APOE genotype, AD-Polygenic Risk Scores (AD-PRS), amyloid-β pathology and decline in cognitive functioning over time in a large sample of cognitively unimpaired older individuals.What combination of risk factors for Alzheimer’s disease (AD) are most predictive of cognitive decline in cognitively unimpaired individuals remains largely unclear. We studied associations between APOE gene was not included in the PRS and was analyzed separately. Baseline amyloid-β status was assessed by visual read of [18F]flutemetamol-PET standardized uptake value images. At baseline and follow-up (2.0 ± 0.4 years), the cognitive domains of memory, attention, executive function, and language were measured. We used generalized estimating equations corrected for age, sex and center to examine associations between APOE genotype and AD-PRS with amyloid-β status. Linear mixed models corrected for age, sex, center and education were used to examine associations between APOE genotype, AD-PRS and amyloid-β status, and their interaction on changes in cognitive functioning over time.We included 276 cognitively unimpaired older individuals  from the EMIF-AD PreclinAD cohort. An AD-PRS was calculated including 83 genome-wide significant variants. The APOE ε4 allele. APOE genotype and AD-PRS were both associated with abnormal amyloid-β status. Increasingly more risk-full APOE genotype, a high AD-PRS and an abnormal amyloid-β status were associated with steeper decline in memory functioning in separate models . A model including 4-way interaction term (APOE×AD-PRS×amyloid-β×time) was not significant. When modelled together, both APOE genotype and AD-PRS predicted steeper decline in memory functioning (APOE β(SE)=-0.05(0.02); AD-PRS β(SE)=-0.04(0.01)). Additionally, when modelled together, both amyloid-β status and AD-PRS predicted a steeper decline in memory functioning (amyloid-β β(SE)=-0.07(0.04); AD-PRS β(SE)=-0.04(0.01)). Modelling both APOE genotype and amyloid-β status, we observed an interaction, in which APOE genotype was related to steeper decline in memory and language functioning in amyloid-β abnormal individuals only (β(SE)=-0.13(0.06); β(SE)=-0.22(0.07), respectively).Fifty-two participants (19%) had abnormal amyloid-β, and 84 participants (31%) carried at least one APOE genotype is related to steeper decline in memory and language functioning in individuals with abnormal amyloid-β only. Furthermore, independent of amyloid-β status other genetic risk variants contribute to memory decline in initially cognitively unimpaired older individuals.Our results suggest that The online version contains supplementary material available at 10.1186/s12883-022-02925-6. Alzheimer’s disease (AD) is the most common cause of dementia. The development of AD starts with a preclinical stage where pathophysiological changes in brain amyloid-β are present, –4 while apolipoprotein E (APOE) ɛ4 allele. APOE ɛ4 heterozygotes are at three-fold greater risk of AD and ɛ4 homozygotes at eight- to fifteen-fold compared to APOE ɛ4 non-carriers. flutemetamol. , per allele; OR 1.43 [95% CI 1.02 to 2.00], respectively)  = -0.03(0.01), p = 0.04). Higher APOE dose was associated with steeper decline in memory functioning (β(SE) = -0.05(0.02), p = 0.008; Table APOE genotype and AD-PRS on the rate of memory decline, which was not significant. Repeating analyses including APOE genotype and AD-PRS as main effects in the same model, we found that both higher APOE genotype dosage and a high AD-PRS were associated with steeper decline in memory over time   = -0.13(0.06), p = 0.03), and no association was found in individuals with normal amyloid-β (β(SE) = -0.02(0.02), p = 0.46; Table APOE×AD-PRS×amyloid-β×time) was not significant. After removing ɛ2ɛ4 carriers from the sample, the results and interpretation did not change (Table S2).At baseline, no significant associations were observed between  -0.050.0, p = 0.0 -0.050.0, p = 0.0 -0.050.0, p = 0.0 -0.050.0, p = 0.0 -0.050.0, p = 0.0 -0.050.0, p = 0.0APOE dose showed steeper decline in language functioning (β(SE) = -0.07(0.03), p = 0.04; Table S3, Figure S1G). When examining interactions of APOE genotype and AD-PRS as main effects in the same model, we found that higher APOE genotype dosage was associated with steeper decline in language over time, while AD-PRS was not (Table S3). We then examined interactions of APOE genotype dosage and amyloid-β status on the rate of language decline, and we found a significant interaction effect (p-interaction = 0.003) . Repeating analyses after stratifying for amyloid-β status, we found that higher APOE dose was associated with steeper language decline in individuals with abnormal amyloid-β (β(SE) = -0.22(0.07), p = 0.002), and no association was found in individuals with normal amyloid-β (β(SE) = -0.02(0.04), p = 0.62; Table S3). No significant associations were found for APOE genotype, AD-PRS and amyloid-β status with attention and executive function performance over time.Examining composite attention, executive function and language scores, we found across the whole group all scores improved over two years (attention: β(SE) = 0.05(0.02), p = 0.02; executive function: β(SE) = 0.06(0.02), p = 0.004; language: β(SE) = 0.11(0.02), p < 0.001). However, individuals with higher APOE genotype dosage and higher AD-PRS were related to