12% (P < 0.05), 45.08% (P < 0.05), and 32.78% (P < 0.05), respectively, at the same dosages  was performed in FLS cells stimulated by TNF-α and then incubation with varying concentrations of ebosin. Results in κB-DNA binding in a dose-dependent manner.Once released from its inactive form complexed with Icription . To confκB, we detected the NF-κB protein level in the cytoplasm and nucleus using the NF-κB p65 antibody. Western blot analysis showed (α by 51.72% (P < 0.01), 46.84% (P < 0.05), 40.33% (P < 0.05), 30.34% (P < 0.05), 23.12% (P < 0.05), and 14.34%, respectively, at dosages 400, 80, 16, 3.2, 0.64, and 0.128 μg/ml. However, it decreased the expression of NF-κB in the nucleus of cells by 80.11% (P < 0.001), 77.02% (P < 0.001), 53.77% (P < 0.001), 19.25% (P < 0.05), 10.07% (P < 0.05), and 5.25%, respectively, at same dosages , 33.63% (P < 0.05), and 26.94% (P < 0.05), respectively, at dosages of 80, 16, and 3.2 μg/ml of ebosin, and meanwhile, the productions of MMP-3 in FLS were suppressed by 83.01% (P < 0.01), 70.81% (P < 0.01), and 32.63% (P < 0.05), respectively , 57.52% (P < 0.001), and 57.28% (P < 0.001), respectively (P < 0.001), 29.94% (P < 0.01), and 19.93% (P < 0.01) separately  that has no significant influence on the cell viability by cytotoxicity assay as described in our previous study [ERK1/2, JNK, and p38 MAPKs are the three major members of the MAPK family that respond to distinct signaling cascades . The empus study . Meanwhius study .κB, which initially exists in the cytoplasm in an inactive complex with IκB, is a pivotal regulator in the regulation of inflammation and immune responses by eliciting the transcriptional responses following different stimuli such as TNF-α or IL-1β [κB appears to precede disease onset, which suggested that inhibition of NF-κB by different means may contribute to reduce the severity of disease [κB in FLS induced by TNF-α. Our results showed decreased levels of phosphorylated IKKα and IKKβ after treating with ebosin. Besides, ebosin was capable of downregulating the levels of phosphorylated IκBα. EMSA indicated that ebosin decreased the DNA-binding activity of NF-κB in the nucleus, probably through affecting the phosphorylation of NF-κB itself. Furthermore, we also demonstrated that ebosin can significantly inhibit the NF-κB nuclear translocation process using western blot analysis and fluorescence microscopy. All of the results mentioned above suggest that ebosin is an inhibitor of the NF-κB-driven signaling pathway, which may be responsible for its anti-inflammatory effects in vivo [The transcription factor NF-or IL-1β . It has  disease . In this in vivo .κB and major collagenolytic enzymes involved in tissue destruction, have been reported to be significantly elevated in the synovial fluid of RA patients [α. These results suggested that inhibition of ebosin on NF-κB activation contributes to reduce the secretion of MMPs and chemokines, thereby protecting RA patients from joint destruction.MMP-1 and MMP-3, as targets of NF-patients . In addipatients . As a mepatients . Anotherα in isolated FLS, acting as an effective in vitro inhibitor of the MAPKs and NF-κB signaling pathways (In conclusion, this study has demonstrated that ebosin is capable of inhibiting the inflammatory responses induced by TNF-pathways . It is a


Objectives: Lycium barbarum polysaccharide (LBP) is a natural polysaccharide extracted from Lycium barbarum that has anti-inflammatory, anti-apoptotic and anti-aging effects, and plays a role in the prevention and treatment of various diseases. In this study, we investigated the therapeutic effect of LBP on particulate matter 2.5 (PM2.5)-induced skin damage.Methods: Cell viability was analyzed by MTT and LDH assays. Apoptosis was analyzed by Annexin V-FITC/PI staining. Oxidative stress/damage were assessed by intracellular ROS levels, MDA content and SOD activity. The intracellular protein expression was analyzed by Western blot. Mitochondrial damage was assayed by mitochondrial membrane potential with JC-1 probe. LC3-GFP adenovirus was transfected into HaCaT cells to analyze intracellular autophagosome levels.Results: In PM2.5-treated HaCaT cells, LBP pretreatment reduced PM2.5-induced cytotoxicity, ameliorated cell morphology and reduced cell apoptosis. LBP also inhibited the expression levels of GRP78 and CHOP, reduced the conversion of LC3I to LC3II, inhibited Bax protein and activated Bcl-2 protein. Furthermore, LBP inhibited PM2.5-induced mitochondrial autophagy (mitophagy) and mitochondrial damage. PM2.5-induced autophagy was regulated by endoplasmic reticulum (ER) stress.Conclusion: LBP protects skin cells from PM2.5-induced cytotoxicity by regulating the oxidative stress-ER stress-autophagy-apoptosis signaling axis, revealing that LBP has a great potential for the skin protection.  Many studies have shown that PM2.5 has many effects on human health, including provoking respiratory diseases, asthma, cardiovascular and cerebrovascular diseases . Not onlIn the study of Li et al., when HaCaT cells  are exposed to PM2.5, cell viability decreased and inflammation was induced . Hu et aThe ER is the main place for protein folding and processing, which maintains cell homeostasis and function . When ceLycium barbarum polysaccharide (LBP) is the main active component of Lycium barbarum, and it has been widely reported to have anti-aging, anti-oxidant, anti-apoptotic and anti-inflammatory effects . LBP has2.2.1.2+ probe Fluo-4 AM were also purchased form Beyotime .Particulate matter in the air was sampled by an air sampler  in Lanzhou, Gansu province for 3 months. After the collection was completed, PM2.5 was obtained by filtering through an ultra-fine glass fiber net. After that, PM2.5 was sterilized, then freeze-dried and concentrated and stored at −80°C for later use. HaCaT cell line was purchased from the Cell Bank of the Chinese Academy of Sciences . LBP was purchased from Natural Field Bio-Technique Co., Ltd . DMEM medium was obtained from Hyclone . Fetal Bovine Serum (FBS) was purchased from Si-Ji-Qing Biotechnology . 2’,7’-Dichlorofluorescin diacetate was purchased from Sigma-Aldrich . Anti-LC3B and anti-P62 antibodies were purchased from Cell Signaling Technology . Anti-GRP78 and anti-CHOP antibodies were purchased from Beyotime . Ad-GFP-LC3B adenovirus, Annexin V-FITC/PI apoptosis detection kit, Mitochondrial membrane potential detection kit (JC-1) and the Ca2.2.2. HaCaT cells were pretreated with LBP (or 3-MA or 4-PBA) for 2 h and incubated with PM2.5 for 24 h, and then the cells were collected for subsequent experiments.HaCaT cells were cultured in DMEM high glucose medium supplemented with 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin at 37°C in air containing 5% CO2.3.The viability of HaCaT cells was detected by MTT method, consistent with our previous research . In addi2.4.5 cells/well) were seeded in 6-well plates for 24 h. Then cells were pretreated with or without LBP (or 3-MA) for 2 h, and subsequently treated with or without PM2.5 (100 μg/ml) for another 24 h. Apoptosis was detected by a confocal microscope , using an Annexin V- FITC/PI Apoptosis Kit.HaCaT cells (1 × 102.5.HaCaT cells (1 × 105 cells/well) were seeded in 6-well plates for 24 h. Cells were pretreated with or without LBP for 2 h, and then co-incubated with or without PM2.5 for 24 h. Subsequently, fluorescent probes DCFH-DA and DHE-labeled intracellular ROS, with Hoches33342-counterstained the nucleus, were observed under a fluorescent microscope.2.6.5 cells/well) were seeded in 6-well plates for 24 h. Then cells were pretreated with or without LBP for 2 h, and subsequently treated with or without PM2.5 (100 μg/ml) for another 24 h. MDA content was detected with the kit provided by the Nanjing Jiancheng Bioengineering Institute , according to the manufacturer's instructions. In brief, MDA content is determined by its reaction with thiobarbituric acid (TBA) to form a colorimetric product, which can be detected at the 532 nm wavelength.HaCaT cells (3 × 102.7.5 cells/well) were seeded in 6-well