Many studies have shown effective protection from myocardial ischemia–reperfusion injury (MIRI) in animal models, but few, if any, treatments have yielded a substantial reduction in clinical. Several studies showed significant therapeutic effects for the Chinese patent medicine Suxiao Jiuxin Pill (SJP) in MIRI, although the specific molecular mechanisms remain undefined. Recently, increasing evidence indicates an important role for m6A modification in autophagy regulation in MIRI, and SJP has not been investigated in this regard.In vivo experiments were performed in a Wistar rat MIRI model. In vitro assays were conducted in hypoxia/reoxygenation (H/R)-treated H9c2 cells. H9c2 cells with ALKBH5 and GSK3β silencing were constructed by lentivirus transfection. TUNEL and Annexin V/PI assays were carried out for apoptosis detection. Then, m6A modification was detected with the EpiQuik m6A RNA methylation quantification kit, and GFP-RFP-LC3B was used to observe dynamic changes in autophagy. The autophagosome structure was assessed by Transmission electron microscopy. qPCR and immunoblot were performed for mRNA and protein analyses, receptively.SJP significantly mitigated MIRI in rats, reducing infarct size and myocardial apoptosis, and improving left ventricular function. In addition, SJP inhibited autophagy through the GSK3β/mTOR pathway in MIRI rats. In cultured H9c2 cells, SJP significantly inhibited H/R- related apoptosis and autophagic activity through the GSK3β/mTOR pathway. Additionally, SJP enhanced ALKBH5 expression in H/R cardiomyocytes, which is important in impaired m6A modification. Interestingly, ALKBH5 knockdown enhanced autophagy and apoptosis in H/R-induced cells, whereas SJP reversed these effects. Further experiments showed that autophagic activity and apoptosis enhanced by ALKBH5 deficiency are GSK3β/mTOR pathway dependent in H/R-treated H9c2 cells. After SJP administration the above effects were alleviated, suggesting SJP inhibited autophagy through the ALKBH5/GSK3β/mTOR pathway in H/R-induced cardiomyocytes. These effects of SJP were common to its two main constituents, including tetra-methylpyrazine (TMP) and borneol (BOR).SJP improves MIRI in rats and alleviates autophagy and apoptosis in H9c2 cells through the ALKBH5/GSK3β/mTOR pathway, thanks to its two major constituents TMP and BOR.The online version contains supplementary material available at 10.1186/s13020-023-00736-6. Approximately 17.3 million individuals die from acute myocardial infarction (AMI) worldwide each year, which is estimated to exceed 23.6 million by 2030 .Cardiomyocyte stress and reperfusion may lead to abnormal mitochondrial function, oxidative stress, and inflammation that lead to autophagy . The eveLigusticum Chuanxiong  and Dryobalanops aromatica C.F.Gaertn. (Bing pian). Chemical studies have revealed that the 36 components of SJP include 6 phenolic acids (such as ferulic acid and vanillic acid), 28 phthalides (such as ligustilide and senkyunolide A), ligustrazine and Borneolum Syntheticum were identified through the ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF MS) [Numerous experimental studies have shown effective protection from MIRI in animal models, but translation into clinical practice has been less successful. The clinical application of natural products or traditional Chinese medicine (TCM) compounds may open a new avenue for MIRI therapy. Suxiao jiuxin pill (SJP) is composed of Rhizome -TOF MS) , 10. We -TOF MS) , 12. Pha-TOF MS) . Our pre-TOF MS) , suggest-TOF MS) . Despite-TOF MS) , and theHere, we used an integrated approach in a rat model of MIRI, together with a hypoxia/reoxygenation (H/R) model in H9c2 cells, to examine the important role of SJP in alleviating MIRI. Furthermore, unveiling the molecular mechanism of SJP in reversing MIRI through m6A methylation modification-mediated autophagy may be a promising target for MIRI treatment.SJP was obtained from the Sixth Chinese Drugs Factory of Tianjin Zhongxin Pharmaceutical . The SJP quality control adhered to the specifications and test procedures as described in the Pharmacopoeia of the People’s Republic of China , and the quality control report were provided in Additional file For MIRI surgeries, Wistar wild-type rats  were utilized. Male rats underwent anesthesia with 1% phenobarbital sodium and were positioned supine on a heating pad (37 ℃). Following left thoracotomy (between the 4th and 5th ribs), left anterior descending (LAD) coronary artery ligation was carried out with a 7–0 nylon suture. Regional ischemia was reflected by the discoloration of the myocardium distal to the occlusion and elevated ST segment detected by electrocardiography. Ischemia induction used a 30-min coronary occlusion and a 2-h reperfusion. All animal protocols had approval from the Shanghai University of Traditional Chinese Medicine Medical Center Institutional Animal Care and Use Committee .−1 d−1), SJP medium-dose , and SJP high-dose  groups. Sham animals underwent intragastric saline administration and the above surgical procedure except for LAD occlusion; the I/R group had intragastric saline administration and I/R surgery. According to previous studies, clinical dosing, and the Meeh-Rubner equation of dose conversion between humans and rats, the SJP dose groups received SJP intragastrical each day for 14 days before I/R surgery.Rats were divided into 5 groups of 6 animals, including the sham, I/R, SJP low-dose  was carried out via the aortic cannula. Then, the LAD ligature was tied again. The heart was weighed and sliced horizontally into five slices. After weighing them individually, these slices underwent incubation with 1% 2,3,5-triphenyltetrazolium chloride  in 1 × PBS for 8 to 15 min at 37 ℃ and fixation with 10% neutral buffered formalin for 24–48 h. Images were captured under a microscope. TTC—positive  and negative  areas were assessed digitally. Infarct size (%) was calculated by the following formula: infarct sizes (%) = IR / left ventricular (LV) area × 100 .Apoptosis was assessed with a TUNEL staining kit , as instructed by the manufacturer. In brief, myocardial tissue sections underwent a 20-min fixation with 4% paraformaldehyde and incubation with 20 µg/mL proteinase K for 40 min at ambient. Upon treatment with 0.1% Triton X-100 for 30 min, incubation was carried out with the TUNEL reaction mixture for 1 h, shielded from light. Finally, a BX51 inverted fluorescence microscope (Olympus) was used to count apoptotic cells in 4 random high-power fields per specimen.Echocardiograms were obtained on a Vevo 2100 system (MS400C probe) in the conscious state after gently restraining the rats followed by 24 h reperfusion, by sonographers blinded to grouping. Left ventricular end-diastolic (LVEDD) and end-systolic (LVESD) dimensions were derived from M-mode images. FS (in %) was derived as (LVEDD − LVESD)/LVEDD. Measurements were performed at the level of papillary muscles per standard protocols.6/well and incubated in DMEM containing 10% fetal bovine serum  and penicillin/streptomycin . Following a 24-h culture, complete medium was replaced with freshly prepared DMEM supplemented with cardiomyocyte growth supplements at 37℃ in presence of 5% CO2. Next, H9c2 cells were cultured with reoxygenation (95% air and 5% CO2) for 4 h after hypoxia  for 2 h . Then, the cells were seeded in a 6-well plate at 2 × 10n = 3 per group) were examined, including the blank, H/R (H/R + 0.1% ethanol), SJP (H/R + 50 mg/mL SJP), CHIR99021 (H/R + 50 mg/mL SJP + 1 µM CHIR99021), rapamycin (H/R + 50 mg/mL SJP + 0.1 µM rapamycin), TMP (H/R + 100 mg/mL TMP), and BOR (H/R + 25 mg/mL BOR) groups. The dosages of all drugs for cell culture are based on cell counting kit 8 (CCK8) assay  and borneol (BOR) . To asse H/R + 0.% ethanoln = 3 per group) were examined, including the blank, H/R (H/R + 0.1% ethanol), SJP (H/R + 50 mg/mL SJP), TMP (H/R + 100 mg/mL TMP), and BOR (H/R + 25 mg/mL BOR) groups. H9c2 cells in each 6 well plate were treated with 0.1% ethanol, 50 mg/mL SJP, 100 mg/mL TMP or 100 mg/mL BOR for 1 h before H/R modeling.To define the function of m6A modification in SJP-regulated autophagy in H/R-induced H9c2 cells, 5 groups (n = 3 per group) were examined, including the shRNA-Ctrl + H/R, shRNA-Ctrl + SJP (shRNA-Ctrl + H/R + 50 mg/mL SJP), shRNA-Ctrl + TMP (shRNA-Ctrl + H/R + 100 mg/mL TMP), shRNA-Ctrl + BOR (shRNA-Ctrl + H/R + 25 mg/mL BOR), shRNA-ALKBH5 + H/R, shRNA-ALKBH5 + SJP, shRNA-ALKBH5 + TMP, shRNA-ALKBH5 + BOR, shRNA-GSK3β + H/R, shRNA-GSK3β + SJP, shRNA-GSK3β + TMP, and shRNA-GSK3β + BOR groups. The drugs were added in the incubation medium of transfected cells for 1 h before H/R modeling in H9c2 cells.To determine whether ALKBH5 and GSK3β/mTOR signaling played crucial roles in SJP-dependent autophagy, 12 groups (n = 3 per group) were examined, including the shRNA-ALKBH5 + H/R, shRNA-ALKBH5 + SJP, shRNA-ALKBH5 + TMP, shRNA-ALKBH5 + BOR, shRNA-ALKBH5 + GSK3β-inhibitor (1 µM CHIR99021) + H/R group, shRNA-ALKBH5 + GSK3β-inhibitor + SJP, shRNA-ALKBH5 + GSK3β-inhibitor + TMP, and shRNA-ALKBH5 + GSK3β-inhibitor + BOR groups. Both CHIR99021 and the drugs were added to the transfected cells for 1 h before H/R modeling in H9c2 cells.To further confirm whether GSK3β is crucial in ALKBH5-dependent autophagy after SJP treatment, 8 groups , as directed by the manufacturer. In brief, after staining of H9c2 cells with Annexin V and PI for 30 min at ambient, a FACScan flow cytometer  was utilized for analysis within 1 h.H9c2 myocytes underwent infection 72 h post-seeding with adenovirus harboring RFP-GFP-tagged LC3 (designed by the Hill Lab at UT Southwestern), at a multiplicity of infection (MOI) of 30 PFU/cell. Subsequently, the cells underwent culture under reoxygenation for 4 h after hypoxia for 2 h.Harvested cells underwent PBS washing and were fixed with 2.5% glutaraldehyde at 4 °C overnight. This was followed by three washes with 0.1 M PBS and incubation with 1% citric acid  at 4 °C for 3 h. After another PBS wash, dehydration was performed with an ethanol gradient; next, propylene oxide was added. The Spurr resin  was utilized for embedding, with polymerization occurring at 70 °C. Next, 70-nm sections were obtained on an ultrathin slicer , and underwent staining with uranyl acetate and lead citrate . A transmission electron microscope  was utilized for analysis.6/well in 6-well plates. At 24 h post-plating, cardiomyocytes underwent incubation with short hairpin RNA (shRNA) negative control , shRNAs targeting ALKBH5 , and shRNAs targeting GSK3β  as directed by the manufacturer.H9c2 cardiomyocytes were isolated and seeded at 1.2 × 10Total RNA isolation utilized TRIzol  as instructed by the manufacturer, followed by deoxyribonuclease I  treatment. A NanoDrop was employed to assess RNA quality. The altered m6A amounts in mRNA were assessed with a colorimetric EpiQuik m6A RNA Methylation Quantification Kit  as proposed by the manufacturer. Poly-A-purified RNA (200 ng) was utilized to analyze every specimen [−ΔΔCt method was employed for analyzing triplicate data normalized to GAPDH expression. The primers used were: ALKBH5-F 5′-GACCTGCGTGAGAAGCTCAA-3′and ALKBH5-R 5′-TGGTACTTCCGTTTGGTGGTC-3′.Total RNA underwent reverse transcription with the Script One-Step RT-PCR kit . Then, qRT-PCR was performed with SYBR Premix EX Taq kit  on an ABI 7500 instrument (Applied Biosystems). The 2Primary antibodies targeting LC3B , Atg5 , Atg7 , p62 , ALKBH5 , p-GSK3β , GSK3β , p-mTOR , mTOR , and Beclin-1  were