amyloid-β abnormality. Furthermore, we observed in separate models that AD-PRS, APOE genotype dosage and abnormal amyloid-β were associated with steeper decline in memory functioning over time in separate models. The association of higher APOE genotype dosage with steeper memory decline was dependent on amyloid-β abnormality, whereas AD-PRS explained variance in memory decline over time independently of APOE genotype dosage and amyloid-β status. In addition, APOE genotype dosage was associated with steeper decline in language functioning over time, and this association was dependent on amyloid-β abnormality. These results suggest that amyloid-β status, APOE genotype and AD-PRS all explain parts of cognitive decline in older individuals with initially normal cognition.In a cognitively unimpaired older population, we found that both higher APOE genotype dosage and higher AD-PRS were associated with amyloid-β pathology replicates previous findings in cognitively unimpaired individuals. [APOE ɛ4 other genes may contribute to amyloid-β abnormality. Pathway-enrichment analyses [Our findings that both higher viduals. , 65 Thisanalyses  of the Sanalyses APOE genotype dosage was related to decline in memory functioning over time. This observation is largely consistent with earlier studies in cognitively unimpaired individuals. [APOE genotype dosage and high AD-PRS explained variance in memory decline independently. Further research is needed to understand whether specific risk genes contribute to the rate of cognitive decline in preclinical AD. While the effect of high APOE genotype dosage was specific for individuals with abnormal amyloid-β , the hius study  endocytous study , 75 Futudomains.  As all cia risk.  We previia risk.  For futuAPOE genotypes, we placed both APOE ɛ2ɛ4 and ɛ3ɛ4 carriers into the same category of the APOE genotype variable even though previous studies have shown that ɛ3ɛ4 carriers may have worse outcomes than ɛ2ɛ4 carriers. [Table S2). Since it has been shown that predictive performance of AD-PRS based on European samples is lower in non-European ancestry samples, [APOE ɛ4 carriers compared to the 226 individuals who were assessed at follow-up (see Table S4). The follow-up duration of an average of two years may have been too short to observe decline or clinical progression in initially cognitively unimpaired individuals, who take on average 5–6 years to show decline or clinical progression. [A strength of the present study is the relatively large sample of cognitively unimpaired older individuals with a substantial age range (60–94), AD biomarkers, genetic risk data, and repeated neuropsychological assessment available. Possible limitations are that by using a dichotomous variable for amyloid-β burden, more sensitive information could have been lost, because binary visual reads disregard the potential significance of quantifying early, pathological, amyloid-β accumulation.  In relatarriers.  However,samples,  our resuression.  For thisAPOE genotype, the AD-PRS is also associated with memory decline. Future research should further investigate which specific genetic variants contribute to memory decline and through which mechanism, because this will be important for developing treatments that may prevent decline in cognition.Our findings provide further support among cognitively unimpaired older individuals that, in addition to abnormal amyloid-β and Below is the link to the electronic supplementary material.Supplementary Material 1: Figure S1.Supplementary Material 2: Figure S2.Supplementary Material 3: Supplementary Materials.


Nature Communications 10.1038/s41467-019-13962-0, published online 21 January 2020.Correction to: In the original version of the manuscript, the image shown in Figure 4g, bottom row (Aβ1–42 + rAggregatin), under “6h” was incorrect. This image incorrectly showed the same sample as shown in the original Figure 4g, top row (Aβ1–42), under “0.5h”.The correct version of figure 4g is as follows:which replaces the previous incorrect version:


T1 CAs, commercial magnetic iron oxide nanoparticles (MIONs)-based T2 CAs, and reported exceedingly small MIONs (ES-MIONs)-based T1 CAs, in this study, a facile co-precipitation method was developed to synthesize biodegradable and biocompatible ES-MIONs with excellent water-dispersibility using poly (aspartic acid) (PASP) as a stabilizer for T1-weighted MRI of tumors. After optimization of the synthesis conditions, the final obtained ES-MION9 with 3.7 nm of diameter has a high r1 value (7.0 ± 0.4 mM−1 s−1) and a low r2/r1 ratio (4.9 ± 0.6) at 3.0 T. The ES-MION9 has excellent water dispersibility because of the excessive –COOH from the stabilizer PASP. The pharmacokinetics and biodistribution of ES-MION9 in vivo demonstrate the better tumor targetability and MRI time window of ES-MION9 than commercial Gd chelates. T1-weighted MR images of aqueous solutions, cells and tumor-bearing mice at 3.0 T or 7.0 T demonstrate that our ES-MION9 has a stronger capability of enhancing the MRI contrast comparing with the commercial Gd chelates. The MTT assay, live/dead staining of cells, and H&E-staining indicate the non-toxicity and biosafety of our ES-MION9. Consequently, the biodegradable and biocompatible ES-MION9 with excellent water-dispersibility is an ideal T1-weighted CAs with promising