plates for 24 h. Then cells were pretreated with or without LBP for 2 h, and subsequently treated with or without PM2.5 (100 μg/ml) for another 24 h. The SOD activity in HaCaT cells was detected by the kit provided by Nanjing Jiancheng Bioengineering Institute , according to the manufacturer's instructions. The intracellular SOD activity was detected at a wavelength of 450 nm.HaCaT cells .2.9.HaCaT cells were pretreated with or without LBP (or 4-PBA) for 2 h and then treated with or without PM2.5 (100 μg/ml) for 24 h. Cells were collected and then lysed on ice using RIPA lysate. One percent of PMSF was added into lysate to prevent protein degradation. Further operation is consistent with our previous research .2.10.2+ probe Fluo-4 AM was added and co-incubated in the dark for 15 min. The intracellular Ca2+ level was detected by a fluorescent microscope.HaCaT cells were pretreated with LBP for 2 h, and then incubated with PM2.5 for 24 h. After cells were washed with PBS three times, the Ca2.11.To measure autophagic vesicles, HaCaT cells were transfected with GFP-LC3 adenoviruses at a 20 multiplicity of infection (MOI). After cultured for 24 h, HaCaT cells were pretreated with LBP (or 4-PBA) for 2 h and then were treated with PM2.5 for 24 h. Furthermore, Mito tracker Red was incubated with HaCaT cells for 20 min to detect mitophagy. Then the fluorescent signals were detected by a confocal microscopy .2.12.The decrease of mitochondrial membrane potential is an important marker of mitochondrial damage. HaCaT cells were pretreated with 3-MA or LBP for 2 h, and then incubated with PM2.5 for 24 h. After washed with PBS three times, cells were incubated with JC-1 probe in darkness for 30 min, and then mitochondrial membrane potential was detected by a confocal microscopy. When the mitochondrial membrane potential is normal, JC-1 is at the state of aggregates (red fluorescence); when the mitochondrial membrane potential is decreased, JC-1 can be de-aggregated into monomers (green fluorescence), indicating mitochondrial damage.2.13.p < 0.05 were considered statistically significant.Data were collected from at least three independent experiments. Statistical analysis was performed as the mean ± standard deviation (SD). Data analysis was expressed using Prism 9.0 software (GraphPad Software) or Origin 8.0 software. The data were analyzed using student's t-test. Differences with 3.3.1.HaCaT cells were pretreated with LBP  for 2 h and then exposed to PM2.5 (100 μg/ml) for 24 h. After that, we evaluated the protective effect of LBP on PM2.5-induced cytotoxicity by MTT and LDH assay. The results showed that PM2.5 induced cell proliferation inhibition and led to cell death, while LBP (2.5 or 5 mg/ml) pretreatment alleviated PM2.5-induced cytotoxicity (3.2.We further studied the effect of LBP on PM2.5-induced oxidative stress and oxidative damage in HaCaT cells. HaCaT cells were pretreated with LBP (2.5 mg/ml) for 2 h, and then incubated with PM2.5 for 24 h. Intracellular ROS were labeled by DCFH-DA and DHE, and observed with a fluorescent microscope. The results showed that LBP significantly inhibited the increase of intracellular ROS  and CHOP (2.85 ± 0.39), while LBP pretreatment could significantly inhibit the expression of GRP78 (1.12 ± 0.66) and CHOP (1.53 ± 0.41)  and death (PI positive cells) , thereby lessening oxidative damage, improving cell survival and reducing cell apoptosis. These results reveal the protective effect and mechanism of LBP against skin damage and degeneration.


However, controversial findings also exist, and its underlying molecular mechanisms are largely unknown. Recently, the byproducts of H2S, per-/polysulfides, emerged as biological mediators themselves, highlighting the complex chemistry of H2S. In this study, we characterized the biological effects of P*, a slow-releasing H2S and persulfide donor. To differentiate between H2S and polysulfide-derived effects, we decomposed P* into polysulfides. P* was further compared to the commonly used fast-releasing H2S donor sodium hydrogen sulfide (NaHS). The effects on oxidative stress and interleukin-6 (IL-6) expression were assessed in ATDC5 cells using superoxide measurement, qPCR, ELISA, and Western blotting. The findings on IL-6 expression were corroborated in primary chondrocytes from osteoarthritis patients. In ATDC5 cells, P* not only induced the expression of the antioxidant enzyme heme oxygenase-1 via per-/polysulfides, but also induced activation of Akt and p38 MAPK. NaHS and P* significantly impaired menadione-induced superoxide production. P* reduced IL-6 levels in both ATDC5 cells and primary chondrocytes dependent on H2S release. Taken together, P* provides a valuable research tool for the investigation of H2S and per-/polysulfide signaling. These data demonstrate the importance of not only H2S, but also per-/polysulfides as bioactive signaling molecules with potent anti-inflammatory and, in particular, antioxidant properties.Hydrogen sulfide (H The ATDC5 cells were grown on glass coverslips in a 12-well plate (8 × 104 cells/well) for 24 h. The cells were washed once with FBS-free DMEM/F12 and incubated with 10 µM WSP-5 (in FBS-free DMEM/F12 + 100 µM hexadecyl (trimethyl)ammonium bromide) for 30 min. After washing the cells with phosphate-buffered saline (PBS), they were incubated with 1 mM P* (in FBS-free DMEM/F12 + 100 µM hexadecyl (trimethyl)ammonium bromide) for 30 min. Afterwards, fluorescence imaging was performed at room temperature using a Zeiss Axiovert 200 equipped with a xenon lamp, polychromator, Chroma filters , and a CoolSNAP fx-HQ CCD-camera . For each condition, 8 images were taken randomly and analyzed using ImageJ . For background correction, fluorescence was measured in cell-free areas.We monitored the H4 cells/well) for 48 h, followed by 24 h of incubation with or without NaHS or P*. Cells were washed once with PBS and trypsinized. After centrifugation at 500× g for 2 min, the cell pellets were resuspended in 1 mL of fresh medium. Cell suspensions were diluted 1:1 in 0.4% trypan blue solution and counted using the DeNovix CellDropTM Cell Counter . For a positive control, the cell suspensions were boiled for 5 min at 95 °C.The number of viable cells was assessed using trypan blue staining. The ATDC5 cells were grown in 12-well plates (5 × 103 cells/well) until 80% confluency was reached. After 24 h of treatment with NaHS or P*, an MTT assay was performed according to manufacturer protocol .MTT was used to measure cell viability in isolated primary human chondrocytes. Cells were grown in a 96-well plate (5 × 105 cells/well) and grown for 48 h. Following different treatments, the cells were washed twice with PBS and directly lysed in 1-fold Laemmli buffer. To minimize viscosity, cell lysates were treated with a short impulse of ultrasound (15 s) and boiled for 5 min at 95 °C afterwards. Equal amounts of protein were run on 10% SDS-PAGE mini gels and electro-transferred to PVDF membranes . After blocking for 1 h at room temperature with 5% non-fat dried milk in PBS + 0.1% Tween 20, the membranes were incubated with primary antibodies overnight at 4 °C. After 2 h of incubation with horseradish peroxidase coupled anti-rabbit secondary antibody, the proteins were detected using the enhanced chemiluminescence (ECL) WesternBright detection system  and a GeneGnome detection device . Relative density was determined with Syngene GeneTool software and adjusted to tubulin or the corresponding unphosphorylated protein as a reference protein.ATDC5 cells were seeded in 6-well plates (1 × 105 cells/well) and grown for 48 h. After 1 h of incubation with 0.5 mM NaHS or 0.5 mM P*, the cells were washed once with PBS and were mechanically scraped off using 0.85 mL of HEN-buffer (pH 7.8) composed of 100 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 1 mM EDTA, 0.1 mM neocuproine, proteinase inhibitors , and phosphatase inhibitors . After centrifugation (1000× g for 5 min at room temperature), the cells were incubated on ice in 0.85 mL of HEN-buffer supplemented with 0.2% Igepal