used. Horseradish peroxidase (HRP)-linked anti-mouse IgG and anti-rabbit IgG  were used, respectively. Data were normalized to β-actin expression. Immunoreactive bands were quantified by densitometry and computer-assisted image analysis.P < 0.05 was deemed to be statistically significant. GraphPad Prism 7.0  was utilized for graphing.Data are mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) was carried out for comparing multiple groups, with post hoc Bonferroni test, using SPSS 21.0 . P < 0.05), but no significant differences were observed in SJP groups while the SJP medium-dose group reduced the I/R zone markedly. The therapeutic effect of drugs on MIRI is also reflected in the improvement of cardiac function, and hence, the effect of SJP was evaluated through an electrocardiograph. The SJP groups significantly increase the levels of LVEF and LVFS in MIRI rats compared to the I/R group . Taken together, these results indicated that SJP inhibits MIRI in rats.As shown in Fig. P < 0.05). Compared to the I/R group, the SJP groups significantly decreased the levels of LC3B II/I and Beclin-1 (P < 0.05). However, Atg5 did not show the same trend. Together, these results suggested that SJP inhibits autophagy in MIRI rats.Under the condition of cardiac I/R injury, the process of autophagy is activated in response to the energy crisis and oxidative stress. Then, we assessed the effect of SJP on the autophagy marker LC3B and autophagy-related molecules, including Atg5 and Beclin-1 Atg12) in MIRI rats by western blot. As shown in Fig.  in MIRI GSK-3 is a serine/threonine kinase involved in many cellular functions in the heart, including gene expression, hypertrophy, and apoptosis , 19. AlsP < 0.05). Thirdly, tandem fluorescent mRFP-GFP-LC3 transfection was performed in H9c2 (AdLC3-H9c2) [Firstly, to determine whether the effect of SJP and its active components (TMP and BOR) inhibiting autophagy were involved in H/R-induced cardiomyocytes injury, H9c2 cells were exposed to H/R. Consistent with the results in MIRI rats, the levels of LC3B II/I, Atg5, and Beclin-1 were elevated after H/R but were reversed under the intervention of SJP  . Normal C3-H9c2) . The autC3-H9c2) . SJP redConsistent with the results of I/R in rats Fig. a, the trTo further explore the role of GSK3β and mTOR in H/R-induced H9c2 cells with SJP administration, we used CHIR99021 and rapamycin to reduce the expression of GSK3β and mTOR, respectively, in H9c2 cells. In the current experiments, the addition of CHIR99021 further inhibited p-mTOR/mTOR, LC3B II/I, Atg5, Beclin-1, and p62 levels, as well as autolysosomes and autophagosomes compared to the SJP group  . Next, wGSK3β mRNA and autophagy-related genes were dramatically downregulated and mTOR mRNA was upregulated in H/R-induced cardiomyocytes with SJP pretreatment . Fig S2. HPLC fingerprint of Suxiao Jiuxin Pills. Fig S3. HPLC fingerprint of Ferulic acid. Fig S4. HPLC fingerprint of Senkyunolide I. Fig S5. HPLC fingerprint of Senkyunolide A. Fig S6. Establishment of hypoxia-reoxygenation model and determination of reoxygenation time. Fig S7. Determination of drug concentration. Fig S8. SJP and ALKBH5 overexpression inhibit excessive autophagy (n=3). *, #, P < 0.05 versus NC + H/R group. Fig S9. Virus interference efficiency of ALKBH5 and GSK3β by qPCR (n=3). Table S1. Specific situation of main components of SJP (1). Table S2. Primer information.


The nadifloxacin oxalic acid co-crystal is stabilized by intermolecular hydrogen bonds. FT–IR, DSC and XRD studies were carried out to confirm the structure and a Hirshfeld surface analysis was performed to investigate the intermolecular interactions. H,5H-pyridoquinoline-2-carboxylic acid] with oxalic acid, C19H21FN2O4·0.5C2H2O4, was prepared by slow evaporation from a chloroform:acetone solvent system. Nadifloxacin belongs to the group of antibacterial drugs. The co-crystal is stabilized through an intramolecular O—H⋯O bond and intermolecular hydrogen bonds. It was studied by FT–IR spectroscopy, differential scanning calorimetry and X-ray diffraction. Hirshfeld surface analysis indicated that the major contribution to the packing is from O⋯H/H⋯O interactions.The 2:1 co-crystal of nadifloxacin [systematic name: 9-fluoro-8-(4-hydroxypiperidin-1-yl)-5-methyl-1-oxo-6,7-dihydro-1 A larger ring motif is formed with two molecules of nadifloxacin and two molecules of oxalic acid having an In the crystal, O4—H4⋯O1ds Fig. 2 forms ch) Fig. 3. The hydif Fig. 4.4.44(16) ring motif is observed. Both adducts are connected through tetrameric clusters of water molecules  and OA  were dissolved in a mixed chloroform–acetone solvent (5 ml:5 ml), heated on a water bath for 15–20 min and then kept undisturbed for slow evaporation. Crystals were obtained at room temperature after 24-48 h. They were characterized by FTIR, DSC, and single crystal XRD.Omnic software  was used to analyze the data. Each sample was scanned in the range 400-4000 cm−1Infrared spectra of NAD·OA crystals were recorded using FT–IR spectroscopy (Thermo-Nicolet 6700 FTIR–NIR spectrometer) with the samples made in KBr pellets. −1 and 1682 cm−1, respectively, while in the co-crystal, the former now appears at 1734 cm−1. Differential Scanning Calorimetric (DSC) analysis indicated the melting points of NAD and OA to be 478.9 K and 387.8 K, respectively, while the melting point of the co-crystal is 438.8 K.In the IR spectrum, the C=O stretching frequencies for NAD (carboxylic acid group) and OA were observed at 1716 cm6.Crystal Explorer17 –(c), respectively  = 1.2–1.5Ueq(C). C-bound O atoms were freely refined.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989023002244/mw2196sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989023002244/mw2196Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989023002244/mw2196Isup3.cmlSupporting information file. DOI: 2194837CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


N-phenyl-1,4-dihydropyridine-3-carboxamide were obtained by slow evaporation of a solution of the title compound in acetone. The crystal structure is sustained by hydrogen bonds between the NH and the carbonyl O function of the 4-oxo-1,4-dihydropyridine ring of the molecules, forming infinite chains along the b-axis direction.Single crystals of 4-oxo- 12H10N2O2, shows a nearly planar conformation. The crystal structure is sustained by hydrogen bonds between the NH and the carbonyl O function of the 4-oxo-1,4-dihydropyridine ring of the molecules, forming infinite chains along the b-axis direction.The title compound, C N-phenylnicotinamide, which is an efficient molluscicide , 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride  and hydroxybenzotriazole  were dissolved in 6 ml of DMF and stirred at 0°C for 1 h. Then diisopropylethylamine  and aniline  were added and the reaction was completed under continuous stirring at 50°C for 12 h. Then 20 ml of deionized water were added to the reaction mixture, which was placed into a refrigerator at 5°C overnight. The resulting precipitate was collected by filtration and washed with deionized water to obtain 0.306 g (39% based on 4-hydroxynicotinic acid) of the title compound Fig. 3. The obtCrystal data, data collection and structure refinement details are summarized in Table 210.1107/S2414314623006016/im4020sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2414314623006016/im4020Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2414314623006016/im4020Isup3.cmlSupporting information file. DOI: 2280191CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Pteridium revolutum and their antioxidant and antiglycated activities. Three novel water-soluble heteropolysaccharides, named PRP0, PRP1, and PRP2, were isolated from P. revolutum. The average molecular weight was determined by high performance gel permeation chromatography analysis as 1.04 × 106, 8.39 × 105, and 7.37 × 105 Da, respectively. Their structures were characterized using physicochemical and spectroscopic methods. The antioxidant and antiglycated activities were assayed in vitro. PRP0, PRP1, and PRP2 consist of l-Ara, l-Rha, d-Man, d-Xyl, d-Fuc, d-Gal, and d-Glc in different proportions. PRP1 mainly has a backbone of -linked d-Man and (1 → 3)-linked d-Gal on main chain. PRP2 is mainly composed of -linked d-Man and (1 → 3)-linked d-Gal on main chain. All polysaccharides have strong scavenging power on 2,2-difenil-1-picril-hidrazil and hydroxyl radicals and significantly antiglycated activity in Bovine serum albumin-Glucose model, which showing that the polysaccharides have potential application value on the functional food.This study aims to analysis the structures of polysaccharides isolated from  It has the functions of removing dampness, diuresis, antipyretic and insect repellent.Bracken ferns have more than 12,000 species widely distributed in uplands and marginal areas throughout Asia, Australia, Europe, North and South America, with about 2600 species in China . Certainountries . Many chum genus . PteridiP. revolutum still unclear. Therefore, the present work aimed to investigate the extraction, separation, structure characterization and the bioactivity of polysaccharides from this plant. The results exhibit that the novel polysaccharides have strong antioxidant and antiglycated activities in a concentration dependent manner. It could be used as a functional ingredient in food to reduce the formation of glycosylation products of protein.Plant polysaccharides have drawn considerable attention from the world because of their potential application value in various industries . Previou22.1P. revolutum was collected from Suichang County, Zhejiang province, China. A voucher specimen was deposited in Zhejiang Gongshang University, Hangzhou, China. The dried aerial part of P. revolutum was ground to pass a 60-mesh sieve and extracted by refluxing ethanol  to remove liposoluble compounds. Then, the sample was extracted with hot distilled water . The extracts obtained were vacuum concentrated at 55 °C by a rotary evaporator  and precipitated with ethanol . The sample were centrifuged . The precipitate was re-dissolved in ultra-pure water to remove the protein using the Sevage method . The aqueous phase was concentrated, dialyzed ) against distilled water for at least 2 d. Finally, the solution was vacuum concentrated (55 °C) and lyophilized to offer the crude polysaccharide (cPRP).The sample of The cPRP sample was re-dissolved in ultra-pure water, filtered  and chromatographed using DEAE Sepharose Fast Flow  chromatography column , eluted with ultra-pure water and the NaCl (0.1 ∼ 0.5 M) successively. The eluent (10 mL/tube) was checked using the phenol–sulphuric acid method and then pooled, dialyzed, and lyophilized. The fractions eluted by ultra-pure water, 0.1 and 0.2 M NaCl were further purified on a Sephadex G-200 gel  CC (5.0 cm × 50 cm), eluted with ultra-pure water to obtain three polysaccharides PRP0, PRP1, and PRP2 .2.2T-series Dextran  (The molecular weight (MW) and homogeneity analysis of the three purified polysaccharides were achieved by high performance gel permeation chromatography  2424, Waters Corporation. USA) using a Ultra- hydrogel 2000 column (7.8 × 300 mm), eluted by ultra-pure water (0.30 mL/min). The average MW was determined using the calibration curve, which was prepared with standards  50, 10) .l-arabinose (l-Ara), l-rhamnose (l-Rha), d-mannose (d-Man), d-xylose (d-Xyl), d-fucose (d-Fuc), d-galactose , and d-glucose (d-Glc)] and inositol were acetylated with the same method. All the resulting aldononitrile acetates were detected by Gas Chromatography  using a temperature program of 120 °C (maintain 3 min), raised to 210 °C . Carrier gas: N2, 1.0 mL/min. Injector/Detector temperature: 250/280 °C.The monosaccharide compositions of PRP0, PRP1, and PRP2 were analyzed according to the method reported before . Briefly2.3Section 2.2.The purified polysaccharides (each 10 mg) were hydrolyzed by TFA  . The pro2.44 , respectively. Ethylene glycol (1 mL) was used to destroy the excess NaIO4. The consumption of periodate was detected by Ultraviolet spectroscopy . The generated products of formic acid were titrated by NaOH (0.01 M). After reduced by NaBH4 and neutralized with HOAc to pH 7.0, the solution was then hydrolyzed in TFA  and analyzed the monosaccharide compositions by GC  were methylated with the method reported  and dete2.6−1) was tested using a Nicolet iS 10 Spectrometer . 