translational possibility to compete with the commercial Gd chelates.Magnetic resonance imaging (MRI) has been widely using in clinical diagnosis, and contrast agents (CAs) can improve the sensitivity MRI. To overcome the problems of commercial Gd chelates-based The online version contains supplementary material available at 10.1186/s12951-022-01562-y. T1-weighted CAs  can shorten the proton’s longitudinal relaxation time (T1) to produce brighter images, while T2-weighted CAs  can shorten proton’s transverse relaxation time (T2) to generate darker images . Th. Thr1 va1/T2 relaxation rate (1/T1 or 1/T2) is plotted versus concentration for contrast agents, and the r1 and r2 values are calculated from the slopes of the corresponding fitting lines. T1 CAs increase signal intensity of T1 images by shortening the longitudinal relaxation time (T1) of protons, which leads to high r1 values. The Fe3O4 nanoparticles with size below 5.0 nm have low Ms values causing low r2 values according to Eq.  could be utilized for T1 MRI [The Tr T1 MRI , 36.h) of ES-MION9 is 13.7 nm . Although Gd3+ can cause nephrogenic systemic fibrosis and can be deposited in the human brain and body [3+ leads to long-term toxicity, which cannot be revealed in the short-term MTT assay.To evaluate the biosafety of ES-MION9, its cytotoxicity was examined by thiazolyl blue tetrazolium bromide (MTT) assay on MCF-7 cells (Human breast cancer cells) and 4T1 cells (Mouse breast cancer cells). Figure and body , Fig. 4ATo further demonstrate the non-cytotoxicity of ES-MION9, live/dead cytotoxicity analysis was used to evaluate the toxicity of ES-MION9 to 4T1 cells and MCF-7 cells  . Compared with controls, ES-MION9-treated mice showed no obvious pathological abnormalities in major organs , indicating that our ES-MION9 does not lead to systemic toxicity.Additional file T1 CAs, commercial MIONs-based T2 CAs, and reported ES-MIONs-based T1 CAs, a facile method based on co-precipitation was developed to synthesize biodegradable and biocompatible ES-MIONs with excellent water-dispersibility for T1 MRI of tumors using PASP as the stabilizer. After optimization of the synthesis conditions, the final obtained ES-MION9 with a diameter of 3.7 nm has a high r1 (7.0 ± 0.4 mM−1 s−1) and a low r2/r1 (4.9 ± 0.6) at 3.0 T. The ES-MION9 has excellent water dispersibility due to the excessive carboxyl groups from PASP. The physical properties of ES-MION9 were further characterized by TEM, XRD, EDS, XPS, UV–vis, FT-IR, TGA, and magnetization curve. LSCM images and flow cytometry results prove the cellular uptake of ES-MION9 by endocytosis. The pharmacokinetics, and biodistribution of ES-MION9 in vivo demonstrate the better tumor targetability and MRI time window of ES-MION9 than commercial Gd chelates. T1-weighted MR images of aqueous solutions, cells and tumor-bearing mice at 3.0 T or 7.0 T demonstrate that our ES-MION9 has a stronger MRI capability than the commercial Gd chelates. The MTT assay, live/dead staining of cells, and H&E-staining indicate the non-toxicity and biosafety of our ES-MION9. Consequently, the biodegradable and biocompatible ES-MION9 with excellent water-dispersibility is an ideal T1-weighted CAs with promising translational possibility to compete with the commercial Gd chelates.In summary, in order to surmount the problems of commercial Gd chelates-based 2, 20.0 mL and 0.5–4.0 mg mL−1 of PASP (Mw = 7000) solution was first bubbled using N2 for 60 min. After that, the solution was heated to 100 °C under reflux. A Fe solution  was then rapidly charged to the above-mentioned PASP solution. Subsequently, NH3.H2O  was added under magnetic stirring. After 1.0 h, the reaction was stopped by cooling off. Finally, the synthesized ES-MIONs were purified via dialysis (Mw cut-off 8–14 kDa) in pure water for purification. An ICP-OES  was used to determine the CFe of the ES-MIONs.In order to eliminate OCFe = 2.8 mM), and the mixture was magnetically stirred for 24.0 h. The prepared R6G@ES-MION9 solution was then centrifugally ultra-filtrated  and washed utilizing ultrapure water for purification. Finally, the obtained R6G@ES-MION9 was resolved in ultrapure water (4.0 mL) and kept in 4.0 °C of refrigerator.At room temperature, 70.0 µL of Rhodamine 6G (100.0 μM) was added into 4.0 mL of ES-MION9 (Additional file 1. The online version contains supplementary material available at https://jnanobiotechnology.biomedcentral.com.


These NMR assignments will serve as a useful tool in the discovery of small-molecule therapeutics targeting PD-L1 and in the characterisation of functional interactions with other protein partners, such as CD80.The co-inhibitory immune checkpoint interaction between programmed cell death-protein 1 (PD-1) and programmed cell death-ligand 1 (PD-L1) serves to regulate T-cell activation, promoting self-tolerance. Over-expression of PD-L1 is a mechanism through which tumour cells can evade detection by the immune system. Several therapeutic antibodies targeting PD-L1 or PD-1 have been approved for the treatment of a variety of cancers, however, the discovery and development of small-molecule inhibitors of PD-L1 remains a challenge. Here we report comprehensive sequence-specific backbone resonance assignments ( Human PD-L1 is a 272 amino acid, single-pass transmembrane protein, with two N-terminal