CA-630 for 3 min. After centrifugation (1000× g for 5 min at 4 °C), the supernatants were carefully collected (cytosolic fraction). Pellets (nuclear fraction) were incubated on ice in 15 µL RIPA buffer  for 10 min. After the addition of 5-fold Laemmli buffer, the cytosolic and nuclear fractions were homogenized by sonication for 10 s and boiled for 5 min at 95 °C afterwards.ATDC5 cells were seeded in 6-well plates (1 × 104 cells/well) and transfected the following day with Nrf2 siRNA  or scrambled negative control siRNA  at a final concentration of 10 nM using the siTran 2.0 transfection reagent  or Lipofectamine 3000  according to the manufacturer’s instructions. At 6 h post-transfection, the transfection complex-containing medium was replaced with fresh complete medium, and the cells were left to rest overnight before treatment.ATDC5 cells were grown in 12-well plates (4 × 104 cells/well) for 24 h. Cells were incubated with increasing concentrations of either NaHS or P* (0.25–1 mM) for 1 h at 37 °C, washed, and further incubated with an incubation buffer containing 10 µM hydroethidine and increasing concentrations of NaHS or P* (0.25–1 mM) with menadione (100 µM) for 4 h at 37 °C. In another experiment, the ATDC5 cells were incubated with NaHS or P* (0.25–1 mM) for 24 h, washed, and incubated with an incubation buffer containing 10 µM hydroethidine and menadione (50 µM) for 4 h at 37 °C. Afterwards, fluorescence imaging was performed at room temperature using a Zeiss Axiovert 200 equipped with a xenon lamp, polychromator, Chroma filters , and a CoolSNAP fx-HQ CCD-camera . For each condition, 10 images were taken randomly and analyzed using ImageJ . For background correction, fluorescence was measured in cell-free areas.Production of superoxide was assessed as previously described . First, IL-6 levels in cell culture supernatants were quantified using a commercially murine IL-6 ELISA kit .® Universal PCR Master Mix and pre-designed TaqMan® Gene Expression Assays  as follows: Nrf2 , IL-6 , Nrf2 , and cyclophilin D . Reactions were carried out using a StepOnePlusTM Real-Time PCR System (Thermo Fisher Scientific). Cycling conditions were as follows: 2 min 50 °C, 10 min 95 °C, 40 cycles of 15 s 95 °C, and 1 min 60 °C. Data were analyzed according to the 2−∆∆Ct method using cyclophilin D as a reference gene.Total RNA was isolated using the AurumTM Total RNA Mini Kit  according to the manufacturer protocols. Total RNA quality was determined by measuring OD260/OD280 by means of UV/Vis spectroscopy using a Nanodrop 2000 spectrophotometer . The cDNA synthesis was performed with the iScript cDNA Synthesis Kit (Bio-Rad Laboratories). The qPCR analysis was performed using TaqManIn primary human chondrocytes from OA patients, isolation of total RNA, cDNA synthesis, and SYBR Green-based qPCR were conducted as previously described . Succinap-values. p-values less than 0.05 (*), 0.01 (**), 0.001 (***) or 0.0001 (****) were considered statistically significant.All data are expressed as means ± SEM. Experiments were performed independently at least three times. Statistical analyses were performed with GraphPad Prism 9.0 . If not otherwise stated, one-way ANOVA with Tukey’s post hoc test was used for statistical comparison. Only the statistical results for the highest concentrations used are shown in the figures for the purpose of clarity. 2S in the presence of thiols, such as cysteine or glutathione, reaching the maximum level after approximately 10 min . After 24 h of incubation, both NaHS and P* induced NQO1 expression; again, the effect of P* appeared more pronounced . As per-ydration ,43,44,45p-value < 0.0001) was validated by qPCR , ERK1/2 (inhibitor of the upstream activator MEK1/2-U0126) and PI3K/Akt (LY294002), followed by incubation with P* for 6 h. Indeed, inhibition of p38 MAPK and PI3K/Akt, but not ERK1/2, clearly reduced P*-mediated HO-1 induction e. Since HO-1, as a stress-inducible enzyme, is generally induced by reactive oxygen species (ROS) . Therefop-value = 0.028), , c. RepresApart from protecting cells against oxidative stress, HO-1 also exerts anti-inflammatory effects. Hence, we examined the effects of NaHS and P* on IL-6 expression in ATDC5 cells and in human OA chondrocytes.2S donors before stimulation (p = 0.54). In contrast, P* reduced IL-6 protein levels by approximately 55% (p < 0.01). In line with this finding, P* decreased IL-6 at the transcriptional level by approximately 70% (p < 0.0001), , b. We fur0.0001), c. In add0.0001), a.p < 0.001), whereas NaHS did not affect MMP-13 expression , , a. This ep < 0.0001) and P* further augmented the observed decline (p = 0.0012), (p > 0.5) , b. In addp > 0.5) c, althoup > 0.5) f.2S release or mediated by the mixture of persulfides and polysulfides formed by the decomposition of P* [p < 0.0001), decomposed P* reduced IL-6 levels to only 74.4 ± 3.6% (p < 0.01), (We aimed to clarify whether the anti-inflammatory effects of P* were dependent on its immediate Hon of P* . Therefo< 0.01), d.2S donors blocked the activation of NF-κB p65 (p-NF-κB) or the degradation of IκBα, the negative regulator of NF-κB , (p < 0.05), while co-incubation with P* further enhanced this decrease by approximately 40% (p = 0.06), , c. Blocki= 0.06), d. Howeve2S and persulfide donor P* in the murine chondrocyte-like cell line ATDC5 on oxidative stress and inflammatory markers. We found that P* (i) induced the cytoprotective enzyme HO-1 via activation of Nrf2, PI3K/Akt and p38 MAPK pathways, (ii) exerted antioxidative effects via Nrf2 activation, and (iii) decreased IL-6 levels more efficiently than NaHS . Clearly, further studies are needed to fully understand the mechanism behind P*-mediated biological effects. In vivo administration of P* might further pave the way for the development of Hmparable . Therefo2S and persulfide donor. By decomposing P* into polysulfides, P* provides a valuable new research tool to study H2S and per-/polysulfide signaling in cells and tissues. Our work demonstrates the importance of not only H2S but also polysulfides as bioactive signaling molecules with potent antioxidant and anti-inflammatory effects.In this study, we characterized the biological effects of P*, a H


Meanwhile, the structurally homologous SRO precursor, tryptophan (TRP), does not affect HSA’s affinity to monomeric Aβ, despite slowdown of the association and dissociation processes. Crosslinking with glutaraldehyde and dynamic light scattering experiments reveal that, compared with the TRP-induced effects, SRO binding causes more marked changes in the quaternary structure of HSA. Furthermore, molecular docking reveals distinct structural differences between SRO/TRP complexes with HSA. The disintegration of the serotonergic system during AD pathogenesis may contribute to Aβ release from HSA in the central nervous system due to impairment of the SRO-mediated Aβ trapping by HSA.Prevention of amyloid β peptide (Aβ) deposition via facilitation of Aβ binding to its natural depot, human serum albumin (HSA), is a promising approach to preclude Alzheimer’s disease (AD) onset and progression. Previously, we demonstrated the ability of natural HSA ligands, fatty acids, to improve the affinity of this protein to monomeric Aβ by a factor of 3 , 248–253). Using plasmon resonance spectroscopy, we show here that another HSA ligand related to AD pathogenesis, serotonin (SRO), increases the affinity of the Aβ monomer to HSA by a factor of 7/17 for Aβ Human serum albumin HSA; 66.5 kDa) is the most abundant blood and cerebrospinal fluid (CSF) protein and is vital to maintaining the osmotic pressure of blood and the transport of numerous substances, including fatty and amino acids, hormones, metal ions, and over 250 drugs . HSA  kDa is tBased on the Aβ-buffering role of plasma HSA, removal of plasma Aβ via plasma exchange and replacement with therapeutic HSA were suggested for facilitation of Aβ efflux from the brain of AD patients to plasma . The cliSerotonin  is a HSA ligand closely related to AD pathogenesis . It Numerous studies have demonstrated