1D and 2D-NMR (Nuclear Magnetic Resonance) spectra were recorded with a Bruker AVANCE III 500 spectrometer  at 60 °C (333 K) dissolved in D2O (99.9%).UV absorption spectrum (200–400 nm) was detected on a Shimadzu UV-1880 spectrophotometer . IR spectrum  on 2,2-difenil-1-picril-hidrazil (DPPH) and hydroxyl radicals (•OH) was evaluated according to previous methods .2.82.8.13), BSA (10 mg/mL), glucose (0.5 M), and samples with different concentrations (0.25 ∼ 2.0 mg/mL). Negative control: no aminoguanidine (AG) and polysaccharide. Positive control: AG instead of polysaccharide. All the mixtures were reacted in dark . The mixture is cooled in ice water bath and stored at 4 ℃ for the following experiments. The fluorescence of the solution was measured using at an excitation/emission wavelength of λ380/λ450 nm . The inhibition activity was calculated as following equation (Eq).F0: Fluorescence intensity of negative control. F1: Fluorescence intensity of sample/AG solution.The anti-glycation ability of PRPs was assayed using Bovine serum albumin-Glucose (BSA-Glu) model according to Hafsa’s method  with som2.8.2λ530 nm on a Syhergy H1 Microplate Reader .The amadori products were determined by method reported before . 0.1 mL 2.8.3λ294 nm . Glyoxal was used a standard to make a calibration curve.The content of dicarbonyl compounds were measured by Girard-T assay . 0.4 mL 2.8.4λ335/λ385 nm. The effect on pentosidine was calculated as following Eq.F0: Fluorescence intensity of negative control. F1: Fluorescence intensity of sample/AG solution.The content of pentosidine was determined by a LS55 spectrofluorometer . The flu2.8.5λ370 nm. The PCO content (nmol carbonyl/mg protein) was calculated using coefficient .The level of protein carbonyl group (PCO) was evaluated using reported method . Briefly2.8.6λ412 nm. The content of thiol group (nmol thiol group/mg protein) was calculated using extinction coefficient of the DTNB .The content of thiol group was measured by Ellman method . 1.0 mL 2.9Statistical analysis was analyzed by T-test to assess the significance (SPSS 20.0) of difference between groups and all data were given as mean ± SD.33.1P. revolutum by hot distilled water extraction, precipitated in 75% EtOH (v/v), deproteinated and freeze-dried. cPRP gave four peaks on the DSFF CC for purification , and 1.00: 2.38: 11.56: 5.84: 10.65: 1.06: 3.34 (PRP2-I), respectively. These results demonstrated that almost d-Man was present as the main chain and d-Gal was both in main chain and side chain in PRP1 and PRP2. Therefore, the backbone of PRP0 was composed of d-Man and d-Glc (3:2) because these two monosaccharides were mainly found in PRP0-I after 2 M TFA hydrolysis. While the backbones of PRP1 and PRP2 were consisted of d-Man and d-Gal in 1: 1.2 and 1.1: 1, respectively.The three polysaccharides were partially hydrolyzed with TFA and dialyzed with MWCO 3,500 member. The monosaccharide compositions of the out and in the dialysis bag were collected and analyzed separately. The results , Table 14 . 0.935, 1.012, or 0.805 mol periodate was consumed per mole of sugar residue to produce 0.400, 0.364, or 0.238 mol HCOOH for PRP0, PRP1 or PRP2, respectively  bonds amounted to 40.0% in PRP0, 36.4% in PRP1, 23.8% in PRP2, with the (1 → 2)-/(1 → 4)-/-/-linked glycosyl bonds amounted to 13.5% (PRP0), 28.4% (PRP1), 32.9% (PRP2), and (1 → 3)-/-/-/-/-/-linked glycosyl bonds amounted to 46.5% (PRP0), 35.2% (PRP1), 43.3% (PRP2), respectively. The GC–MS spectra of Smith degradation products indicated the existence of erythritol, glycerol, rhamnose, arabinose, fucose, mannose and galactose -linked d-Man were the major residues of the main-chain in PRP1. (1 → 5)-Linked l-Ara, (1 → 3)-linked l-Rha, (1 → 2)- linked d-Man, (1 → 2)- and (1 → 4)-linked d-Xyl, (1 → 3)-linked d-Fuc, (1 → 6)-linked d-Glc, - and (1 → 6)-linked d-Gal joined to position C-3 of -linked d-Man as branches. l-Ara, l-Rha, d-Xyl, and d-Glc were at the termini of the side-chains. PRP2 mainly consisted of a backbone of -linked d-Man and (1 → 3)-linked d-Gal, with (1 → 5)-linked l-Ara, (1 → 3)-linked l-Rha, (1 → 2)- and (1 → 4)-linked d-Xyl, (1 → 3)-linked d-Fuc, (1 → 6)-linked d-Gal as branches. d-Glc and d-Gal were located at the end of side-chains.The above results made clear that (1 → 3)-linked 1H (13C NMR spectrum (C 98 to 112 ppm (C 101.70 ppm was were attributed to the (1 → 6)-linked d-Galp residue, the corresponding anomeric proton signal appeared at δH 5.02 ppm, which affirmed the (1 → 6)-linked d-Galp was α-d-conformation (H/δC 5.39/98.65 and 4.82/101.88 ppm belonged to H1/C1 of (1 → 3)- and -linked dα--Galp residues, respectively -α-lAraf--αManp--α-dManp--α-dXylp--α-dXylp--α-LRhap--α-DFucp--β-dlcp- -β-D- Glcp--dα- -Manp--dα--Xylp-(1→/→4)-dα--Xylp-(1→ were downfield shifted to 80.95/76.93 ppm from 74.53/72.87 ppm in →4)-dα--Xylp--dα--Galp--lα--Rhap- in →6)-α-D-Galp- in →3,6)-dα--Galp- in →3, 6)-dα--Manp--α-dGalp--α-dXylp--α-dylp- -αManp--α-lRhap--α-dGalp--α-dFucp--lα--Rhap--dα--Galp--lα--Rhap- in →5)-lα--Araf and C6 (66.67 ppm) in →6)-dα--Galp--dα--Manp-(1→ were observed at δC 79.10 (C2) and 80.22 (C4) ppm. All the linkage positions of the desired glycosidic bonds are verified by the chemical shifts moved to lower field of the corresponding carbon atoms on the sugar ring. The NMR data of the ten main residues were listed in Like PRP1, the 2-By comprehensive analyses of the information of partial acid hydrolysis, periodate oxidation, methylation, and NMR spectra, the predicted structures of two novel polysaccharides PRP1 and PRP2 are shown in 3.33.3.1The scavenging activity of four polysaccharides on DPPH free radicals were shown in P. aquilinum , and pentosidine is a representative compound of fluorescent AGEs. As shown in The contents of PCO and free thiol group were used to evaluate the protein oxidation induced by AGEs. As shown in The effects of PRPs on protein thiol group level in glucose-glycated BSA were shown in Many factors can induce the formation of AGEs and protein damage. The production of reactive oxygen species and free radical is one of the mechanisms . In our 4P. revolutum polysaccharides were reported for the first time. The spectrometry and spectroscopy analyses indicated that PRP1 consists of -linked d-Man and (1 → 3)-linked d-Glc, while PRP2 consists of -linked d-Man and (1 → 3)-linked d-Gal on main chain.The preparation and structure characterization from l-Arabinose, l-Rhamnose, and d-Galactose may be an important factor for the activity. Moreover, polysaccharide with smaller MW usually has better performance on antioxidant activity because of better solubility. Consequently, in future research, a comprehensive understanding of the structure–activity relationships of P. revolutum polysaccharides will be studied.Interestingly, compare the antioxidant activities between PRP1 and PRP2, PRP1 has better hydroxyl radical scavenging ability, while PRP2 has better DPPH scavenging ability. These differences may be resulted in the physiochemical and structural properties of the two polysaccharides, such as the molecular weight, monosaccharide contents, glycosidic linkage, main-chain, branched-chain, and water solubility. The difference in the content of In the BSA-Glu model, PRPs displayed inhibitory activity on the formation of Amadori products, dicarbony compounds, pentosidine, fluorescent AGEs, PCO and have a protective effect on protein thiol group. These results suggest that PRPs can be used as an natural antioxidants and the inhibitors of AGEs.Kui-Wu Wang: Conceptualization, Methodology, Writing – review & editing, Supervision. Xin-Yuan Sheng: Writing – original draft, Investigation, Data curation. Bin Wu: Data curation, Writing – review & editing. Hong Wang: Writing – review & editing. Jian-Bo Chen: Resources. Shi-Wei Wang: Investigation, Validation, Data curation.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


TEF1-α sequences), in this study, the wild boletes in Jiangxi Province were investigated, and five new species are described: Austroboletus albus, Xanthoconium violaceipes, Xanthoconium violaceofuscum, Xerocomus rutilans and Xerocomus subsplendidus. Descriptions and hand drawings of the new species are presented.Fungi of the order Boletales are extremely important in both ecology and economy, since most of them are ectomycorrhizal fungi, which play vital roles in maintaining forest ecosystems, water and soil protection, vegetation restoration and so on. Although previous studies have shown that this order has a very high species diversity in China, there are few reports on the species diversity of boletes in Jiangxi Province, China. Based on morphological  and phylogenetic analyses (ITS, LSU, and  The order Boletales is one of the largest groups of Basidiomycota and most of them are not only ectomycorrhizal fungi but also edible fungi. They are economically and ecologically important . They coIn the past few decades, over 500 species of Boletales, including nearly 100 new species, have been reported from China ,26,27,28Jiangxi has the second highest forest coverage in China, which is dominated by evergreen broad-leaved forests with a subtropical warm and humid monsoon climate and abundant rainfall. The climatic conditions and vegetation types are very suitable for the growth and reproduction of macrofungi. It is predicted that there are more than 12,000 species of fungi in Jiangxi Province, including more than 1000 species of macrofungi .Until 2016, 659 species had been reported in Jiangxi Province , and theIn this study, 140 boletes specimens were collected in Jiangxi Province. All specimens were completely dug out with a shovel after taking photos of the habitat and related characteristics of the basidiomata, then a part of the context was cut from the connection between pileus and stipe and dried with silica gel to extract DNA, and the remaining specimens were dried at 41–56 °C by a food-grade fruit dryer.Twelve specimens of five new boletes species were stored in the Herbarium of Fungi of Jiangxi Agricultural University (HFJAU). The macroscopic morphological characteristics mainly come from field records and photographs of basidiomata. Color codes were obtained from Kornerup & Wanscher . MicromoThe number of measured basidiospores is given as n/m/p, which means that the measurements were created on n basidiospores from m basidiomata of p collections. Dimensions of basidiospores are given as (a)b–c(d), where the range b–c represents a minimum of 90% of the measured values (5th to 95th percentile), and extreme values (a and d), whenever