Ig-like domains forming the extracellular region (residues 19–239). PD-L1 is the ligand for PD-1 (265 residues), which is also a single-pass transmembrane protein, with a single Ig-like domain forming the extracellular region (amino acids 24–170). The interaction between PD-1 and PD-L1 has been shown to be facilitated by the most N-terminal IgV-like domain of PD-L1 (D1) and the extracellular Ig-like domain of PD-1 , which appears to be limited in vivo to when both proteins are on the surface of the same cell  and 2H/15N/13C-labelled PD-L1 (D1D2) were expressed as insoluble inclusion bodies in appropriately transformed Ecoli BL21 (DE3) cells grown in M9 minimal medium containing 3 g/l 13C glucose and 1 g/l 15N ammonium sulphate. Deuterated samples were prepared from cells grown in minimal media prepared using > 99% D2O. The BL21 (DE3) cells were cultured at 37° C and protein expression was induced with 1 mM IPTG at an optical density of 0.8–1.0 at 600 nm. The cells were then cultured for a further 5 hours before harvesting by centrifugation. For refolding and purification of PD-L1, cell pellets were resuspended in PBS at pH 7.2 and lysed by sonication before the inclusion bodies were collected by centrifugation. Inclusion bodies were washed twice with 50 mM tris-HCl, 200 mM NaCl, 0.5% triton-X100, 10 mM EDTA and 10 mM DTT at pH 8.0 and once in the same buffer without triton-X100. Washed inclusion bodies were then resolubilised in 50 mM tris-HCl, 5 M guanidine-HCl, 200 mM NaCl and 20 mM DTT at pH 8.0 prior to refolding by drop-wise 100-fold dilution into 0.1 M tris-HCl, 1 M arginine, 0.25 mM oxidised glutathione, and 0.25 mM reduced glutathione at pH 8.0 for PD-L1 (D1)  and PD-L1 (D1D2: 19–239) were synthesised and cloned into pET28a by GenScript, with codon usage optimised for expression in 15N/13C labelled PD-L1 (D1) and 2H/15N/13C labelled PD-L1 (D1D2) respectively, in a 25 mM potassium phosphate, 20 mM sodium chloride, 10 μM EDTA and 0.02% sodium azide (w/v) buffer at pH 7.5, containing 5% D2O. All NMR experiments for PD-L1 (D1) were collected at 298 K whereas NMR experiments for PD-L1 (D1D2) were recorded at 303 K. Sequential backbone resonance assignments for PD-L1 (D1) were determined using a combination of 15N/13C/1H HNCACB CACB NH and HNCO experiments for PD-L1 (D1) and in the TROSY-HNCO for PD-L1 (D1D2). The acquisition parameters used for all NMR experiments are summarised in Table 3D NMR experiments were acquired from 190 μM and 240 μM samples of uniformly N, 90% N, 90% C’, 92% Cα and 92% Cβ) were obtained for PD-L1 (D1) with backbone amide assignments made for 101 of the 112 non-proline residues  under accession number 51412.Comprehensive sequence-specific backbone resonance assignments , with backbone amide assignments made for 167 of the 211 non-proline residues   and PD-L1 (D1D2) using primarily the NMR assignments obtained (Shen and Bax It is our hope that these backbone NMR assignments will be a useful tool for the study of the interactions of PD-L1 with functional partners as well as in the development of small-molecule therapeutics targeting PD-L1.


We also studied the role of several NOX isoforms during mild TBI (mTBI) and demonstrated that NOX2 and, to a lesser extent, NOX1 deficient mice are protected from mTBI pathology, whereas injury is exacerbated in NOX4 knockouts. Given the pathogenic role played by NOX2 in mTBI, we treated mice transcranially with NCATS-SM7270 after injury and revealed a dose-dependent reduction in mTBI induced cortical cell death. This inhibitor also partially reversed cortical damage observed in NOX4 deficient mice following mTBI. These data demonstrate that NCATS-SM7270 is an improved and specific inhibitor of NOX2 capable of protecting mice from NOX2-dependent cell death associated with mTBI.NADPH oxidases (NOX's), and the reactive oxygen species (ROS) they produce, play an important role in host defense, thyroid hormone synthesis, apoptosis, gene regulation, angiogenesis and other processes. However, overproduction of ROS by these enzymes is associated with cardiovascular disease, fibrosis, traumatic brain injury (TBI) and other diseases. Structural similarities between NOX's have complicated development of specific inhibitors. Here, we report development of NCATS-SM7270, a small molecule optimized from GSK2795039, that inhibited NOX2 in primary human and mouse granulocytes. NCATS-SM7270 specifically inhibited NOX2 and had reduced inhibitory activity against xanthine oxidase   •A new NOX2 inhibitor, NCATS-SM7270, protects mice from mild traumatic brain injury.•Knockout of NOX1 or NOX2 protects from injury while deletion of NOX4 increases it.