degeneration of the serotonin system in AD, a decline in the cortical/serum SRO level, and negative consequences of disruption in serotonergic signaling ,16. The 40 or Aβ42 sample was immobilized on the surface of the SPR sensor chip by amine coupling, followed by removal from the surface of the non-covalently bound Aβ molecules by a 2% SDS water solution [2, 2.5 mM CaCl2, pH 7.4). The resulting SPR sensograms exhibited a concentration-dependent association–dissociation pattern and were well approximated by the one-site binding model (DK) for intact HSA complexes with Aβ40 and Aβ42 were 0.6 × 10−7 M and 1.2 × 10−7 M, respectively : Y174, E177, S216, K219, Q220, L222, K223, F235, W238, A239, R242, L243, Q245, R246, F247, L258, L262, H266, R281, L284, A285, I288, L299, K310, S311, H312, I314, A315, E316, E318, D320, P363, Y365, S366, V367, V368, K468, P471, C472, D475, Y476, S478, V479. Molecular docking of HSA with SRO/TRP using AutoDock Vina software  revealedTo explore the conformational flexibility of the established SRO/TRP-binding pocket of HSA, we conducted a multiparametric evaluation of the protein predisposition for intrinsic disorder using a set of commonly utilized per-residue disorder predictors. In these analyses, a threshold of 0.5 is typically used to identify disordered residues and regions in a query protein. Residues with the disorder scores (DS) 0.25 ≤ DS < 0.5 are considered to be moderately disordered, whereas residues with 0.15 ≤ DS < 0.25 are flexible. The results of this analysis are shown in The literature data on HSA interaction with monomeric Aβ are contradictory ,36,37,3840/Aβ42 with a 3-fold decrease in the DK value [DK value. Such a dramatic change in HSA’s affinity for Aβ could be achieved via the following mechanisms or their combination: (I) an allosteric mechanism ; or (II) ligand binding to Aβ, affecting its interaction with HSA. In the case of mechanism I alone, the SRO-induced changes in HSA‘s affinity for Aβ would be noticeable at SRO concentrations above 1.6 µM .42 fibrillation by 50–100 µM SRO [42 fibrillation by TRP, compared with SRO [The absence of a marked influence of 10 µM SRO on the HSA–Aβ interaction points out the contribution of mechanism II. This conclusion is in line with the data on the potent inhibition of Aβ0 µM SRO , which cwith SRO , indicatThe revealed influence of SRO on the HSA–Aβ interaction could be relevant to AD progression, considering the disintegration of the serotonergic system during AD, accompanied by a decrease in the SRO level in biofluids ,16,39. TSince chemical structures of agents in clinical studies in AD, including SSRIs aimed at increasing the SRO level at nerve terminals ,43, indi42 produced in E. coli (#A-1163-1) was obtained from rPeptide . Usp2-cc was prepared mainly as described in [4OH and acetic acid were purchased from Component-reaktiv  and Labteh , respectively. Trifluoroacetic acid (TFA) was bought from Fisher Sci . Dimethyl sulfoxide (DMSO) was purchased from Helicon . Ethanolamine and ProfinityTM IMAC Resin were obtained from Bio-Rad Laboratories . A Jupiter C18 column was purchased from Phenomenex® .A fatty-acid-free HSA sample prepared under non-denaturing conditions (#126654)  was purcribed in . The SRO−1cm−1 for HSA and 1490 M−1cm−1 for Aβ40/Aβ42.Protein concentrations were measured spectrophotometrically using molar extinction coefficients at 280 nm calculated according to : 34,445 40 was expressed in E. coli and purified as described earlier [40 fusion protein was dialyzed twice against 50 mM Tris-HCl and 5 mM 2-ME (pH 8.6) buffers containing 6 M and 4 M urea, respectively. Aβ40 was excised from the fusion protein via incubation with Usp2-cc (50-fold molar excess of the fusion over the enzyme) at room temperature for 3 h. Urea was added to the solution up to 8 M. The hydrolysate was passed through a Profinity IMAC column equilibrated with 50 mM Tris-HCl, 5 mM 2-ME, and 8 M urea (pH 8.0–8.5) to remove His-tagged ubiquitin and Usp2-cc. Aβ40 was purified by chromatography on an HPLC Jupiter C18 column . Precise chain cleavage by Usp2-cc was confirmed by electrospray ionization mass spectrometry . The purified Aβ40 samples were freeze-dried and stored at −70 °C.Human Aβ earlier  with som40/Aβ42 samples were pretreated for SPR measurements mainly as described in [Recombinant human Aβribed in . The fre40/Aβ42 in 10 mM sodium acetate, pH 4.5 buffer) was immobilized on a ProteOn GLH sensor chip surface (up to 7000–9000 RUs) by amine coupling. The remaining activated amine groups on the chip surface were blocked by 1 M ethanolamine solution. The non-covalently bound Aβ40/Aβ42 molecules were washed from the chip surface with 2% SDS water solution until stabilization of the signal [20 monoclonal antibody (7N22) as the analyte [2, 2.5 mM CaCl2, pH 7.4) was passed over the chip at a rate of 30 μL/min for 300 s, followed by flushing the chip with the running buffer for 2400 s. The sensor chip surface was regenerated by passage of 10 mM EDTA pH 7.9 solution containing 2% SDS. The influence of HSA ligands on its interaction with Aβ40/Aβ42 was studied in the presence of TRP/SRO . The double-referenced SPR sensograms were globally fitted according to the one-site binding model using Bio-Rad ProteOn Manager™ v.3.1 software. The effective equilibrium (DK) and kinetic (dk and ak) dissociation/association constants were evaluated for each HSA concentration, followed by averaging of the resulting values (standard deviations are indicated).SPR measurements were performed at 25 °C using a Bio-Rad ProteOn™ XPR36 instrument mainly as previously described . Ligand e signal . The mon analyte ; the imm2, 2.5 mM CaCl2, pH 7.4 buffer in the absence/presence of 1 mM SRO/TRP at 37 °C for 1 h, mainly as described in [Crosslinking of HSA (36 μM) with 0.02% glutaraldehyde was performed in 20 mM HEPES-KOH, 140 mM NaCl, 4.9 mM KCl, 1 mM MgClribed in .2, 2.5 mM CaCl2, pH 7.4 buffer in the absence/presence of 1 mM SRO/TRP were performed at 25.0 °C using a Zetasizer Nano ZS spectrometer . The back-scattered light from a 4 mW He/Ne laser (632.8 nm) was collected at an angle of 173°. The acquisition time for a single autocorrelation function was 45 s. The resulting autocorrelation functions are averaged values from 20 measurements. The volume–size distributions were calculated using the following parameters: a refractive index of 1.330 and a viscosity value of 0.8882 cP.DLS measurements of HSA (36 μM) solutions in 20 mM HEPES-KOH, 140 mM NaCl, 4.9 mM KCl, 1 mM MgClhttp://autodock.scripps.edu/resources/adt/index_html, accessed on 28 April 2021). The three-dimensional structures of SRO and TRP were obtained from the PubChem database  and converted into PDB format using PyMOL v.1.6.9.0 software . SRO/TRP-binding pockets of HSA were identified using the DoGSiteScorer algorithm [https://proteins.plus/, accessed on 28 April 2021). AutoDock Vina  [https://plip-tool.biotec.tu-dresden.de/plip-web/plip/index, accessed on 28 April 2021) [The X-ray structure of HSA was obtained from Protein Data Bank  entry 1Ulgorithm  implemenil 2021)  was usedil 2021)  using nu® VLXT [® VSL2 [® VL3 [http://www.pondr.com, accessed on 28 April 2021), PONDR® FIT, which is a meta-predictor that incorporates predictions from several different sources [® VLXT [® VL3 [® VLS2B [® FIT [The intrinsic disorder predisposition of HSA was analyzed by commonly used per-residue predictors, including PONDR® VLXT , PONDR®  [® VSL2 , and PON2 [® VL3 , which a sources , and the sources . We util [® VLXT , PONDR® T [® VL3 , PONDR® [® VLS2B , PONDR® B [® FIT , IUPred2B [® FIT ,34. The The presented example of ligand-induced facilitation of HSA interaction with monomeric Aβ demonstrates the high potential for pharmacological modulation of this interaction aimed at decreasing the free Aβ concentration, leading to the inhibition of malicious Aβ deposits. The revealed effect seems to involve ligand binding to both participants of the reaction. The data deepen our understanding of the SRO-mediated effects reported in animal and clinical studies.