present , are in parentheses. Q represents the ratio of length/width of the spores. Qm refers to the average Q of basidiospores ± sample standard deviation .TEF1-α)  (12–)13–17(–17.5) × (6–)6.5–7.5(–8.5) μm, Q = (1.63–)1.75–2.2(–2.5), Qm = 2.10 ± 0.21, including ornamentation, subfusiform to amygdaliform and slightly angular; surface with intricate reticulum or irregular pits becomes shorter to nearly smooth toward the both poles and yellow to golden yellow in KOH. Hymenophoral trama boletoid is composed of thin- to slightly thick-walled hyphae, 3–8 μm wide and hyaline in KOH. Cheilocystidia are 36–67 × 7.4–10.3 μm, subfusiform to subfusoid-mucronate, sometimes narrowly mucronate, rostrate, thin-walled and hyaline to grayish brown in KOH. Pleurocystidia are absent. Pileipellis is a trichoderm 65–160 μm thick, composed of hyaline to grayish yellow in KOH, thin-walled, and 3–7.5 μm diam; terminal cells are 22–60 × 3–10 μm, clavate or subterete, with an obtuse apex. Pileus trama is composed of thin-walled hyphae 4–11 μm in diameter. Clamp connections are absent in all tissues.Fagus longipetiolata) and Pinaceae (Tsuga chinensis and Pinus massoniana).Habitat: Solitary or group on the wet ground under mixed forests of Fagaceae .Xanthoconium violaceipes F. Zhou, H.Y. Song and D.M. Hu, sp. nov. .Description: Basidiomata are medium-sized to large. Pileus 1–10 cm in diameter, initially hemispherical, becomes convex to applanate with maturity, surface is dry, purple-black (11F1) to purple (11B3) when young, brownish green (30B6–30E6) when mature, margin is lighter and deeper in the center. Context is 0.3–0.7 cm in thickness in the center of the pileus, white (1A1) and does not change when injured. Hymenophore adnate around the stipe are quite crowded; pores are circular to angular, 2–3 per mm, white (1A1) when young and yellow (1A5) to yellow-brown (1B5) when old; tubes up to 1 cm long are white (2A1) to pale brown (2B2) and do not change in color when injured. Stipe 2–10.5 × 0.6–1.5 cm, central, cylindrical, solid; surface dry, with obvious dark purple (16D3) stripes, slightly interwoven near the pileus; context is white (16A1) to light pink (16A2), no reaction when bruised; basal mycelium white. Odor indistinct.Basidia are 20–36 × 11.5–16 μm, thin-walled, clavate and four-spored; sterigmata are 3–7 μm long, colorless to hyaline in KOH. Basidiospores [40/2/2] (11.5–)12.5–16.5(–17) × (3.5–)4–5.5(–6) μm, Q = (2.18–)2.27–3.33(–3.71), Qm = 3.00 ± 0.35, ellipsoid to subfusiform to cylindrical, slightly thick-walled (up to 0.5 μm), golden yellow in KOH, smooth. Hymenophoral trama are composed of subparallel hyphae 5–12 μm broad, colorless to hyaline in 5% KOH. Cheilocystidia are 35–55 × 8–12 μm, clavate to subfusiform, rarely mucronate, rostrate, thin-walled and colorless to hyaline in KOH. Pleurocystidia are 45–80 × 21–24 μm, clavate and cystiform. Pileipellis is a trichoderm 60–156 μm thick, composed of hyaline hyphae in KOH, thin-walled, and 4–13 μm in diameter; terminal cells are 14–46.5 × 8–18.5 μm, clavate to pyriform or cystidioid, with obtuse apex. Pileus trama is composed of thin-walled hyphae 5–12 μm in diameter. Stipitipellis is 55–110 μm thick, clavate with an obtuse apex, terminal cells (18–25 × 5–11 μm), and colorless in KOH. Stipe trama is composed of parallel hyphae 5–10 μm wide. Clamp connections are absent in all tissues.Lithocarpus spp., Castanopsis spp. and Quercus spp.Habitat: Scattered on soil in subtropical forests of Fagaceae, including Distribution: Jiangxi Province, China.Additional specimens examined: China, Jiangxi Province: Shangrao City, Yanshan County, Wuyishan Nature Reserve, 117°45′42″ E, 27°50′3″ N, elev. 1750 m, 1 August 2021, F. Zhou, HFJAU12004 (WYS121); Shangrao City, Yanshan County, Wuyishan Nature Reserve, 117°45′44″ E, 27°50′3″ N, elev. 1840 m, 23 August 2022, F. Zhou, HFJAU12005 (WYS619).Xanthoconium violaceofuscum F. Zhou, H.Y. Song and D.M. Hu, sp. nov. .Description: Basidiomata are medium-sized to large. Pileus is 3–13 cm diameter, hemispherical to subhemispherical to applanate, surface is dry, purplish brown (7C2), surface is normally densely covered with dark reddish brown (7E3) tomentose scales. Context are up to 1.5 cm in thickness in the center, pale purple (7B2), change to blue (19B5) first, then black (7E1) when injured. Hymenophore adnate around the stipe, scattered; pores circular to angular, 1–2 per mm, purplish brown (7D4) when young and yellow (5B5) to yellowish brown (5D5) when old; tubes are up to 1.5 cm long, purplish brown (7D4) to yellow (5B5) to yellowish brown (5D5), change to blue (19B5) to black (7E1) when injured. Stipe is 3.5–7 × 1–3 cm, central, cylindrical or clavate, solid; surface is dry, purple-brown (7E3) to black (7E1) when young and black (6F1) when mature. Context is pale purple-brown (6D2), changes to black (6F1) when bruised; basal mycelium is purple-gray (6D1). Odor indistinct.Basidia are 22–34 × 7.5–13 μm, thin-walled, clavate and four-spored; sterigmata are 2–6 μm long. Basidiospores are [40/2/2] 7–9(–11.5) × (3.5–)3.7–5(–6) μm, Q = (1.67–)1.75–2.29(–2.36), Qm = 1.97 ± 0.19, ellipsoid to subfusiform, thin-walled and golden yellow in KOH, smooth. Hymenophoral trama is composed of subparallel hyphae 6–10 μm broad, yellowish white to hyaline in 5% KOH. Cheilocystidia are 37.5–64 × 7–10 μm, subfusiform to subfusoid-mucronate, sometimes narrowly mucronate, rostrate, thin-walled, hyaline to grayish brown in KOH. Pleurocystidia are 33–118 × 12–23 μm, clavate or cystiform. Pileipellis is a trichoderm 30–65 μm thick, composed of hyaline to grayish yellow in KOH, thin-walled, 7–12 μm in diameter; terminal cells are 30–65 × 10–13.5 μm, clavate or subterete, with an obtuse apex. Pileus trama is composed of thin-walled hyphae 7–12 μm in diameter. Stipitipellis ca. is 150 μm thick, with clavate and cystiform terminal cells (37–74 × 8–15.5 μm), colorless and sometimes yellowish brown in KOH. Stipe trama is composed of parallel hyphae 6–12 μm wide. Clamp connections are absent in all tissues.Lithocarpus spp., Castanopsis spp. and Quercus spp.Habitat: Scattered on soil in subtropical forests of Fagaceae, including Distribution: Currently only known from Jiangxi Province, China.Additional specimens examined: China, Jiangxi Province: Shangrao City, Yanshan County, Wuyishan Nature Reserve, 117°45′13″ E, 27°50′14″ N, elev. 1550 m, 27 August 2022, F. Zhou, HFJAU12008 (WYS724).Xerocomus rutilans F. Zhou, H.Y. Song and D.M. Hu, sp. nov. .Description: Basidiomata are medium-sized to large. Pileus is 2.9–9 cm in diameter, initially hemispherical, becomes convex to applanate with maturity and the surface is dry, purple (11A2) to ochre (9B4) when young, brownish yellow (5B6) when mature. Context is up to 1 cm in thickness in the center of the pileus, white (5A1) and does not change when injured. Hymenophore adnate to slightly depressed around the stipe, quite crowded; pores iiragular, 2–3 per mm, yellow (2A6) when young and yellowish brown (5C6) when old, slightly higher around the stipe; tubes are up to 1 cm long, golden (2A7) to yellowish brown (5C6) and do not change in color when injured. Stipe is 4–9 × 1–3 cm, central, cylindrical, solid, dry and covered with white (2A1) tomentose scales; context is white (2A1) and has no reaction when bruised; basal mycelium is white (1A1). Odor indistinct.Basidia are 26–45 × 9–11.5 μm, thin-walled, clavate and four-spored; sterigmata are 3.5–6 μm long. Basidiospores are [40/2/2] (8–)8.5–11.5(–12) × (3.5–)4–5.5(–6) μm, Q = (1.83–)2–2.56(–2.75), Qm = 2.27 ± 0.23, ellipsoidal to elongated to fusiform, thin-walled, pale yellow to yellow-brown in KOH, smooth. Hymenophoral trama is composed of subparallel hyphae 5–13 μm broad, colorless to hyaline in KOH. Cheilocystidia are 22–36 × 9–12 μm, common, clavate to subclavate, thin-walled and hyaline to grayish brown in KOH. Pleurocystidia are 28–48 × 8–11 μm, common, clavate to subfusiform or cystidioid. Pileipellis is a trichoderm 60–140 μm thick composed of hyaline to yellow hyphae in KOH, thin-walled, and 6–11 μm in diameter; terminal cells 26–81 × 6–10 μm, clavate to cylindrical. Pileus trama is composed of thin-walled hyphae 5–12 μm in diameter. Stipitipellis is up to 100 μm thick, with clavate to ventricose terminal cells (25–47 × 7–16 μm), hyaline to grayish brown in KOH. Stipe trama is composed of parallel hyphae 4–12.5 μm wide. Clamp connections are absent in all tissues.Fagus longipetiolata) and Pinaceae (Tsuga chinensis and Pinus massoniana).Habitat: Solitary or group on the wet ground under mixed forests of Fagaceae .Xerocomus subsplendidus F. Zhou, H.Y. Song and D.M. Hu, sp. nov. .Description: Basidiomata are small to medium-sized. Pileus is 2.2–5.6 cm in diameter, subhemispherical to applanate to infundibulate, initially earth-yellow (6B5) to yellowish brown (6B7), and brown (6C7) to dark brown (6E5) later and have lighter margins; surface dry, always covered with yellowish brown (6C5) tomentose when young, cracking into brown (6A4) to dark brown (6E5) scales with age. Context is white (6A1), up to 0.8 cm in thickness in the center of the pileus, and does not change when injured. Hymenophore is slightly depressed and decurrent around the apex of the stipe, quite scattered; pores are irregular, 1 per mm or less, yellow (3A6), changes to blue (22D5) when bruised; tubes are up to 1 cm long, golden (2A6) to yellowish brown (2D7), changes to blue (22D5) when injured. Stipe is 2.5–5 × 0.4–0.7 cm, central, cylindrical or crooked, solid; covered with pale brown (2B3) to reddish brown (9B4) tomentose in the upper part; context is white (3A1) to pale brown (3A2), no reaction when bruised; basal mycelium white. Odor indistinct.Basidia are 27–38.5 × 9–11 μm, thin-walled, clavate and four-spored; sterigmata are 3–7 μm long. Basidiospores are [60/3/3] 9–15.5(–16) × 4–5.5(–6) μm, Q = (1.82–)2–3.11(–3.56), Qm = 2.40 ± 0.32, ellipsoid to subfusiform to cylindrical, pale yellow to yellow-brown in KOH and smooth. Hymenophoral trama is composed of subparallel hyphae 5–13 μm broad, colorless to hyaline in KOH. Cheilocystidia are 39–65 × 8.5–12 μm, lanceolate, clavate to ventricose, thin-walled and hyaline to grayish brown in KOH. Pleurocystidia are 31–82 × 10–13 μm, common, similar to cheilocystidia. Pileipellis is a trichoderm 70–230 μm thick composed of hyaline hyphae in KOH, thin-walled, 5–13 μm in diameter; terminal cells are 27.5–54.5 × 10–16 μm, clavate or cystidioid. Pileus trama is composed of thin-walled hyphae 5–12 μm diam. Stipitipellis is 45–140 μm thick, with clavate to pyriform terminal cells (18–28.5 × 8.5–15 μm) and hyaline to grayish brown in KOH. Stipe trama is composed of parallel hyphae 6–18 μm wide. Clamp connections are absent in all tissues.Fagus longipetiolata) and Pinaceae (Tsuga chinensis and Pinus massoniana).Habitat: Solitary or group on the wet ground under mixed forests of Fagaceae ; Shangrao City, Yanshan County, Wuyishan Nature Reserve, 117°45′43″ E, 27°50′29″ N, elev. 1740 m, 27 August 2022, F. Zhou, HFJAU12011 (WYS718).Austroboletus albus conforms to the characteristics of Austroboletus. It shared similar morphological characteristics with A. subflavidus, A. albidus and A. roseialbus. However, A. subflavidus has larger basidia [27–49(51) × 12–19 μm] and basidiospores [(13.1)15.9 ± 1.15(19.5) × (5.5)7.0 ± 0.58(8.7) μm], slightly shorter and coarser stipe [(2.9)4.5–7.5(10.2) × (0.4)0.6–1.8(2.0) cm], pileus surface often cracks with matures , smaller pleurocystidia [(47)52–60 × 