•NCATS-SM7270 partially reversed mTBI in NOX4KO mice. Much woGSK2795039, a putative NOX2 inhibitor, was shown previously to reduce inflammation  and TBI 22.150 of 0.18 μM and required 100x greater concentrations to inhibit xanthine oxidase palladium(II) CHCl adduct  were purchased from Strem Chemicals. Analytical analysis was performed on an Agilent LC/MS  and both were performed at a flow rate of 1 mL/min. Method 1: A 7-min gradient of 4%–100% acetonitrile  in water (containing 0.05% TFA) was used with an 8 min run time on a Phenomenex Luna C18 column  at a temperature of 50 °C. Method 2: A 3-min gradient of 4%–100% acetonitrile (containing 0.025% TFA) in water (containing 0.05% TFA) was used with a 4.5 min run time on a Phenomenex Gemini Phenyl column  at a temperature of 50 °C. Purity determination was performed using an Agilent Diode Array Detector for both Method 1 and Method 2. Mass determination was performed using an Agilent 6130 mass spectrometer with electrospray ionization in the positive mode. 1H NMR spectra were recorded on Varian 400 MHz spectrometers. Chemical shifts are reported in ppm with undeuterated solvent (DMSO‑d6 at 2.49 ppm) as an internal standard for DMSO‑d6 solutions. All compounds tested in the biological assays had a purity greater than 99%, based on both analytical methods. High-resolution mass spectrometry was recorded on Agilent 6210 Time-of-Flight LC/MS system. Confirmation of molecular formula was accomplished using electrospray ionization in the positive mode with the Agilent Masshunter software (version B.02).All air or moisture-sensitive reactions were performed under a positive pressure of argon with oven-dried glassware. Anhydrous dioxane was purchased from Sigma-Aldrich. Cesium carbonate, K4.21 , 1-methyl-6--1H-indole 2 , tri-2-furylphosphine , and potassium phosphate  in 13 mL dioxane/water (4/1 mixture) was bubbled with argon for 5 min then added palladium(II) acetate . The reaction mixture was capped and stirred at 90 °C for 2 h. The reaction mixture was allowed to cool to room temperature before adding a Pd scavenger resin. The reaction was further stirred for another 30 min, diluted with dichloromethane, and filtered through a Celite pad. The filtrate was concentrated and the residue was directly loaded into a flash silica column. The crude product was purified on a flash system eluting with 0–60% ethyl acetate in hexanes over 32 column volumes using a 25 g silica column to yield 0.496 g (49%) of 4-bromo-1-cyclopropyl-3-(1-methyl-1H-indol-6-yl)-1H-pyrrolo pyridine 3. LCMS Retention time: (Method 2) = 3.68 min (M + H) = 366.A vial containing a mixture of 4-bromo-1-cyclopropyl-3-iodo-1H-pyrrolo pyridine 2CO3  and 1-methyl-1H-pyrazole-3-sulfonamide  in dry dioxane was bubbled with argon for 5 min. To the above reaction mixture methanesulfonatopalladium(II) CHCl adduct [39 1445085-87-9]  was added, capped, and heated in a microwave reactor for 1 h at 150 °C. The reaction was diluted with excess DCM and stirred with a Pd scavenger resin for 1 h. The reaction was filtered through a Celite pad to remove solid suspensions. The filtrate was concentrated and purified on a flash system using a 25 g silica column eluting with 20–100% ethyl acetate in hexanes for 20 column volumes after that at 100% ethyl acetate until the product elutes from the column. The slightly colored solid obtained after evaporating the solvent was suspended in ethanol and allowed to stand overnight at room temperature. The clear solid was collected by filtration. The white solid obtained was dried under a high vacuum to yield a pure product N-(1-cyclopropyl-3-(1-methyl-1H-indol-6-yl)-1H-pyrrolo pyridin-4-yl)-1-methyl-1H-pyrazole-3-sulfonamide (NCATS-SM7270). LC-MS Retention Time: (Method 1) = 7.018 min and (Method 2) = 3.871 min 1H NMR  δ 13.13 , 8.30 , 7.74–7.65 , 7.62–7.52 , 7.47 , 7.24 , 7.20 , 7.08 , 6.83 , 4.14 , 2.78–2.63 , 2.47 , 1.80 , 0.78 , 0.55 to −0.05 ; HRMS (ESI) m/z (M + H)+ calcd. For C33H28FN4O4S3 659.1251, found 659.1282.An oven-dried microwave vial containing the above product 4-bromo-1-cyclopropyl-3-(1-methyl-1H-indol-6-yl)-1H-pyrrolo pyridine 3 , Cs4.3The rat liver microsomal stability , paralle4.45 cells/well in Varioskan Lux luminometer for 30 cycles of 1 s per well (approximately 1 h). As published previously, patients with mutations in the gp91 component of NOX2 do not show significant ROS production in this assay [Human blood was obtained after informed consent by the Department of Transfusion Medicine  and provided as de-identified specimens. Human granulocytes were isolated using Percoll density fractionation as described  and NOX2NOX2 activity was also measured using an undifferentiated leukemic cell line, K562, that was stably transfected with gp91phox, p47phox, and p67phox . Cells w4.5Given concerns about previous assays for specific NOX family members  we here After the times indicated in Bovine Xanthine Oxidase (Sigma) was dialyzed in PBS to remove ammonium sulfate and stored at 4 °C. Hypoxanthine was prepared by heating in DMSO. Reactions were prepared by diluting xanthine oxidase in HBSS  that contained 50 μM luminol and were preincubated 5 min with control or inhibitor before the addition of hypoxanthine at a final concentration of 0.5 mM. Luminescence was measured at 24 °C as described above.4.6C57BL/6 J (B6), B6.129 S-Cybbtm1Din/J (NOX2 KO) , B6.129X4.7Mouse granulocytes were isolated from bone marrow essentially as described  except b4.8We used a focal cortical model of mTBI to assess the role of NOX family members and NOX2 inhibitors as described previously ,10,32. M4.8.1+ “surfaces” were created, and dead cells were counted as DAPI+ cells that colocalized with propidium iodide as described [One hour prior to sacrifice (5–6 h post-injury), aCSF with inhibitor or DMSO was washed off the exposed skull, and 200 μL propidium iodide  was applied transcranially to label dead cells as described . Mice thescribed . Dead ceThe 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.