To assess possible antitumor action mechanisms, we focused on the comparative analysis of pro- and antioxidant behaviors. Pro-oxidant activity, in which the reactive oxygen species (ROS) formed by the reaction of the complexes with H2O2 produce oxidative damage to 2-deoxy-d-ribose, was evaluated using the TBARS method. Additionally, the antioxidant action was quantified through the superoxide dismutase (SOD)-like activity, using a protocol based on the inhibitory effect of SOD on the reduction of nitrobluetetrazolium (NBT) by the superoxide anion generated by the xanthine/xanthine oxidase system. Our findings show that Cu–amino acid complexes are strong ROS producers and moderate SOD mimics. Conversely, Cu–dipeptide–phen complexes are good SOD mimics but poor ROS producers. The activity of Cu–dipeptide complexes was strongly dependent on the dipeptide. A DFT computational analysis revealed that complexes with high SOD-like activity tend to display a large dipole moment and condensed-to-copper charge, softness and LUMO contribution. Moreover, good ROS producers have higher global hardness and copper electrophilicity, lower copper softness and flexible and freely accessible coordination polyhedra. In a search for new antitumoral agents, a series of homoleptic copper(II) complexes with amino acids and dipeptides, as well as heteroleptic complexes containing both dipeptides and 1,10-phenanthroline, were studied. Furthermore, a single-crystal structure containing alanyl-leucinato ([Cu Metal-based drugs have played an important role in cancer treatment and in the expansion of research groups focused on the development of new antitumor drugs. It is well-known that cisplatin and subsequent generations  have shown high effectiveness for the treatment of tumors in clinical practice but also present several toxic effects . In spitIn the search for new drugs with lower toxic effects, several research groups have focused their work on complexes with endogenous metals; for instance, copper, the coordination compounds of which were found to be promising antitumor agents by Krasnovskaya et al. . AntitumDue to the redox activity of copper complexes, they can present a dual role. They can act as protective antioxidants destroying, for example, free radicals. For this purpose, the human organism has several endogenous enzymatic systems to deal with the oxidative stress. The superoxide dismutases are one set of detoxifying systems, which, as their name implies, catalyze the superoxide radical dismutation. In this regard, several copper complexes have shown evidence that they play a protective role in oxidative processes, acting as good mimics of the Cu/Zn SOD (SOD1) ,11,12,13Another aspect related to the redox behavior is the ability of copper to transport coordinated active molecules inside the cell, where they are released upon Cu(II) bioreduction before they can act .In recent decades, our group has synthesized and characterized several copper complexes with amino acids, dipeptides and 1,10-phenanthroline ,28,29,30d-ribose (using the TBARS method) was performed. Even though there are some records of the antioxidant and pro-oxidant activities of the selected copper(II) complexes, the information has never been methodically analyzed. To this end, a comparative study of the experimental evidence collected by our group and others was carried out, with special emphasis on the relationship between the activities and the available structural information. In order to shed light on the structural and electronic basis behind the redox behavior, a DFT computational analysis was performed on selected systems.In this article, the crystal structure for the copper(II)–AlaLeu complex is presented for the first time. Interestingly, it constitutes the first homotrinuclear carbonato-bridged copper(II) complex bearing a dipeptide ligand. Furthermore, and following the research on promising antitumor compounds, the antioxidant behavior of a series of copper complexes with amino acids, dipeptides and 1,10-phenanthroline was compared by measuring the SOD-like activity. To learn more about their reactivity, evaluation of the pro-oxidant activity by means of an assessment of the oxidative damage to 2-deoxy-−1)2] nH2O, [Cu(dipH−2)]⋅nH2O and heteroleptic [Cu(dipH−2)phen]⋅nH2O, where AA = amino acid, dipH = L-dipeptide and phen = 1,10-phenanthroline. The charge neutrality in the complexes was achieved by the monodeprotonation of each AA and bideprotonation of each dipeptide, both in the homoleptic and the heteroleptic complexes.The copper complexes included in this article were selected on the basis that they are chemically related but present different copper environments in solution, and some of them exhibit cytotoxic activities. They were synthesized with high purity and good yields. The general stoichiometries were [Cu(AA2]·H2O (Cu–Gly), [Cu(Ala)2] (Cu–Ala), [Cu2] , [Cu(Ser)2] (Cu–Ser), [Cu(Ile)2]·H2O (Cu–Ile), [Cu]·1/2H2O , [Cu] , [Cu(AlaGly)] (Cu–AlaGly), [Cu(AlaPhe)]·1/2H2O (Cu–AlaPhe), [Cu(PheAla)]·1/2H2O (Cu–PheAla), [Cu(AlaLeu)3(H2O)(CO3)]·PF6·H2O (Cu–AlaLeu), [Cu(AlaGly)(phen)] (Cu–AlaGly–phen), [Cu(PheAla)(phen)] (Cu–PheAla–phen), [Cu(phen)] . The code used was [Cu(Gly)3(H2O)(CO3)]·PF6·H2O where the carbonate anion acts as a bridging ligand, which was not observed for the other complexes synthesized and crystallized following the same procedure.Almost all the homoleptic copper dipeptide complexes presented similar stoichiometry, except for ..conceptu2− dismutation. According to 2− anion along the direction of the dipole moment vector (μ) towards the metal center , which has (i) a metal center that is more electrophilic and softer, (ii) an adequate location for the LUMO and (iii) stabilizing interactions with the superoxide anion.Regarding the dipeptide complexes, the experimental evidence indicated that the redox activity was highly variable . The thrfragment f. Striki2− fragment (50 = 32.3 μM).In contrast, the homoleptic Cu–AA compounds proved to be moderate SOD mimics, with activities that were almost independent of the complex chemical nature . Accordifragment h. All th2O2, probably via the formation of an adduct with H2O2 and the subsequent formation of the intermediate Cu(ligands)-OOH− upon deprotonation .All the complexes tested produced OH from Htion see  53]. Ho. Ho2O2, tendency . Among t2O2 and OOH−, some interesting aspects arose  ion. Upon protonation, the flexibility of the coordination polyehdra seems to have been a crucial factor in stabilizing the Cu(ligands)-OOH− species. The homoleptic complexes with amino acids can twist the coordination scheme, allowing the OOH− species to remain pentacoordinated (OOH)phen] species, the phen ligand was displaced by the OOHlyhedron h, similalyhedron g,i,j. ThAnalytical-grade chemicals and solvents needed for the complex synthesis were purchased from SIGMA and used as commercially available without further purification.The copper complexes with amino acids were obtained following well-known synthetic procedures ,55.3Cu(OH)2 was used, while in the case of the complexes with Val and Ile, an aqueous solution with CuSO4·H2O was added.In the case of the complexes with Gly, Ala and Ser, CuCOThe analytical results were:2]·H2O, blue crystals, (FW: 229.64), yield: 46%, Anal. Calcd; found (%) C 20.9, N 12.19, H 4.35; C 21.28, N 12.15, H 4.32; λmax (nm)/εM (M−1cm−1)(water) = 632/44.5.For [Cu(Gly)2], blue-violet crystals, (FW: 328.77), yield: 65%, Anal. Calcd; found (%) C 30.04, N 11.68, H 5.01; C 30.43, N 11.87, H 5.12; λmax (nm)/εM (M−1cm−1)(water) = 618/54.6.For [Cu(Ala)2], blue powder, (FW: 271.68), yield: 60%, Anal. Calcd; found (%) C 26.31, N 10.23, H 4.38; C 26.81, N 9.63, H 4.59; λmax (nm)/εM (M−1cm−1)(water) = 636/42.For [Cu(Ser)2], blue crystals, (FW: 295.80), yield: 30%, Anal. Calcd; found (%) C 40.57, N 9.46, H 6.76; C 40.81, N 9.60, H 6.75; λmax (nm)/εM (M−1cm−1)(water) = 612/53.3.For [Cu2]·H2O, blue crystals, (FW: 341.84), yield: 93%, Anal. Calcd; found (%) C 42.12, N 8.19, H 7.60; C 42.64, N 8.36, H 7.88; λmax (nm)/εM (M−1cm−1)(water) = 618/53.3.For ·1/2Hmax (nm)/εM (M−1cm−1)(water) = 636/68. For [Cu], blue crystals, (FW: 235.73), yield: 80%, Anal. Calcd; found (%) C 35.67, N 11.88, H 5.13; C 35.92, N 11.96, H 5.58; λ2O, blue sheets, (FW: 226.7), yield: 70%, Anal. Calcd; found (%) C 26.45, N 12.36, H 4.90; C 26.59, N 12.23, H 5.12; λmax (nm)/εM (M−1cm−1)(water) = 635/65. For [Cu(AlaGly)])]·Hmax (nm)/εM (M−1cm−1)(water) = 625/60. For [Cu(AlaPhe)])], blue crystals, (FW: 297.5), yield: 80%, Anal. Calcd; found (%) C 48.35, N 9.40, H 4.70; C 48.15, N 9.30, H 4.81; λ2O, blue crystals, (FW: 306.8), yield: 60%, Anal. Calcd; found (%) C 46.93, N 9.13, H 4.89; C 47.12, N 9.19, H 4.23; λmax (nm)/εM (M−1cm−1)(water) = 634/86. For [Cu(PheAla)])]·1/2H3(H2O)(CO3)]·PF6·H2O, blue crystals, (FW: 1065.35), yield: 70%, Anal. Calcd; found (%) C 31.40, N 7.85, H 5.54; C 32.23, N 8.17, H 6.09; λmax (nm)/εM (M−1cm−1)(water) = 626/70. For )]·5H2O, blue-purple crystals, (FW: 549.98), yield: 60%, Anal. Calcd; found (%): C 52.40, N 10.19, H 5.50; C 52.11, N 10.22, H 5.68; λmax (nm)/εM (M−1cm−1)(water) = 634, 