7–10 μm] and longer cheilocystidia [(26)37–67 × 7–10 μm], slightly larger basidiospores [(14.0)15.5 ± 0.8(17.8) × (5.0)5.6 ± 0.3(6.2) μm], larger stipitipellis terminal cells [20–40 × (4)8–15(18) μm] with rare tw- spored and four-spored basidia [Xa. separans can be easily distinguished by larger pileus [60–200(220) mm] and stipe [60–150 × 10–30 mm], longer basidia [(27)32–41(43) × 10–12 μm], smaller pleurocystidia [(52)55–70 × 11–14 μm] and larger cheilocystidia [(24)30–65(80) × 8–15(18) μm]  .Xa. violaceipes are clustered on one branch within Xanthoconium with 100% bootstrap support, and sister to Xa. separans with 82% statistical support, but they have a long genetic distance.Based on the phylogenetic analyses, the collections of Xa. violaceofuscum has similar pileus and stipe colors to Boletus violaceofuscus [B. violaceofuscus has larger basidiospores [(14.0)15.5 ± 0.8(17.8) × (5.0)5.6 ± 0.3(6.2) μm] and basidia [(29)32–40(43) × 10.5–13.5 μm], has smaller pleurocystidia [(47)52–60 × 7–10 μm], and the tubes are white or whitish at first, stipe has net–like ornamentation and at the base has white tomentum, has a few two-spored and four-spored basidia in stipitipellis, and the flesh is white.Morphologically, eofuscus ,71. HoweXa. violaceofuscum are clustered on an independent branch within Xanthoconium with 100% bootstrap support.In phylogenetic analyses, the collections of Xerocomus rutilans is similar to Aureoboletus zangii , larger cheilocystidia (24–51 × 9–16 μm) and pleurocystidia [40–68(77) × 11.5–18 μm].Morphologically, s zangii ,72 by itXerocomus rutilans are clustered into an unique branch within Xerocomus with high statistical support (BS = 100%), and related to X. rugosellus  and pleuro- and cheilocystidia (45–80 × 10–14 μm).Phylogenetically, the samples of ort BS = 00%, and gosellus  with 50%Xerocomus subsplendidus is similar to X. fraternus, X. subparvus and X. yunnanensis , and the shape of pileipellis terminal cells is subcylindrical. X. subparvus has cream to yellowish pileus context and a red tinge near pileipellis, staining bluish slowly or indistinctly when injured, context of the stipe is pale yellow at the upper part, staining bluish slowly when injured and pale brown to pale-red brown at the lower part; X. subparvus has smaller basidiospores [(8.5)9–10.5(11.5) × (3)3.5–4(4.5) μm], and the shape of pileipellis terminal cells is subcylindrical. X. yunnanensis is characterized by the tubes staining red-brown slowly when young and becoming bluish when mature on injury, the fresh yellow context of pileus near the pileipellis, and the white to yellowish context of stipe, bluing indistinctly when cut [X. yunnanensis has smaller basidiospores [(9)10–11.5(13) × 4–4.5(5) μm], and the terminal cells of pileipellis are tapered or bullet-shaped.Morphologically, nanensis . HoweverXerocomus subsplendidus form an independent branch within Xerocomus with 100% bootstrap support, and are sistered to Xerocomus sp.  with 58% statistical support, but they have a long genetic distance.According to our phylogeny, the sequences of


As a result of the different nature of the involved guest species, the crystal components in 1a create a three-dimensional supramolecular architecture, while the crystal structure of 1b consists of two-dimensional supramolecular aggregates extending parallel to the crystallographic ab plane.In the crystal structures of the formamide monosolvate ( In addition to the development of acyclic receptor molecules, the triethylbenzene scaffold was found to be valuable for the construction of macrocyclic systems. The possibilities for functionalization of acyclic and macrocyclic molecules of this type are manifold, allowing the synthesis of a whole range of compounds for systematic binding studies. Some examples of suitable functional groups, which can act as recognition units and have been considered in our studies, are heteroaromatic units such as pyridine-, pyrimidine- (Lippe 2.1a) and the n-propanol/H2O solvate/hydrate (1b) of the title compound, C38H50N4O3, crystallize in the triclinic system (PZ = 2). The model for the least-squares refinement includes positional disorder for one of the amino moieties of the structure of 1b with occupancies of 0.78/0.22. The perspective views of the host–guest complexes shown in Fig. 11a and 61.3 (1), 81.3 (1), 80.7 (1)/88.4 (3)° for 1b. Despite the large number of strong donor/acceptor sites in the host molecule, only three intramolecular C—H⋯O hydrogen bonds  and a weak C—H⋯N bond  = 2.59 Å]. With the exception of the amino hydrogen H3, which for sterical reasons seems to be excluded from non-covalent bonding, all other strong donors participate in molecular association comprising O—H⋯O [d(H⋯O) = 2.26 Å], O—H⋯N [d(H⋯N) = 2.00 (1) Å] and N—H⋯O type [d(H⋯O) = 2.46 (2) Å] hydrogen bonds. The pattern of intermolecular bonding is completed by C—H⋯O interactions [d(H⋯O) = 2.52–2.63 Å], C—H⋯π contacts  = 2.71–2.81 Å] and π–π stacking  Å], the latter formed by the hydroxymethyl-substituted aromatic rings of inversion-related molecules. Within this three-dimensional supramolecular network, the solvent molecules form N—H⋯O bonded dimers [d(H⋯O) = 2.01 (1) Å] of the graph-set motif et al., 1995In the complex structure of 1a Fig. 3, the forn-propanol proved to be an inclusion compound of 1 with n-PrOH and H2O possessing a host/guest stoichiometric ratio of 1:0.78:0.22. The model for least-squares refinement assumes partial occupancies for the alcohol and water molecules, i.e. the solvent species are distributed in a statistical manner in the voids of the host lattice. Despite the presence of strong donors/acceptors, the disordered moiety of the host hardly participates in molecular association. Only the minor disorder component of this residue is involved in any intermolecular interactions, by forming a weak C—H⋯O bond to the water oxygen [d(H⋯O) = 2.16 Å] . As shown in Fig. 4a, the oxygen atom of the alcohol molecule is linked to one of the hydroxy hydrogens of the host . In an analogous way, this hydrogen acts as a donor site for hydrogen bonding to the water molecule . Unfortunately, the positions of the water hydrogen atoms could not be obtained from the difference electron-density map, so that the complete pattern of hydrogen bonding in the crystal of 1b could not be elucidated. Nevertheless, a striking motif of hydrogen bonds is present, involving a total of three hydroxy groups of the host and the propanol molecules . They form chain-like synthons in the direction of the b axis, bounded by an amine H and a ring N atom, and can be described by the graph set ab plane. As the surfaces of the two-dimensional aggregates are defined by the non-polar molecular parts, interlayer interactions are restricted to van der Waals forces.The colourless rod-like crystals obtained from 0) Fig. 5. Taking 0) Fig. 5 can be r4.et al., 2016β-d-glucopyranoside in the solid state aminomethyl]-3,5-bis[aminomethyl]-2,4,6-triethylbenzene diethyl ether solvate trihydrate  was stirred for 30 minutes. Subsequently, a solution of 1-(bromomethyl)-3,5-bis[aminomethyl]-2,4,6-triethylbenzene  in 30 mL of THF/CH3CN  was added dropwise and the resulting mixture was stirred at room temperature and under the exclusion of light (the progress of the reaction was monitored by TLC). After filtration, the solvents were evaporated at reduced pressure and the yellow oil was treated with THF/water. The oil was separated from the aqueous phase and dissolved again in THF, dried over MgSO4 and the solvent was removed. By treating the oily residue with diethyl ether/n-hexane, the product was obtained as a white solid in 88% yield . Crystals of the title compound suitable for single crystal X-ray diffraction were grown by slow evaporation of an ethyl acetate/formamide  solution (1a) or a n-propanol solution (1b) at ambient temperature.A suspension of 2,6-bis(hydroxymethyl)-4-methylphenol  and potassium carbonate  in 30 mL of THF/CHAnalysis data: m.p. = 472 K; 1H NMR : δ = 1.12 , 1.23 , 2.23 , 2.29 , 2.34 , 2.69 , 2.74 , 4.33 , 4.46 , 5.20 , 6.10 , 6.34 , 7.11 ; 13C NMR : δ = 16.6 (CH2CH3), 16.8 (CH2CH3), 20.8 (ArCH3), 21.1 (ArCH3), 22.6 (CH2CH3), 23.0 (CH2CH3), 24.0 (ArCH3), 40.6 (CH2NH), 60.8 (CH2OH), 70.3 (CH2OAr), 103.6 (ArC), 113.9 (ArC), 129.5 (ArC), 132.0 (ArC), 133.1 (ArC), 133.7 (ArC), 134.0 (ArC), 144.5 (ArC), 144.7 (ArC), 149.0 (ArC), 152.6 (ArC), 156.5 (ArC), 158.2 (ArC); IR : 3326, 2961, 2903, 1610, 1567, 1488, 1452, 1202, 1080, 1041, 972, 818; MS (ESI): m/z calculated for C38H51N4O3: 611.3956 [M+H]+, found 611.3961; fR = 0.46 .6.Uiso(H) = 1.2–1.5Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989023009155/ex2075sup1.cifCrystal structure: contains datablock(s) 1a, 1b. DOI: 10.1107/S2056989023009155/ex20751asup2.hklStructure factors: contains datablock(s) 1a. DOI: 10.1107/S2056989023009155/ex20751bsup3.hklStructure factors: contains datablock(s) 1b. DOI: 2301884, 2301883CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Correction: Journal of Rare Diseases (2023) 18:283 10.1186/s13023-023-02899-9Following publication of the original article , we haveThe authors’ names were initially published as per below:Masárová Lucia1,2, Panovský Roman1,2*, Pešl Martin1,2,3, Mojica-Pisciotti Mary Luz1,2, Holeček Tomáš1,5,7, Kincl Vladimír1,2, Juříková Lenka6, Máchal Jan1,4, Opatřil Lukáš1,2 and Feitová Věra1,5They should be as follows:Lucia Masárová1,2, Roman Panovský1,2*,Martin Pešl1,2,3, Mary Luz Mojica-Pisciotti1,2, Tomáš Holeček1,5,7, Vladimír Kincl1,2, Lenka Juříková6, Jan Máchal1,4, Lukáš Opatřil1,2 and Věra Feitová1,5Results section text was as follows (bold and incorrect spacing):DMD-FC and Fig. 4f or healthy volunteer.The mean global native T1 relaxation time was similar for DMD-FC and CG  , as well as the mean global ECV value  . The representative native and post-gadolinium T1 maps are presented in Fig. 3f or The mean global native T1 relaxation time was similar for DMD-FC and CG  , as well as the mean global ECV value  . The representative native and post-gadolinium T1 maps are presented in Fig. 3 for DMD-FC and Fig. 4 for healthy volunteer.Figure 3 was incorrect and with incorrect caption as per below:Fig. 3 A representative picture of the native and post-gadolinium T1 map of DMD-FC. DMD-FC- Female carriers of Duchenne muscular dystrophy gene mutationsFigure 3 and its caption should be as follows:Fig. 3 A representative picture of the native and post-gadolinium T1 map of DMD-FC. DMD-FC—Female carriers of Duchenne muscular dystrophy gene mutationsFigure 4 was incorrect, and it is shown below:Fig. 4 A representative picture of the native and post-gadolinium T1 map of a healthy volunteerFigure 4 should be as follows:Fig. 4 A representative picture of the native and post-gadolinium T1 map of a healthy volunteerCMR acquisition section last paragraph had some incorrect spacing, it was as follows:1 mapping was performed as described previously [17] using a Modified Look-Locker Inversion recovery sequence (MOLLI) with a 5(3) 3 scheme to measure native T1 (pre-contrast) and a 4(1) 32 scheme for T1 post-gadolinium (15 min after contrast agent administration).TCMR acquisition section last paragraph text should be as follows:1 mapping was performed as described previously [17] using a Modified Look-Locker Inversion recovery sequence (MOLLI) with a 5(3)3 scheme to measure native T1 (pre-contrast) and a 4(1)3(1)2 scheme for T1 post-gadolinium (15 min after contrast agent administration).TThe original article was updated.