Heterosis or hybrid vigor is the improved performance of a desirable quality in hybrid progeny. Hybridization between high-productive Assam type and high-quality Chinese type clone-cultivar is expected to develop elite tea plant progenies with high quality and productivity. Comparative transcriptomics analyses of leaves from the F1 hybrids and their parental clone-cultivars were conducted to explore molecular mechanisms related to catechin content using a high-throughput next-generation RNA-seq strategy and high-performance liquid chromatography (HPLC). The content of EGCG  and C (catechin) was higher in ‘Kiara-8’ × ‘Sukoi’, ‘Tambi-2’ × ‘Suka Ati’, and ‘Tambi-2’ × ‘TRI-2025’ than the other hybrid and clone-cultivars. KEGG (Kyoto Encyclopedia of Genes and Genomes) and GO (Gene Ontology) analysis found that most pathways associated with catechins content were enriched. Significant differentially expressed genes (DEGs) mainly associated with phenylpropanoid, flavonoid, drug metabolism-cytochrome P450, and transcription factor  pathways appeared to be responsible for the high accumulation of secondary metabolites in ‘Kiara-8’ × ‘Sukoi’, ‘Tambi-2’ × ‘Suka Ati’, and ‘Tambi-2’ × ‘TRI-2025’ as were detected in EGCG and catechin content. Several structural genes related to the above pathways have been obtained, which will be used as candidate genes in the screening of breeding materials. Camellia sinensis (L.) O. Kuntze) is a perennial plantation crop grown in more than 60 countries for the production of the most popular non-alcoholic beverage, tea. Global statistics indicate that approximately five million tons of tea leaves are harvested annually, and more than two billion cups are consumed daily [Tea . Each TF has a specific DNA binding domain that recognizes a 6–10 base pair motif in the DNA . TranscrThe study of morphological characteristics , biochemThe phenotypes of the ‘TRI-2025’ (green leaf), ‘Tambi-2’, ‘Tambi-1’ × ‘Kiara-8’, ‘Tambi-2’ × ‘Suka Ati-40’, ‘Tambi-2’ × ‘Cinyiruan-143’, ‘Tambi-2’ × ‘TRI-2025’, and ‘Kiara-8’ × ‘Sukoi’ were recorded . ‘TRI-202 > 0.99) in a relatively wide concentration range. Moreover, distinct changes in the levels of flavor-related metabolites were detected in parents and the five hybrids. Analysis of individual catechins identified that EGCG is the most abundant catechin in all leaf samples. The content of EGCG was the highest for the ‘Tambi-2’ × ‘Suka Ati-40’ (53.302 mg/g), and it was not significantly different from ‘Kiara-8’ × ‘Sukoi’ (52.014 mg/g) and ‘Tambi-2’ × ‘TRI-2025’ (51.456 mg/g). ‘Tambi-2’ had lower EGCG content (50.703 mg/g), which was not significantly different from ‘Tambi-2’ × ‘Cinyiruan-143’ (49.466 mg/g), while ‘TRI-2025’ (46.365 mg/g) and ‘Tambi-1’ × ‘Kiara-8’ (44.667 mg/g) had the lowest EGCG content. The content of C in ‘Kiara-8’ × ‘Sukoi’ was the highest (1.34 mg/g), and it was not significantly different from ‘Tambi-2’ × ‘Suka Ati-40’ (1.21 mg/g), ‘Tambi-2’ × ‘TRI-2025’ (1.19 mg/g), and ‘Tambi-2’ (0.99 mg/g). ‘Tambi-1’ × ‘Kiara-8’ (0.67 mg/g) had the lowest C content  are well known to play a crucial role in secondary metabolite biosynthesis in plants . We searp < 0.005). De novo transcriptome filtered by Corset was used as a reference [To understand gene expression between samples, we measured transcript levels in terms of fragments per kilo-base exon per million reads (FPKM) based on RNAseq data. Genes were called Differentially Expressed Genes (DEGs) if they exhibited at least a 2-fold change in transcript abundance using edgeR  showed up-regulation in ‘Kiara-8’ × ‘Sukoi’, ‘Tambi-2’ × ‘Suka Ati-40’, ‘Tambi-2’, and ‘Tambi-2’ × ‘TRI-2025’ in the formation of cinnamic acid from phenylalanine. 4CL (Cluster-19805.50504) showed up-regulation in ‘Kiara-8’ × ‘Sukoi’, ‘Tambi-2’ × ‘Suka Ati-40’, ‘Tambi-2’ × ‘TRI-2025’, and ‘Tambi-2’ in the formation of cinnamoyl-CoA from cinnamic acid. SHT  showed up-regulation in ‘Kiara-8’ × ‘Sukoi’ in the formation of caffeoyl-CoA from p-coumaroyl-CoA. CCoAOMT (Cluster-19805.19420) showed up-regulation in ‘Tambi-2’ × ‘Suka Ati-40’, ‘Kiara-8’ × ‘Sukoi’, and ‘Tambi-2’ in the formation of feruloyl-CoA from caffeoyl-CoA. Mannitol dehydrogenase isoformX1 (Cluster-19805.43964) showed up-regulation in ‘Tambi-2’ × ‘Suka Ati-40’ in the formation of p-coumaryl alcohol from p-coumaraldehyde . Figure Highly expressed structural genes involved in flavonoid biosynthesis were identified in analyzed samples, chalcone synthase (CHS), flavonoid 3′,5′-hydroxylase (F35H), anthocyanidin synthase (ANS), anthocyanidin reductase (ANR), caffeoyl-CoA O-methyltransferase (CCoAOMT), leucoanthocyanidin dioxygenase (LDOX), spermidine hydroxycinnamoyl transferase (SHT), cinnamate-4-hydroxylase (C4H), UDP glycosyltransferase (UGT), flavonol synthase (FLS), and leucoanthocyanidin reductase (LAR) b. PAL, 4CHS (Cluster-19805.50241) showed up-regulation in ‘Tambi-2’ × ‘Suka Ati-40’, ‘Tambi-2’ × ‘TRI-2025’, ‘Tambi-2’, and ‘Kiara-8’ × ‘Sukoi’, and the other CHS (Cluster-19805.45299 and