850sh/96. For [Cu(Phe-Ala)(phen)]·4H2O, Anal. Calcd; found (%) C 53.19, N 9.54, H 6.01; C 53.21, N 9.53, H 5.45; λmax (nm)/εM (M−1cm−1)(water) = 630, 850sh/113.For [Cuphen]·4.5H−1 range on a Shimadzu Prestige 21 spectrometer. Electronic spectra were measured on a Shimadzu UV 1603 spectrophotometer, using 1 cm path-length quartz cells. Light-atom elemental analysis of the coordination compound was performed on a Thermo Scientific Flash 2000 analyzer. FT-IR spectra were registered as solid solutions (1%) on KBr in the 4000 to 400 cm3(H2O)(CO3)]·PF6·H2O were obtained by slow evaporation. A suitable crystal was selected and measured using graphite monochromated MoKα radiation (0.71073 Å) at 120.0(2) K on a KAPPA-CCD diffractometer. Using Olex2 ·PF6·H2O.d-ribose, 3.00 mM H2O2 and 40–200 µM copper(II) complex at a final volume of 1 mL. The solution was incubated at 37.0 °C for 40 min, and then 1 mL of 1%(w/v) 2-thiobarbituric acid was added and the solutions incubated again at 90.0 °C for 15 min. After cooling, the absorbance of the solutions was measured at 532 nm. The pro-oxidant activity is expressed as MDA equivalents (μM) using a calibration curve. All experiments were performed in duplicate.Technique: The reactions were performed with a pH 7.4 phosphate buffer containing 50 mM 2-deoxy-4EDTA, 1.5 mg/mL bovine albumin, phosphate buffer pH = 7, 10 mg/mL xanthine oxidase, 6 mM CuCl2 and the compound under study were prepared in a range of concentrations of 1 × 10−3–1 × 10−7 M.Technique: Aqueous solutions of 3 mM Xanthine, 0.75 mM NBT, 3 mM Na2. The absorbance of each tube was measured at 560 nm.In each reaction tube, 0.2 mL of xanthine solution, 0.1 mL of NBT solution, 0.1 mL of albumin solution, 1mL of the corresponding dilution of the study compound and 1.8 mL of phosphate buffer were added. Each tube was incubated at 25 °C and 0.1 mL of xanthine oxidase was added. It was allowed to react at 28 °C and the reaction was stopped with CuClThe corresponding blanks were made without the complex and contained only the complex, and it was verified that the complex did not affect the activity of xanthine oxidase. 4·5H2O was also considered. For comparative purposes, the activity of native superoxide dismutase from bovine erythrocytes and of the CuSO2O2, OOH− or O2−, the input geometries were constructed with the oxygenated ligand initially coordinated along the direction indicated by the electric dipole moment vector (μ) and the electrostatic potential. The initial structures were optimized in aqueous solution by means of density functional theory (DFT) ·PF6·H2O was presented and discussed. The dipeptide coordinated in a similar way to other dipeptide complexes, acting as a tridentate ligand through the terminal amino and carboxylate groups, in addition to the amidic nitrogen atom. Remarkably, it displayed a supramolecular structure significantly different from those previously obtained under the same synthetic and crystallization protocols, as it was the first homotrinuclear carbonato-bridged copper(II) complex with a dipeptide moiety.In this work, the first crystal structure of [CuOn the other hand, the redox behaviors of a series of homoleptic copper(II) complexes with amino acids and dipeptides, as well as heteroleptic complexes containing both dipeptides and 1,10-phenanthroline, were characterized through the evaluation of the pro-oxidant  and antioxidant (SOD-like activity) activities. A comparative analysis of the results showed that the Cu–AA complexes were high ROS producers and moderate SOD mimics, while the Cu–dipeptide–phen complexes were good SOD mimics and poor ROS producers. Meanwhile, the activity of Cu–dipeptide complexes was strongly dependent on the ligand.The DFT computational analysis showed that the SOD-like ability was modulated by the electrostatic interactions with the superoxide anion, which increased with the positive charge at the metal center and the magnitude of the dipole moment. The inclusion of phen as coligand proved to be an efficient way to enhance the electrostatic interactions, as well as to stabilize the superoxide-complex intermediate, enhancing the antioxidant activity. Another way to increase the SOD-like activity was to position a phenyl ring near the metal center (as in Cu-AlaPhe), since it led to a higher contribution of the copper ion to the LUMO, in conjunction with greater condensed local softness and electrophilicity in the metal center. 2O2 was not modulated by electrostatic interactions. Rather, a high level of ROS production was in fact associated with higher global hardness and copper electrophilicity, along with lower condensed-to-copper local softness. Another important feature that had a profound impact on the pro-oxidant ability of the copper complexes was the nature of the coordination environment. When it lacked bulky groups, it allowed the H2O2 to easily reach the labile coordination sites, thermodynamically favoring the reaction. This is why the phen coligand or the phenyl substituent in the dipeptide ligands precluded the approach of the H2O2 molecule, hampering the OH production. Finally, the flexibility of the coordination polyhedra proved to be a crucial factor in stabilizing the Cu(ligands)-OOH− species, giving rise to better pro-oxidant profiles. These findings reveal, for the first time, the structural and electronic bases behind the high ROS production displayed by homoleptic complexes with amino acids, for which the flexibility of the coordination sphere and the absence of bulky ligand substituents make them the best pro-oxidant candidates.Concerning the pro-oxidant ability, the computational evidence indicated that the OH production from H


T was isolated from mine tailings in the Harz Mountains near Goslar, Germany. Cells of BGR 140T were Gram-stain-positive, endospore-forming, motile and rod-shaped. BGR 140T grew aerobically at 25–55 °C (optimum 45 °C) and at pH 1.5–5.0 (optimum pH 3.0). The results of analysis of the 16S rRNA gene sequences indicated that BGR 140T was phylogenetically related to different members of the genus Sulfobacillus, and the sequence identities to Sulfobacillus acidophilus DSM 10332T, Sulfobacillus thermotolerans DSM 17362T, and Sulfobacillus benefaciens DSM 19468T were 94.8, 91.8 and 91.6 %, respectively. Its cell wall peptidoglycan is A1γ, composed of meso-diaminopimelic acid. The respiratory quinone is DMK-6. The major polar lipids were determined to be glycolipid, phospholipid and phosphatidylglycerol. The predominant fatty acid is 11-cycloheptanoyl-undecanoate. The genomic DNA G+C content is 58.2 mol%. On the basis of the results of phenotypic and genomic analyses, it is concluded that strain BGR 140T represents a novel species of the genus Sulfobacillus, for which the name Sulfobacillus harzensis sp. nov. is proposed because of its origin. Its type strain is BGR 140T (=DSM 109850T=JCM 39070T).A mixotrophic and acidophilic bacterial strain BGR 140 Sulfobacillus was first described in 1978 [S. benefaciens (type strain BRGM2T=DSM 19468T=ATCC BAA–1648T) [S. thermosulfidooxidans (type strain AT-1T=VKM B-1269T=DSM 9293T) [S. acidophilus (type strain NALT=ATCC 700253T= DSM 10332T) [S. thermotolerans (type strain Kr1T=VKM B-2339T=DSM 17362T) [S. sibiricus (type strain N1T=VKM B-2280T=DSM 17363T) [S. disulfidooxidans [Alicyclobacillus disulfidooxidans [Sulfobacillus have been tentatively assigned to Clostridiales Family XVII Incertae sedis [Sulfobacillus are moderately thermophilic or thermotolerant acidophiles [Sulfobacillus can obtain energy by oxidizing ferrous iron, elemental sulfur and sulfide minerals in the presence of small amounts of yeast extract. Owing to the sulfur- and iron-oxidizing activity, microorganisms of this genus are important in the oxidative dissolution of sulfide minerals [S. thermosulfidooxidans improves chalcopyrite bioleaching by maintaining a favourable redox potential [S. thermosulfidooxidans was not inhibited in the presence of 200 mM NaCl [T as the type strain of a novel species of the genus Sulfobacillus.The genus dophiles . Cells ominerals . For insMine tailings samples were obtained by coring from 15 to 26 m depths of the Rammelsberg sulfidic mine tailings in the Harz Mountains near Goslar, Germany (51°52′ N 10°25′ E) . The maiT (NCBI Taxonomy ID: 2729629) was isolated from the enrichment culture by means of the overlay technique [o solid medium was prepared according to methods described previously [o solid plates. The isolate was grown in liquid medium containing 20 mM ferrous iron and 0.02 % yeast extract or 5 mM glucose and 0.02 % yeast extract at variable pH and temperature as described below.Bioleaching experiments at low pH in basal salts (BS) medium were carried out for metal extraction from the tailings samples . A mixedechnique . Brieflyeviously . Cells fg, and the pellet was washed twice with 10 mM Tris buffer, pH 8. DNA extraction was achieved by using the FastDNA Spin Kit for Soil  according to a modified protocol [T was sequenced by Microsynth . Sequences were manually edited and curated with BioEdit 7.2.5 [T was phylogenetically related to members of the genus Sulfobacillus with sequence