However, mechanisms underlying opioid-induced changes in endolysosome iron homeostasis and downstream-signaling events remain unclear.Opioids including morphine and DAMGO activate mu-opioid receptors (MOR), increase intracellular reactive oxygen species (ROS) levels, and induce cell death. Ferrous iron . Deferoxamine, an endolysosome-iron chelator, inhibited opioid agonist-induced increases in cytosolic and mitochondrial Fe2+ and ROS. Opioid-induced efflux of endolysosome Fe2+ and subsequent Fe2+ accumulation in mitochondria were blocked by the endolysosome-resident two-pore channel inhibitor NED-19 and the mitochondrial permeability transition pore inhibitor TRO.Morphine and DAMGO de-acidified endolysosomes, decreased endolysosome Fe2+ and ROS as well as cell death appear downstream of endolysosome de-acidification and Fe2+ efflux from the endolysosome iron pool that is sufficient to affect other organelles.Opioid agonist-induced increases in cytosolic and mitochondrial Fe SH-SY5Y cells were added at a density of about 6 × 105 cells/well to 12-well plates and incubated at 37 °C and 5% CO2 overnight prior to preincubation with MOR antagonists and other blockers for 30 min and then various treatments for 1 h. Following treatments, cells were washed three times with PBS and incubated with 100 nM RDA for 20 min in PBS in a 37 °C and 5% CO2 incubator. Cells were washed with PBS to remove extracellular dye. A minimum of 10,000 cells were collected and RDA fluorescence was analyzed at an excitation of 562 nm and an emission of 598 nm. Unstained cells were used as negative controls and cells treated with FeCl3 were used as positive controls. Mean fluorescence intensity (MFI) was determined by flow cytometry .Mitochondrial ROS: SH-SY5Y cells (6 × 105 cells/well) were added into 12-well plates and incubated at 37 °C and 5% CO2 overnight prior to being taken for experimentation. Cells were preincubated with MOR antagonists and other blockers for 30 min, then cells were treated with respective drugs for 1 h, washed gently three times with PBS, and incubated with 2.5 µM of MitoSox  for 20 min in a 37 °C and 5% CO2 incubator. Cells were rinsed three times with PBS, fresh PBS was added to the wells, and a minimum of 10,000 cells per condition were placed into Eppendorf tubes for determination of MitoSox fluorescence at an excitation of 510 nm and an emission of 580 nm by flow cytometry .Mitochondrial membrane potential: SH-SY5Y cells were added to 12-well plates at a density of 6 × 105 cells/well and were incubated overnight at 37 °C and 5% CO2. Cells were preincubated with MOR antagonists and other blockers for 30 min, then treated with respective drugs for 1 h, washed gently three times, and treated with 100 nM MitoTracker™ Red CMXROS  for 30 min in a 37 °C and 5% CO2 incubator. Cells were washed three times with warm PBS, re-suspended in 1 mL PBS, and fluorescence was determined using a flow cytometer  at an excitation of 579 nm and an emission of 599 nm. For confocal microscopy, SH-SY5Y cells were added to 35 mm2 dishes and incubated overnight in an incubator kept at 37 °C and 5% CO2. Cells were treated with respective drugs for 1 h, washed gently three times with PBS, and incubated with 100 nM Mito-Tracker Red CMXROS and 200 nM MitoTrackerTM Green FM  for 30 min in a 37 °C and 5% CO2 incubator. Cells were washed three times with PBS, stained with 1 μg/mL Hoechst 33342 for 10 min, washed with PBS three times, and imaged using confocal microscopy  at an excitation of 579 nm and an emission of 599 nm.Cell death: Cell viability was determined using propidium iodide (PI)  and flow cytometry. SH-SY5Y cells were added to 12-well plates at a density of about 60,000 cells/well, incubated overnight at 37 °C and 5% CO2, and cells were treated with respective drugs for 24 h. MOR antagonists, DFO, NED-19, and TRO were added to the cells 30 min prior to adding morphine and DAMGO. After 24 h, cells were stained with PI (3 µM) at room temperature for 15 min, and fluorescence was determined using a flow cytometer . For determination of apoptotic and necrotic cell death, Apoptosis/Necrosis Detection kits  were used. This method simultaneously determines numbers of apoptotic, necrotic and live cells; 7-AAD labels the nucleus of necrotic cells (red fluorescence), Apopxin Green labels phosphatidylserine (PS) on the cell surface of apoptotic cells (green fluorescence), and CytoCalcein Violet labels the cytoplasm of living cells (blue fluorescence). SH-SY5Y cells were seeded into 35 mm2 glass-bottom dishes at a density of about 1 × 104 cells and incubated overnight at 37 °C and 5% CO2. Cells were treated with respective drugs for 24 h and then incubated with a mixture of 200 µL assay buffer, 2 µL Apopxin Green Indicator, 1 µL CytoCalcein 450, and 1 µL 7-AAD at room temperature for 45 min in the dark. For determination of ferroptotic cell death, treated SH-SY5Y cells were stained with 10 µM of C11-BODIPY 581/591  for 30 min at 37 °C as a measure of lipid peroxidation. Cells were washed two times with PBS and images were acquired using a spinning disk confocal laser microscope .Reagents: Chemical reagents were obtained from ThermoFisher Scientific except as follows; TRO-19622 , naloxone hydrochloride , NED-19 , morphine sulfate , β-funaltrexamine hydrochloride , DAMGO , auranofin , nicotinic acid adenine dinucleotide phosphate tetrasodium salt , and bafilomycin A1 .Statistical analyses: All data were reported as means ± standard deviation (SD). Statistical significance between controls and treatments (two groups) were analyzed with a two-tailed Student’s t-test, and statistical significance between 3 groups or more (multiple groups) were analyzed with one-way ANOVA plus a Tukey’s multiple comparison post-hoc test. A p-value less than 0.05 was considered to be statistically significant. GraphPad Prism 9.3.1 software was used to perform all statistical analyses. No pre-determined exclusion criteria were performed. Normal distribution of data was assessed for statistical analysis by using the Shapiro-Wilk test . The Kruskal–Wallis non-parametric test was used to compare data that had a skewed distribution. The number of cells used for this study was determined based on previous studies [ studies –40. No oEthical statement: Institutional ethical approval was not required for this study.Morphine- and DAMGO-induced endolysosome de-acidification was blocked by naloxone and β-FNA: Because endolysosome de-acidification is known to affect the concentration of Fe2+ in endolysosomes ([Fe2+]el), we first determined the effects of MOR agonists on endolysosome pH. The MOR agonists morphine (1 µM) and DAMGO (1 µM) significantly (p<0.0001) de-acidified endolysosomes  and β-FNA (0.4 µM) .ysosomes –43. Accoeviously , 44, mor(0.4 µM) . Images (0.4 µM)  demonstr(0.4 µM) . Quantitectively . Pre-trey  because of their known ability to transport Fe2+ [2+]el by 22% (2+]el by 48% and pre-treatment with NED-19 blocked significantly (p<0.0001) NAADP-induced decreases in [Fe2+]el  and ROS (2+ ([Fe2+]mito) and ROS (2+]cyt and [Fe2+]mito and significantly (p<0.0001) increased levels of cytosolic and mitochondrial ROS (2+]cyt and [Fe2+]mito and significantly (p<0.0001) decreased cytosolic and mitochondrial levels of ROS; pre-treatment with DFO significantly (p<0.0001) blocked morphine- and DAMGO-induced increases in [Fe2+]cyt and [Fe2+]mito as well as increases in cytosolic and mitochondrial ROS  significantly decreased [Fe2+]cyt and [Fe2+]mito and significantly (p<0.0001) blocked NAADP- as well as morphine- and DAMGO-induced increases in [Fe2+]cyt and [Fe2+]mito . Qualitatively, treatment of SH-SY5Y cells for 1 h with the MOR agonists morphine (1 µM) and DAMGO (1 µM) decreased CMXROS fluorescence staining (depolarization of ∆ψm) and these effects were blocked by the MOR antagonists naloxone and β-FNA  increased (polarization) ∆ψm (m (m (m and the TPC antagonist NED-19 significantly (p<0.0001) blocked NAADP-, morphine- and DAMGO-induced decreases in ∆ψm (nd β-FNA . Morpholnd β-FNA . Images nd β-FNA  demonstrnd β-FNA . Cells pion) ∆ψm . Pre-tre) ∆ψm  significantly (p<0.01) decreased cell death and significantly (p<0.0001) blocked morphine- and DAMGO-induced cell death  and DAMGO (1 µM) significantly (p<0.0001) increased levels of mitochondrial Fe2+ and ROS  significantly (p<0.0001) blocked morphine- and DAMGO-induced decreases in ∆ψm  and their activation by opioid-agonists inhibits G-protein-mediated intracellular effectors including adenylate cyclase (AC) and decreases cAMP levels –53. Intrnd β-FNA , 57 as wMORs are functionally active on the cell surface but have been shown to be active once endocytosed . The opi2+ was necessary and sufficient to increase cytosolic and mitochondrial Fe2+ levels because these effects were blocked by the endocytosed iron chelator DFO. Moreover, naloxone and β-FNA blocked the morphine- and DAMGO-induced efflux of endolysosome Fe2+ and the increases in cytosolic and mitochondrial Fe2+, which further supports the opioid agonist-induced subcellular effects on Fe2+ levels. Additionally, decreased mitochondrial iron levels induce mitochondrial swelling el  and β-FNA (0.4 µM) alone were somewhat surprising in that they caused Fe[Fe2+]el . Howeverll death . CorrespNaloxone and β-FNA have anti-inflammatory effects, suppress superoxide production, and can be neuroprotective –87. Incr2+ that is sufficient to increase cytosolic and mitochondrial Fe2+ levels, as well as increase oxidative stress and cell death. Thus, inter-organellar signaling of Fe2+ may be key to better understanding opioid pharmacotherapeutics and adverse effects of opioid analgesics.Overall, our study demonstrates that opioids induce an MOR-mediated efflux of endolysosome FeSupplementary Material DetailsClick here for additional data file.