Cluster-19805.46386) showed up-regulation in ‘Kiara-8’ × ‘Sukoi’, ‘Tambi-2’ × ‘TRI-2025’, and ‘Tambi-2’ × ‘Suka Ati-40’ in the formation of naringenin chalcone from p-coumaroyl-CoA. F35H (Cluster-19805.49082) showed up-regulation in ‘Kiara-8’ × ‘Sukoi’, ‘Tambi-2’ × ‘Suka Ati-40’, ‘Tambi-2’, and ‘Tambi-2’ × ‘TRI-2025’ in the formation of eriodictyol and dihydroquercetin from naringenin and dihydrokaempferol, respectively, and dihydrotricetin and dihydromyricetin from eriodictyol and dihydroquercetin, respectively. ANS (Cluster-19805.41144) showed up-regulation in ‘Kiara-8’ × ‘Sukoi’, ‘Tambi-2’ × ‘Suka Ati-40’, and ‘Tambi-2’ in the formation of cyanidin and delphinidin from leucocyanidin and leucodelphinidin, respectively. ANR (Cluster-19805.52453) showed up-regulation in ‘Kiara-8’ × ‘Sukoi’, ‘Tambi-2’ × ‘Suka Ati-40’, and ‘Tambi-2’ in the formation of (-)-epigallocatechin and (-)-epicatechin from delphinidin and cyanidin, respectively. LDOX (Cluster-6630.0) showed up-regulation in ‘Kiara-8’ × ‘Sukoi’ in the formation of cyanidin and delphinidin from leucocyanidin and leucodelphinidin, respectively (Anthocyanin biosynthesis). SHT (Cluster-19805.49537) showed up-regulation in ‘Kiara-8’ × ‘Sukoi’ in the formation of caffeoyl-CoA from p-coumaroyl-CoA. UGT (Cluster-19805.51749) and naringenin-3-dioxygenase (Cluster-19805.49197) showed up-regulation in ‘Kiara-8’ × ‘Sukoi’ in the formation of dihydrokaempferol, dihydroquercetin, and dihydromyricetin from naringenin, eriodictyol, and dihydrotricetin, respectively .CsTPS (terpenoid synthase gene) [Highly expressed structural genes involved in sesquiterpenoid biosynthesis were identified in analyzed samples, (-)-germacrene D synthase-like, alpha-farnesene synthase (AFS), squalene epoxidase 3 (SQE3), squalene monooxygenase (SM), and terpene synthase 3 (TPS3). (-)-Germacrene D synthase-like (Cluster-19805.49859 and Cluster-19805.52025) were up-regulated in ‘Kiara-8’ × ‘Sukoi’, while (-)-germacrene D synthase-like (Cluster-19805.37369 and Cluster-19805.36016) were up-regulated in ‘Tambi-2’ in the formation of (-)-germacrene D. AFS (Cluster-19805.47819 and Cluster-19805.38956) were up-regulated at ‘Tambi-2’ × ‘TRI-2025’ and ‘Kiara-8’ × ‘Sukoi’ in the formation of -α-farnesene. TPS3 (Cluster-19805.19418) was up-regulated in ‘Tambi-2’ × ‘Suka Ati-40’, ‘Tambi-2’, and ‘Kiara-8’ × ‘Sukoi’ in the formation of (-)-germacrene D. (-)-Germacrene D synthase-like isoformX2 (Cluster-19805.49552) . In planse gene) . Methyl In the alpha-linolenic acid biosynthesis, acyl coenzyme A oxidase 4 (ACX) and lipoxygenase (LOX) showed a high expression level in analyzed samples. The number of DEGs of ACX (Cluster-19805.33301) was up-regulated in ‘Tambi-2’ × ‘Suka Ati-40’, ‘Tambi-2’ × ‘TRI-2025’, ‘Tambi-2’, ‘Kiara-8’ × ‘Sukoi’, and ‘Tambi-2’ × ‘Cinyiruan-143’ in the formation of trans-2-Enoyl-OPC6-CoA from OPC6-CoA. LOX  has a high expression in ‘Kiara-8’ × ‘Sukoi’, ‘Tambi-2’ × ‘Cinyiruan-143’, and ‘Tambi-2’ followed by ‘Tambi-2’ × ‘TRI-2025’, ‘Tambi-1’ × ‘Kiara-8’, and ‘TRI-2025’ in the formation of 9 (S)-HpOTrE and 13 (S)-HpOTrE from alpha-linolenic acid . OxylipiC. sinensis were positively correlated with the accumulation of flavonols, anthocyanins, and proanthocyanins in tea tissues and cultivars [DEG enrichment analysis in drug metabolism-cytochrome P450 showed that glutathione S-transferase genes dominated and showed high expression between samples. Cluster-19805.64347 (glutathione S-transferase F12-like) showed a high expression level in ‘Tambi-2’ × ‘TRI-2025’, ‘Kiara-8’ × ‘Sukoi’, ‘Tambi-2’ × ‘Suka Ati-40’, and ‘Tambi-2’, while Cluster-19805. 46266 (probable glutathione S-transferase) showed the highest expression level in ‘Tambi-2’ × ‘Suka Ati-40’ e in the ultivars .Annotation analysis in the analyzed sample and DEG analysis showed a high DEG number of MYB transcription factor families, Cluster-19805.57717 (R2R3-MYB1), Cluster-19805.51520 (R2R3 –MYB transcription factor anthocyanin1), Cluster-19805.37397 (MYB4), Cluster-19805.53207 (MYB5), Cluster-19805.65536 (MYB5a), Cluster-19805.47061 (MYB5c), Cluster-19805.48725 (MYB5d), Cluster-19805.46699 (MYB5e) Cluster-19805.40565 (MYB44), Cluster-19805.44296 (MYB75), Cluster-19805.1600 (MYB111), and Cluster-19805.31168 (MYB114) f. Seven The flavonoid biosynthesis pathway is highly organized. Key structural genes and numerous transcription factors act as regulatory protein modulators of gene expression through sequence-specific DNA binding at the transcriptional level or by protein–protein interactions during chromatin remodeling . In our C. sinensis var. sinensis [Enrichment analysis revealed that 10 genes were involved in the catechin biosynthesis of sinensis . Howeversinensis . F35H issinensis .Phenylalanine is the initial substrate of the phenylpropanoid pathway, which is determined by the catalysis PAL, the first and rate-limiting enzyme that regulates overall carbon flux into phenylpropanoid metabolism due to its unique metabolic position . Flavono2+, 4-coumaroyl-CoA serves as a precursor