identities to S. acidophilusT, S. thermotoleransT and S. benefaciensT of 94.8, 91.8, and 91.6 %, respectively . The low sequence identities to species with validly published names provide proof of the status of the novel strain as representing a novel species within the genus Sulfobacillus. The 16S rRNA gene sequences of related type strains were downloaded from NCBI and aligned using the silva Incremental Aligner (sina v1.2.11) [silva_115NR database, followed by manual editing to remove gaps and positions of ambiguous nucleotides in mega X [mega using the (i) neighbor-joining [Sulfobacillus forms three major clusters, while strain BGR 140T forms a separate clade together with other isolates and clone sequences   and the n mega X . Phyloge-joining  and (ii)-joining  algorith-joining , and . Genomic DNA extraction was carried out using MasterPure Gram Positive DNA Purification Kits from Epicentre Biotechnologies according to the manufacturer’s instructions. Libraries were prepared applying the Nextera XT DNA Library Preparation Kit (Illumina). Samples were sequenced on a NextSeq 550 Sequencing System from Illumina using a NextSeq 500/550 High Output Kit v2.5. The genome was assembled via SPAdes 3.14.0 (http://cab.spbu.ru/software/spades/) on short read genome data, which was recorded in DSMZ. After genome assembling, contigs were annotated via Prokka and finally analysed via the Type Strain Genome Server  [Sulfobacillus annotated by NCBI that are available in NCBI, S. thermosulfidooxidansT, S. thermotoleransT and S. acidophilusT. The former one has a size of 3.86 Mb and the latter two of 3.31 and 3.56 Mb, respectively  under the algorithm ‘coverage’ and distance formula 5d [4d) [T with those of S. thermosulfidooxidansT, S. thermotoleransT and S. acidophilusT are 25.5, 22.2 and 19.5 %, respectively   and the S, DSMZ) . The genectively . In genermula d5 . The phyrmula d5  from GBDrmula d5 . The ove d5 [d4)  of the gectively . The resommittee  are consT. ANI calculations were done using a web-service for ANI computation between a pair of genome sequences [T for S. thermosulfidooxidansT, S. thermotoleransT, S. acidophilusT were 66.8, 66.1 and 68.3 %, respectively. This confirms the classification of BGR 140T as representing a novel species when the species boundary cut-off of 95–96 % is considered [The average nucleotide identity (ANI) was also used to evaluate the phylogenetic position of BGR 140equences . The ANInsidered , althoughttps://www.kegg.jp/) and eggNOG 5.0 [T contains all genes putatively encoding proteins of the complete TCA cycle. Similarly, all genes putatively encoding proteins for a complete pentose phosphate cycle were detected. In addition, putative genes encoding proteins for carbon fixation, including the reductive pentose phosphate cycle , were predicted, nevertheless, the Calvin cycle seemed to be incomplete. Genes for assimilatory sulfate reduction and bacterial sulfide:quinone reductase (SQR) were detected. Genes belonging to the dsrE family (dissimilatory sulfate reduction protein E) were predicted along with TusA genes (sulfur carrier protein TusA), genes of the heterodisulfide reductase complex (hdr) and the SOR (sulfur oxygenase reductase) gene. The SOR gene of BGR 140T is similar to the one from S. acidophilus strain TPY  and exhibits 49.68 % similarity to the SOR gene of Acidianus ambivalens DSM 3772T . SOR seems to be present in all strains of members of the genus Sulfobacillus [To predict protein encoding genes (PEGs) and ribosomal nucleic acids the RAST server  with thegNOG 5.0 . BGR 140bacillus . A multiT was cultivated in BS medium containing 5 mM glucose and 0.02 % yeast extract in a 2 l bioreactor (Electrolab). Cultures were stirred at 100 rpm and aerated (0.5 l min−1). The bioreactor temperature was set at varying temperatures (30–55 °C) at a constant pH of 3.0, or varying pH values (2.5–4.5) at a constant temperature of 45 °C. Additional experiments were done in 100 ml shake flasks  to test for pH and temperature limits for growth. Semi-logarithmic plots of cell growth by monitoring culture OD600 against time were used to identify exponential growth phases, and from them specific growth rates were calculated. Tests for growth with different carbon sources were done according to the methods of Johnson et al. [S. thermosulfidooxidans, S. acidophilus, S. benefaciens, S. sibiricus and S. thermotolerans were comparatively studied for their substrate utilization and anaerobic growth.For characterization of growth optima, BGR 140T and the other described species of the genus Sulfobacillus were carried out by the German Collection of Microorganisms and Cell Cultures GmbH  where cultivation of the reference strains was done on DSMZ medium at 40 °C for 2 days.Chemotaxonomic analyses of strain BGR 140For analysis of DAP , cells were hydrolyzed in 4 M HCl at 100 °C for 15 h. The hydrolysates were subjected to thin-layer chromatography on cellulose plates according to a protocol described previously .−1 to 200 °C, ramp 5 °C min−1 to 270 °C, ramp 120 °C min−1 to 300 °C and hold for 2 min. The inlet temperature was set to 170 °C and then linearly increased with 200 °C min−1 up to 350 °C and hold for 5 min. The MS parameters were set to aux temperature 230 °C, source temperature 230 °C and electron impact ionization at 70 eV with mass range of m/z 40–600 or 40–800, respectively. Peaks were identified based on retention time and mass spectra. The position of single double bounds was confirmed by a derivatization to the corresponding dimethyl disulfide adduct [Cellular fatty acids were analysed after conversion into fatty acid methyl esters (FAMEs) by saponification, methylation and extraction using minor modifications of previously described methods . The fate adduct .2SO4, anisaldehyde/H2SO4) [Polar lipids were extracted from freeze dried cells using a chloroform:methanol:aqueous 0.3 % NaCl mixture  and were analysed by two dimensional silica gel thin layer chromatography . The fire/H2SO4) . Respirae/H2SO4) .T were motile, straight rods, 2.27±0.96 µm long and 0.59±0.13 µm wide as determined by SEM observations . Cells formed biofilms and aggregates on pyrite, as described for other bioleaching microorganisms [T grew aerobically at 25–55 °C  and at pH 1.5–5.0 . No growth was observed at 20 °C or 60 °C.Cell morphology was studied by using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) of thin sections. Cells of BGR 140rvations . Cells frvations  and S1. rganisms . Cells oT was A1γ peptidoglycan with meso-diaminopimelic acid as the diagnostic diamino acid. The major polar lipids were determined to be glycolipid, phospholipids and phosphatidylglycerol . The fatty acid composition of BGR 140T was different to that of other species of the genus Sulfobacillus with validly published names. The predominant fatty acid was 11-cycloheptanoyl-undecanoate, there were also minor amounts of anteiso-C17 : 0, C16 : 0, anteiso-C15 : 0, C12 : 0, iso-C16 : 0 and iso-C15 : 0. BGR 140T shared its minor fatty acids with several other type strains but 11- cycloheptanoyl-undecanoate only occurred in S. acidophilus and BGR 140T (T was demethylmenaquinone (DMK) 6. DMK-6 was also the predominant respiratory quinone in cells of S. acidophilus, S. benefaciens and S. sibiricus. Cells of S. thermotolerans possessed MK-6 (80.3 %) as the main isoprenoid quinone, while a smaller amount of MK-7 (19.7 %) was also detected. In a previous study MK-7 was the only reported respiratory quinone of S. thermotolerans [S. thermosulfidooxidans included DMK-6 (39.8 %), MK-6 (45.1 %) and MK-7 (15.1 %)  . The DNAbacillus . Growth BGR 140T . It sharSulfobacillus harzensis sp. nov. (harz.en′sis. N.L. masc. adj. harzensis of or pertaining to the Harz Mountains).meso-diaminopimelic acid.Cells are 2.27±0.96 µm long and 0.59±0.13 µm wide, motile, Gram-stain-positive rods that form subterminal or central round or oval endospores. Colonies grown on ferrous iron agar are fried-egg-like and round orange-centred and the colony size was 0.2–1 cm after about seven days cultivation. Growth occurs at 25–55 °C (optimum 45 °C) and at pH 1.5–5.0 (optimum pH 3.0). Facultative autotroph capable of autotrophic growth with elemental sulfur, ferrous iron and metal sulfides. Facultative anaerobe, capable of anaerobic growth with ferric iron as an electron acceptor. A suite of organic substrates including glucose, mannose, arabinose, fructose, sucrose, starch, ethanol, mannitol, glutamic acid, alanine and casein can be assimilated. The major polar lipids are glycolipid, phospholipid and phosphatidylglycerol. The most abundant cellular fatty acid is 11-cycloheptanoyl-undecanoate. The respiratory quinone is DMK-6. The cell wall peptidoglycan is A1γ composed of T (=DSM 109850T=JCM 39070T), which was isolated from mine tailings in the Harz mountains, near Goslar, Germany (51°52′ N 10°25′ E). The genomic DNA G+C content of the type strain is 58.2mol%. The unassembled and assembled genome sequencing data (JABBVZ000000000) and 16S rRNA gene (MK951693) were assigned to the NCBI BioProject: PRJNA627582.The type strain is BGR 140Click here for additional data file.