The smallest FWHM reached 23.6 (24.9) meV at a density of 190 (260) μm−2 (low-rate high-T). The mediate rate formed uniform QDs in the traditional β phase, at a density of 320 (400) μm−2 and a spectral FWHM of 28 (34) meV, while size-diverse QDs formed in β2 at a spectral FWHM of 92 (68) meV and a density of 370 (440) μm−2. From atomic-force-microscope QD height distribution and T-dependent PL spectroscopy, it is found that compared to the dense QDs grown in β phase  with the most large dots (240 μm−2), the dense QDs grown in β2 phase (580 μm−2) show many small dots with inter-dot coupling in favor of unsaturated filling and high injection to large dots for PL. The controllable annealing  forms β2 or β2-mixed α or β phase in favor of a wafer-uniform dot island and the faster T change enables optimal T for QD growth.In this work, we developed pre-grown annealing to form β2 reconstruction sites among β or α (2 × 4) reconstruction phase to promote nucleation for high-density, size/wafer-uniform, photoluminescence (PL)-optimal InAs quantum dot (QD) growth on a large GaAs wafer. Using this, the QD density reached 580 (860) μm The QDs were grown on a 3-inch Nribution . The In 10 cm−2, respectively). The sample 2a (c: 1.2 ML) before forming bi-mode large QDs at 1.6 ML . For the rate of 0.1 (0.2) ML/s with slow migration, the optimal amount for a sufficient island is 2.0 ML (θc: 1.6 ML). For the rate of 0.2 ML/s with limited migration (β2), as the In deposition raises from 2.0 to 2.2 ML, the QD density remains at 5.8 × 1010 cm−2 in the center while rising from (7.4 ± 1.2) × 1010 to (8.2 ± 0.9) × 1010 cm−2 at the Surr (the variation is related to the T distribution), with the highest one reaching 9.1 × 1010 cm−2 as presented. The dilute QD growth shows three critical coverages  for large dot  formation /C for N > n and ~ηN/n for N < n, where N denotes the QD number in a sub-μ-region 200 × 200 nme filled : sample e filled i; the sa10 cm−2 with a room-T PL spectral FWHM of 34 meV (41 meV) in wafer center (Surr), grown at a high rate and low T to limit In migration. Low rate growth at high T forms uniform QDs at a density of 1.9 × 1010 cm−2 and a spectral FWHM of ~23.6 meV. Both show wafer-uniform PL spectra, unlike the β phase (T-sensitive). Mediate rate at a low T forms uniform QDs in the β phase  at the center and diverse QDs in β2 . The pre-grown annealing provides a controllable modulation of the surface reconstruction and enables an optimal T to form dense wafer-uniform QDs. In summary, we use pre-grown annealing to form less-arsenic reconstruction β2 and fabricate high-density uniform InAs quantum dots (QDs) on 3-inch GaAs substrates with wafer-uniform PL. The maximum density reaches 5.8 (8.2 ± 0.9) × 10


A supramolecular self-assembly of semiconducting polymers and small molecules plays an important role in charge transportation, performance, and lifetime of an optoelectronic device. Tremendous efforts have been put into the strategies to self-organize these materials. In this regard, here, we present the self-organization of terthiophene and its methyl alcohol derivative with 4,4′-bipyridine (44BiPy). An unexpected 2D layered organization of 5,5″-dimethyl-2,2′:5′,2″-terthiophene (DM3T) and 44BiPy was obtained and analyzed. Single-crystal X-ray diffraction analysis revealed that DM3T and 44BiPy consist of stacked, almost independent, infinite 2D layers while held together by weak hydrogen bonds. In addition to this peculiar supramolecular arrangement of these compounds, the investigation of their photophysical properties showed strong fluorescence quenching of DM3T by 44BiPy in the solid state, suggesting an efficient charge transfer. On the other hand, the methyl alcohol derivative of terthiophene, DM3TMeOH, organized in a closed cyclic motif with 44BiPy via hydrogen bonds. P3HT) and poly:polystyrene sulfonate (PEDOT:PSS)) are widely studied and used materials in the fabrication of organic optoelectronic devices [Over the last several decades, organic π-conjugated compounds have emerged as a new class of semiconducting materials because of their fascinating optical and electronic properties offering a wide range of applications in organic light-emitting diodes (OLEDs), organic solar cells (OSCs), organic field-effect transistors (OFETs), organic lasers, optical or electrochemical sensors, and many more ,4,5,6,7. devices .As we know, without any substituent, polythiophene is a poorly soluble polymer in most common organic solvents, which makes it difficult to process for fundamental study of charge transportation and redundant to use for any practical application. Therefore, for practical purposes, solution-processed poly-(di)alkyl)thiophenes have been obtained by introducing alkyl chains at the backbone polythiophenes and utilized various optoelectronic application. Nevertheless, many well-defined oligothiophenes have been synthesized and studied to serve as the basis for a thorough understanding of the electrical properties of polythiophenes. Among these oligothiophenes, terthiophene and its derivatives, because of their facile synthesis, are widely studied to understand the electronic structure and charge transport mechanism in this class of materials. For example, Birnbaum and Kohler recorded the fluorescence excitation and emission spectra of 2,2′:5′,2″-terthiophene and performed a detailed analysis of the lowest-energy excited singlet state , which wDM3T)  [DM3T with 4,4′-bipyridine (44BiPy) via slow evaporation of the solvent, hoping that these two molecules would organize into a supramolecular self-assembly via chalcogen bonding (ChB). However, this experiment led to a totally unexpected result. The synthesis, purification, and characterization of alkyl-substituted and/or alkyl-capped oligothiophene have been reported in literature ,21. HoweDM3T) . Weak hy bonding  led to t[44BiPy) ,24. To vDM3T–44BiPy is presented. We also present the synthesis of methanol (DM3TMeOH) and its hydrogen-bonded cocrystal structure analysis with 44BiPy. A detailed comparison of structural analysis of DM3T, DM3T–44BiPY, and DM3TMeOH–44BiPy is presented. We then discuss the photophysical properties and fluorescence quenching of DM3T and DM3TMeOH in the presence of 44BiPy. The chemical structures of the DM3T, DM3TMeOH, and 44BiPy are shown in The crystal structure and Hirshfeld surface analysis of DM3T was prepared by a palladium-catalyzed Suzuki cross-coupling reaction [3)4) as a catalyst derivative (DM3TMeOH was prepared starting from 3-thiophenemethanol (TMeOH) via a multistep organic synthesis procedure as depicted in TMeOH with NBS in dichloromethane at room temperature gave a dibromo derivative (DBTMeOH) which was subsequently subjected to protection of –OH group by tert-butyldimethylsilyl chloride (TBDMSCl). Following the same Suzuki cross-coupling reaction conditions and reagents as used to synthesize DM3T except using newly synthesized DBTMeOTBDMS instead of 2,5-dibromothiophene gave the –OH-protected desired terthiophene derivative, DM3TMeOTBDMS, which was easily deprotected by treatment with TBAF in THF at room temperature to yield DBTMeOH  and 44BiPy (3 equiv.), while pale-green cocrystals were obtained for DM3TMeOH–44BiPy from a solution of DM3TMeOH (2 equiv.) and 44BiPy (1 equiv.). All single crystals were subsequently analyzed.Crystal growth was conducted via slow evaporation of a mixture of solvents. Bright-green-yellow single crystals of DM3T. DM3T crystallized in the monoclinic non-centrosymmetric space group P21 with four molecules per unit cell. All the thiophene units adopted an S-trans conformation with slightly curved molecular plane geometry  were evidenced by the S23···π interaction (d = 3.868 Å), as well as chalcogen–chalcogen contacts between S1 and S21 (d = 3.690(3) Å). Although the distances between these noncovalent interactions were slightly higher than the sum of their van der Waals radii [In the transverse arrangement of ls radii , their pDM3T using Hirshfeld surface analysis and fingerprint plots. DM3T, which provide valuable insights into the atom pair interactions and their relative contributions to the Hirshfeld surface.The relative strengths and contributions of intermolecular interactions of a crystalline compound can be visualized by Hirshfeld surface analysis. For this purpose, a software program, CrystalExplorer , was used to generate and analyze the Hirshfeld surface , shape iDM3T arose from three types of interactions: H···H (40.2%), C···H/H···C (29.7%), and S···H/H···S (18.7%). These interactions involved hydrogen bonding, as well as C–H···π interactions between carbon and hydrogen atoms. The significant contribution from H···H interactions suggests that the vast majority of London dispersion forces were acting within the crystal structure. Additionally, the fingerprint plots showed lower proportions of S···C/C···S (7.8%) and S···S (3.6%) interactions. The presence of S···C/C···S interactions indicates the presence of chalcogen bonds, while S···S interactions represent the chalcogen–chalcogen contacts, and both contribute to the overall crystal packing of DM3T. The Hirshfeld surface analysis, combined with the fingerprint plots, provides a comprehensive understanding of the atom pair interactions such as HB, CH···π, ChB, and S···S contacts and highlights their significance in the arrangement of DM3T molecules within the crystal lattice.The fingerprint plots revealed that the major contributions to the Hirshfeld surface of DM3T and 44BiPy, as shown in PDM3T and three 44BiPy molecules. In DM3T, all the thiophene units were in S-trans conformation. These molecules were held together by strong HB, C–H···π, and S···S interactions.Single-crystal X-ray diffraction analysis of the cocrystal agreed well with the expected structure of the constituents, 44BiPy molecules. The details of all noncovalent interactions are summarized in The typical distances of C–H···π interactions were within the range of 2.714–3.094 Å, while the S6···S6 separation was measured at 3.554 Å, which is approximately 1.3% shorter than the sum of van der Waals radii of sulfur atoms . Even stDM3T–44BiPy revealed a remarkable and distinctive layered structure, as depicted in DM3T and 44BiPy compounds self-organized into independent infinite layers, which were stacked on top of one another and connected by weak hydrogen-bonding interactions. The DM3T layer was primarily stabilized by a combination of hydrogen bonding and short contacts between sulfur atoms (S···S). In contrast, the 44BiPy molecules formed an infinite layer characterized by a complex arrangement, primarily stabilized by strong HBs.The supramolecular cocrystal packing of DM3T and 44BiPy was measured to be 15.291 Å and 14.076 Å, respectively. These data highlight the separation between adjacent layers in the crystal structure.DM3T–44BiPy are noteworthy. The distinct characteristics of the individual layers and their stacking provide valuable insights into the crystal packing structure. Understanding these arrangements is crucial for comprehending the properties and potential applications of this cocrystal system , C···H/H···C (28.9%), N···H/H···N (12.9%), and S···H/H···S (9.9%). While the proportions of S···C/C···S (4.0%) and S···S (2.0%) interactions were relatively low, they were still significant enough to confirm their presence in the crystal packing. In contrast, the contributions from C···N/N···C (0.5%) and C···C/C···C (0.3%) interactions were negligible, aligning with the findings of the X-ray structure.DM3T–44BiPy.Notably, the Hirshfeld surface analysis combined with the fingerprint plots revealed a complete absence of N···S/S···N interactions (0%). This finding elucidates the layered crystal packing observed in DM3T–44BiPy. The dominance of hydrogen-bonding interactions, along with the confirmation of other intermolecular contacts, explains the layered organization observed in the crystal structure. Further investigations can delve into the implications of these interactions on the physical and chemical properties of DM3T–44BiPy, facilitating the development of advanced materials and functional devices.The comprehensive understanding of the atom pair interactions and their contributions provided by these analyses enhances our knowledge of the crystal packing of DM3TMeOH and 44BiPy, in the cocrystal  led to the adoption of the S-DM3TMeOH–44BiPy cocrystal. Understanding the intermolecular and intramolecular hydrogen-bonding interactions contributes to our knowledge of the stability and conformational preferences of the cocrystal. These insights can guide further investigations into the properties and potential applications of this cocrystal system  group present in DM3TMeOH, the red spot corresponding to the