Based on morphological and morphometric data, the new species is characterized by a 601-734 µm long body, a prominent perioral labial disc and oblique amphidial slits, 10-11 µm long stylet, vulva at 65-67% of body length, 15.5-17 µm long spicules, and an elongate conoid tail with a pointed terminus. It is the second species of the genus Discopersicus and differs from its counterpart D. iranicus, by its anterior position of vulva, shorter stylet, lower M.B. ratio, different shape of tail tip, and shorter spicules in male specimens. A molecular phylogenetic analysis using the D2-D3 expansion segments of 28S rDNA sequences, placed the new species in close relationship with five sequences of the genus Boleodorus.Discopersicus  Khuzestan province is located in southwest of Iran, and with arid and warm climate, has an important role for production of agricultural products in Iran. A large number of nematodes have been described from this province . Family Discobasiria, but the relevant paper has never been published. Discotylenchus iranicus  in Dezful, Khuzestan province, southwestern Iran. Nematodes were extracted using the tray method  of each forward and reverse primers, 2 μl of DNA template and 11 μl deionised water. Reactions were carried out in a Thermal Cycler  with an initial denaturation step of 95°C for 4 min followed by 33 denaturation cycles of 94°C for 30sec, annealing for 30 sec at 57°C, extension at 72°C for 90sec and a final extension at 72°C for 10 min. The quality of DNA targets were checked by electrophoresis of 4 μl from each of PCR products in 1% agarose gel containing ethidium bromide. The PCR products were visualized and photographed under UV light and the length of each PCR product was measured by comparison with the Low DNA Mass Ladder . The PCR products were purified and sequenced directly for both strands using the same primers with an ABI 3730XL sequencer . The newly obtained sequences were submitted to GenBank database under accession numbers MW202233 and MW202234 as indicated on the 28S phylogenetic tree.Nematode DNA was extracted from single live female individuals of the new species, as described by Tanha Maafi et al. (2003), and used as template for polymerase chain reaction (PCR). The D2-D3 expansion segments of 28S rDNA were amplified using the forward D2A (5′-ACAAGTACCGTGAGGGAAAGTTG-3′) and reverse D3B (5′-TCGGAAGGAACCAGCTACTA-3′) primers . Each PCAtetylenchus longilabiatus . Cephalic framework inconspicuous, weakly sclerotized. Stylet delicate, conus about one-third, 3.3 to 3.7 µm or 32 to 35% of the total stylet length. Dorsal pharyngeal gland opening 1.5 to 2.0 µm from stylet base. Pharynx tylenchoid with procorpus cylindroid. Pharyngeal median bulb oval with distinct valve, 39 to 45 µm from anterior end. Isthmus slender, longer than procorpus. Basal bulb pyriform to saccate-shaped, 8.0 to 10.0 µm in width and 25 to 30 µm in length. Nerve ring nearly at mid of isthmus and located at 74 to 78 µm from anterior end. Pharyngo-intestinal valve hemispherical. Excretory pore slightly sclerotized, located at anterior end of basal bulb. Hemizonid one to three annuli anterior to the excretory pore, 85 to 89 µm from anterior end. Deirids at level or slightly posterior to excretory pore, 88 to 97 µm from anterior end. Reproductive system monodelphic. Vulva with transverse slit, not protruding, without lateral flaps. Vagina 8.7 to 9.5 µm or 48 to 53% of vulva body diameter. Postvulval uterine sac short, 8.7 to 9.5 µm or 47 to 52% of vulval body diameter. Spermatheca offset, oval, 8.0 to 9.5 µm × 23 to 27 µm. Uterus perpendicular to body axis. Ovary outstretched, oocytes arranged in a single row. Rectum very short. Anus minute. Tail elongate-conoid ending to a pointed tip.Male: General characterization similar to female except in genital system. Testis 140 to 170 µm or 24 to 31% of body length. Spicules tylenchoid-shaped. Gubernaculum simple, slightly curved. Bursa adcloacal, 20 to 25 µm long.Holotype, five paratype females and four paratype males were deposited in the nematode collection of the Department of Plant Protection, Faculty of Agriculture, University of Zanjan, Zanjan, Iran. Three paratype females and three paratype males were deposited in the nematode collection of the Department of Animal Biology, Plant Biology and Ecology of the University of Jaén, Spain.Daucus carota L.) plants in Dezful, Khuzestan Province, southwestern Iran, collected by Manouchehr Hosseinvand at February 2013 and May 2019 .Soil around of carrot , shorter stylet (10 to 11 vs 11 to 15 µm), shorter distance from anterior end to valve of median bulb (M.B. ratio = 35-39 vs 39.1-46.0), different tail tip shape (pointed vs rounded) and shorter spicules (15.5-17.0 vs 17.5-22.0 µm).Discopersicus hexagrammatus n. sp., yielded two single fragments of 658bp long. The newly generated sequences (accession numbers: MW202233 and MW202234) showed a very low intraspecific variability with 4 different nucleotides and 0 indel (99.4% similarity). The average nucleotide compositions of the two new sequences were: % A; % T; % C; and % G, respectively. The BlastN search using these sequences showed 87% similarity (100% coverage) with Neopsilenchus magnidens  , 86% similarity (99% coverage) with Boleodorus thylactus  , and 86% similarity (67% coverage) with Discopersicus iranicus . The partial D2-D3 dataset for phylogenetic analysis comprised of 42 sequences, including 38 sequences of different Tylenchidae species, Atetylenchus longilabiatus (MN807620) and Psilenchus hilarulus (EU915489) as outgroups and the two newly obtained sequences. D. hexagrammatus n. sp. formed a sister clade (PP = 100%) with five isolates of the genus Boleodorus  . This clade has fallen into a well supported major clade formed by sequences of ten isolates of the genus Basiria , three isolates of the genus Neothada , two isolates of the genus Neopsilenchus  (JQ005018 and KP313832) and D. iranicus.Amplification and sequencing of D2-D3 expansion segments of 28S rDNA from two different individuals of Discopersicus in the subfamily Boleodorinae , we could observe two major clades A and B. The clade A containing species with non-offset spermatheca except D. iranicus, and the clade B including species with offset spermatheca. Adding the sequences of species having offset spermatheca from the genus Basiria can be used for testing this result.Our 28S rDNA tree supported the position of odorinae  in closesearches . The new