O···H interaction is particularly prominent on the surface, indicating its strength and size.Like tructure . NotablyDM3TMeOH–44BiPy were reversed compared to those in DM3T–44BiPy, where N···H/H···N accounted for 12.9% and S···H/H···S accounted for 9.9%. This reversal was attributed to the extensive complex network of hydrogen bonds observed within the 44BiPy layer of the DM3T–44BiPy crystal, which changed to linear hydrogen-bonding interactions between DM3TMeOH and 44BiPy in the DM3TMeOH–44BiPy cocrystal.The relative contributions of different interactions to the overall surface are as follows: H···H (38.4%), C···H/H···C (32.3%), S···H/H···S (14.1%), N···H/H···N (7.2%), O···H/H···O (3.4%), S···C/C···S (2.6%), S···S (1.4%), and C···C/C···C (0.8%) . Once agDM3TMeOH–44BiPy.Furthermore, the proportion of S···S (1.4%) interactions confirms their presence in the crystal packing, while the insignificant proportion of C···C/C···C (0.8%) interactions ruled out the occurrence of π–π stacking, which aligns with the observations from the X-ray structure of DM3T and DM3TMeOH, and their binding with 44BiPy in solution and solid-state using UV/visible spectroscopy and fluorescence spectroscopy. The UV/visible spectra of all materials were recorded in chloroform solution at room temperature and are presented in In the literature, the photophysical properties of α-oligothiophenes  have been extensively studied both theoretically and experimentally ,29. We aDM3T in chloroform solution was devoid of structural features and exhibited a single broad absorption band at 365 nm –π*(11Bu) transition (S0→S1) of the oligothiophene backbone [44BiPy did not show any noticeable effect on the absorption spectra of DM3T [DM3T and DM3T–44BiPy decreased significantly as the bulk of the material remained unexcited.As anticipated, the UV/visible absorption spectrum of  dioxane ). This cbackbone ,30. ImpoDM3TMeOH was also devoid of structural features and displayed only a single broad absorption band with slight blue-shift by 8 nm compared to that of DM3T. This blue-shift was attributed to the S-cis configuration of the terthiophene units in the ground state. Like DM3T in the solution state, the absorption spectrum of DM3TMeOH remained unaffected by the presence of 44BiPy. In the solid state, DM3TMeOH–44BiPy exhibited similar absorption to that of DM3TMeOH in the solution state.Similarly, Moving on to the emission properties, DM3T exhibited characteristic well-resolved vibronic features at 421 nm, 442 nm, and 473 nm. This is consistent with the previous literature that reported maximum fluorescence at 407 nm and 426 nm in dioxane [DM3T experienced a red-shift of 40 nm in the solid state due to the aggregation-caused quenching (ACQ) effect [In chloroform solution,  dioxane . Compare) effect , while m44BiPy, fluorescence quenching was observed in the solution state, and this quenching became more pronounced in the solid state. This suggests an electron transfer process from DM3T to 44BiPy in the excited state. Interestingly, in the solid state of DM3T–44BiPy, the vibronic band at 465 nm completely disappeared, and the broad shoulder of DM3T’s emission spectrum became a distinct emission band. On the basis of the energetics of DM3T’s electronic states, this indicates that electron transfer occurred from DM3T’s 1Bu electronic state to the lowest unoccupied molecular orbital (LUMO) of 44BiPy.Upon the addition of DM3TMeOH exhibited vibronic bands at 428 nm and 448 nm in its fluorescence spectrum in the solution state. In the presence of 44BiPy, pronounced fluorescence quenching was observed due to the formation of strong hydrogen bonds  and S···H/H···S (14.1%) interactions in DM3TMeOH–44BiPy. This change in proportions reflected the difference in crystal packing, transitioning from an almost independent layered cocrystal to a molecular packing arrangement involving a cyclic motif, although both self-assemblies were directed by hydrogen bonds.The resolved 2D fingerprint plots in all cases revealed that hydrogen bonds were the major contributors to the overall Hirshfeld surface. Interestingly, the relative proportions of N···H/H···N (12.9%) and S···H/H···S (9.9%) interactions in DM3T, DM3T–44BiPy, and DM3TMeOH–44BiPy. The differences observed in their crystal packings and hydrogen-bonding patterns highlighted the influence of molecular composition and functional groups on the supramolecular organization of these cocrystals. The findings of photophysical properties investigations demonstrated the influence of 44BiPy on the fluorescence properties of both DM3T and DM3TMeOH, suggesting electron transfer and fluorescence-quenching effects in the presence of 44BiPy. The solid-state behavior of these systems highlighted the impact of aggregation and hydrogen bonding on their luminescent properties.In summary, the X-ray structures and Hirshfeld surface analyses provided valuable insights into the molecular arrangements and intermolecular interactions in 1H- and 13C-NMR spectra were recorded on JEOL 400 Hz spectrometers at 298 K. CDCl3 was used as received . The residual nondeuterated solvent was used as an internal standard for calibration of 1H- and 13C-NMR spectra. Chemical shifts (δ) are expressed in ppm, and coupling constants values (nJ) are expressed in Hz. Abbreviations used for NMR spectra are as follows: s, singlet; d, doublet; m, multiplet.General experimental information. Solution NMR spectroscopy analyses were performed at the Centre d’Instrumentation en Resonance Magnétique (CIREM) of the Université Libre de Bruxelles in Belgium. ®, 99%, CAS no. 128-08-5) were purchased from Sigma-Aldrich (now known as Merck) . Tetrakis(triphenylphosphine)palladium (0)  was obtained from Apollo Scientific . 4,4′-Bipyridine  was bought from TCI Chemicals . Ethanol absolute  was obtained from VWR Chemicals . Dichloromethane  and N,N-dimethylformamide  were purchased from Chem-Lab NV, . All materials were used as received. The air-sensitive reaction was performed using standard Schlenk techniques under an argon atmosphere. Flash column chromatography was carried out using silica gel  purchased from DAVISIL-Grace GmbH, Germany. Analytical thin-layer chromatography was performed using silica plates with aluminum backings , and the results were visualized using a 254/365 nm UV lamp.Materials. 2,5-Dibromothiophene  was purchased from BLD Pharmatech GmbH . 5-Methylthiophene-2-boronic acid pinacol ester , potassium carbonate anhydrous , and toluene  were bought from Merck . 3-Thiophenemethanol , tetrabutylammonium fluoride (TBAF) solution , and N-bromosuccinimide (NBS) (ReagentPlusDM3T). A mixture of 2,5-dibromothiophene , 5-methylthiophene-2-boronic acid pinacol ester , and Pd(PPh3)4  was dissolved in toluene  and heated to 90 °C. A solution of K2CO3 , bubbled with argon gas for 5 min) was then added to the reaction mixture and stirred at the same temperature for 24 h. After cooling to room temperature, the reaction mixture was diluted with ethyl acetate and washed with water followed by brine solution. The organic layer was dried over anhydrous sodium sulfate and filtered. Solvent was removed and residue was purified by silica gel column chromatography using ethyl acetate/hexane (v/v = 1:4) as an eluent to afford the title compound as a bright-yellow solid . 1H-NMR  δ 7.00–6.88 , 6.65 , 2.47   δ 139.25, 136.13, 135.00, 126.04, 123.58, 123.49, 15.44  . 13C-NMR9, 15.44 .DBTMeOH). The title compound was synthesized according to a slightly modified literature procedure [1H-NMR  δ 6.99 , 4.52 . 13C-NMR  δ 141.53, 130.61, 111.53, 109.33, 59.37.Synthesis of 2,5-dibromo-3-thiophenenemethanol (rocedure . To a soDBTMeOTBDMS). The title compound was synthesized according to a literature procedure [DBTMeOH , tert-butyldimethylsilyl chloride , and imidazole  was dissolved in DMF (10 mL) and heated at 65 °C for 2 h. After cooling to room temperature, the reaction mixture was diluted with ethyl acetate and washed with water. The organic layer was washed with brine, dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by silica gel column chromatography using hexane as eluent to afford the title compound as a colorless liquid . 1H-NMR  δ 6.96 , 4.54 , 0.91 , 0.08 .Synthesis of tert-butylmethoxy)dimethylsilane (rocedure . A mixtuDM3TMeOTBDMS). The title compound was prepared using the well-known palladium-catalyzed Suzuki cross-coupling reaction. A mixture of DBTMeOTBDMS , 5-methylthiophene-2-boronic acid pinacol ester , and Pd(PPh3)4  was dissolved in toluene  and heated to 90 °C. A solution of K2CO3 , bubbled with argon gas for 5 min) was then added to the reaction mixture and stirred at the same temperature for 24 h. After cooling to room temperature, the reaction mixture was diluted with ethyl acetate and washed with water, followed by brine solution. The organic layer was dried over anhydrous sodium sulfate and filtered. The solvent was removed, and the residue was purified by silica gel column chromatography using hexane/dichloromethane (85:15) as an eluent to afford the title compound as a light-green solid . 1H-NMR  δ 7.10 , 6.92 , 6.72–6.69 , 6.66–6.63 , 4.73 , 2.49 , 2.47 , 0.92 , 0.08 . 13C-NMR  δ 140.49, 139.26, 138.57, 135.31, 135.02, 133.13, 130.25, 126.08, 126.02, 125.96, 125.09, 123.50, 60.04, 26.02, 18.46, 15.44, 15.38, −5.12.Synthesis of tert-butylmethoxy)dimethylsilane (DM3TMeOH). To a solution of DM3TMeOTBDMS  in THF (10 mL) at room temperature was added tetrabutylammonium fluoride  and stirred. After 1 h, THF was removed under reduced pressure, and the residue was dissolved in dichloromethane. The organic layer was washed with brine, dried over anhydrous sodium sulfate, and concentrated. The crude product was absorbed on silica gel and eluted with dichloromethane to afford the title compound as green solid . 1H-NMR  δ 7.11 , 6.97 , 6.95 , 6.74–6.70 , 6.68–6.64 , 4.72 , 2.50 , 2.48   δ 140.93, 139.56, 137.61, 136.01, 134.63, 132.55, 132.18, 126.48, 126.11, 126.09, 125.08, 123.74, 59.23, 15.46, 15.38 methanol  . 13C NMR6, 15.38 .DM3T–44BiPy, was performed on a Rigaku Oxford Diffraction Supernova Dual Source (Cu at zero) diffractometer equipped with an Atlas CCD detector using ω scans and Mo Kα (λ = 0.71073 Å) radiation. The images were interpreted and integrated with the program CrysAlisPro [Data collection and refinement. Single-crystal data collection (at 100 K) and evaluation of the cocrystal, sAlisPro . Using OsAlisPro , the strsAlisPro ,38. Non-3T) with or without terminal substituents only and the results were reduced to following 23 CCDC entries: 112398, 232984, 254856, 619628, 644333, 644334, 646597, 699323, 747221, 800037, 836023, 848505, 1005911, 1040724, 1040725, 1221168, 1862144, 1991924, 1991925, 2044752, 2062986, 19862144, and 1981015. Apart from these homo-crystal entries, only one crystal structure is reported as a two-component cocrystal of 3T with 7,7,8,8-tetracyanoquinodimethane (TCNQ) (CCDC 1862143), and only one crystal structure is reported as a three-component cocrystal consisting of 3T, guanidinium, and 4,4’-azobenzenedisulfonate (CCDC 797309).Cambridge structural database survey. A simple search was launched using the string “terthiophene” in the compound name option. The search revealed more than 200 crystal structures including quaterthiophene. The hitlist was manually screened for 2,2′:5′,2”-terthiophene  and its methyl alcohol derivative in combination with 4,4′-bipyridine (44BiPy). Through single-crystal X-ray diffraction analysis, we made the unexpected discovery of a three-dimensional stacked, layered organization of DM3T and 44BiPy. These layers were found to be nearly independent of each other and held together by weak hydrogen bonds. The investigation of the photophysical properties of this system revealed a strong fluorescence quenching of DM3T by 44BiPy in the solid state. This suggests an efficient charge transfer between the two components. Such phenomena are of significant interest in the development of energy-generation devices, particularly in the field of organic solar cells. Furthermore, we observed that DM3TMeOH, the methyl alcohol derivative of DM3T, organized itself in a closed cyclic motif with 44BiPy through strong hydrogen bonds. Overall, our findings shed light on the unique self-organization and supramolecular arrangements of DM3T and its derivatives with 44BiPy. The unexpected 3D stacked, layered organization, along with the efficient charge transfer offers new avenues for future research in self-organized layered materials. Further exploration of the photophysical properties and applications of this system could lead to advancements in the development of energy generation devices.In our study, we presented the synthesis and self-organization of methyl-capped terthiophene (