for the biosynthesis of numerous plant secondary products, such as flavonoids [DEGs of C4H in ‘Kiara-8’ × ‘Sukoi’, ‘Tambi-2’ × ‘Cinyiruan-143’, ‘Tambi-2’ × ‘Suka Ati-40’, and ‘Tambi-2’ × ‘TRI-2025’ were higher compared to ‘TRI-2025’ and ‘Tambi-2’ a. PAL, Cavonoids . DEGs ofavonoids .The DEGs of Caffeoyl CoA O-methyltransferase had the highest expression level in ‘Tambi-2’ × ‘Suka Ati-40’, ‘Kiara-8’ × ‘Sukoi’, ‘Tambi-2’, and ‘Tambi-2’ × ‘TRI-2025’ a. The ovANR, ANS, CHS, CHI, DFR, F3H, F35H, LAR, and UGT detected in the samples were some key enzymes that directly regulated the flavonoid biosynthesis in tea. The DEGs of those genes had higher expression numbers at ‘Kiara-8’ × ‘Sukoi’, ‘Tambi-2’ × ‘Suka Ati-40’, and ‘Tambi-2’ than the other samples. ANR is one of the two enzymes of the flavonoid biosynthesis pathway that produces flavan-3-ols (epicatechin) monomers from anthocyanidin . ANS is The purple leaves of ‘Kiara-8’ × ‘Sukoi’, ‘Tambi-2’ × ‘Suka Ati-40’, and ‘Tambi-2’ × ‘TRI-2025’ showed a high content of anthocyanin. The increase in anthocyanin biosynthesis was mainly due to increased PAL activity . ClusterR2R3-MYB families (Cluster-19805.57717 and Cluster-19805.51520) were identified in the samples that had high DEG numbers in ‘Kiara-8’ × ‘Sukoi’, ‘Tambi-2’, and ‘Tambi-2’ × ‘Suka Ati-40’ f. An R2RThis study showed the catechin heterosis, especially in EGCG content in ‘Tambi-2’ × ‘TRI-2025’ compared with the female (‘Tambi-2’) and male parents (‘TRI-2025’) and ‘Tambi-2’ × ‘Suka Ati-40’ compared with the female parent (‘Tambi-2’). Several gene clusters and TFs that influence the formation of EGCG and C in the biosynthesis of phenylpropanoids and flavonoids have been identified.Transcriptomic variation and expressional regulation of putative functional genes in quality-related pathways between green-leaf and purple-leaf clone-cultivars and selected F1 hybrids from crossing between clone-cultivar parents were detected using a comprehensive analysis of transcriptomic differences that are reflected at the phenotypic level in tea plants. ‘Kiara-8’ × ‘Sukoi’, ‘Tambi-2’ × ‘Suka Ati’, and ‘Tambi-2’ × ‘TRI-2025’ were selected as candidate high-quality tea hybrids based on EGCG and catechin content and DEGs involved in phenylpropanoid, flavonoid, and drug metabolism-cytochrome P450 pathways, and also high expression level of TF  related to the phenylpropanoid and flavonoid pathways. These results are important for determining the relationship between genetic background and the catechin content of tea to improve future breeding materials.For transcriptomic and biochemical analysis, we used an individual plant of parent cultivars ‘Tambi-2’ (Chinese type) and ‘TRI-2025’ (Assam type) and also a series of F1-hybrids: ‘Tambi-2’ × ‘TRI-2025’, ‘Tambi-2’ × ‘Suka Ati’, ‘Tambi-2’ × ‘Cinyiruan-143’, ‘Tambi-1’ × ‘Kiara-8’, and ‘Kiara-8’ × ‘Sukoi’ from crossing between parents with green-leaf tea  and purple-leaf tea  were collected in September 2020 from a polyclonal garden of PT Pagilaran in Batang district, Central Java province. Fresh and healthy leaves were harvested and immediately frozen in liquid nitrogen after collection. These preparations were preserved at −80 °C in a container in the laboratory before being processed further for secondary metabolites and RNA extraction.To determine major constituents in tea, fresh leaves were crushed into a fine powder with liquid nitrogen using a mortar and pestle. About 1 g of fine powder was dissolved in 10 mL methanol. Further, the solution was subjected to a continuous vortex for 24 h and subsequently centrifuged at 2000 rpm (15 min). The supernatant was then filtered through 5C filter paper. The solution was then collected in small bottles and evaporated to receive the crude extract. About 1.5 mg of crude extract was dissolved in 1.5 mL methanol for HPLC. The solution was subjected to a continuous vortex for 5 min. The solution was then filtered through a 0.45-micron syringe filter (nylon) into an HPLC vial screw. The standard solution of EGCG and catechin (C) was diluted in the mobile phase, yielding the standard solutions with different concentrations. Subsequently, the solution was filtered using a 0.45-micron syringe filter into an HPLC vial screw. Sample-extracted solutions and standard solutions were later degassed by sonication for 15 min before injecting into the HPLC system to obtain the chromatograms. The standard and sample-extracted solutions (1 mg/mL) were run to ensure that both analytes were chemically identical with similar molecular structures. This analysis was performed on a high-pressure liquid chromatograph (Shimadzu Prominence Detector SPD-M20A) installed with Analyst software. A C18 column  was used at a flow rate of 1.0 mL/min. The detection wavelength was set to 200–600 nm, and the column temperature was 25 °C. The mobile phase consists of MeOH: 0.05% TFA aq 25:75. The total run time of the analytical method was set to 70 min in the HPLC system. The samples were analyzed in triplicate.®. The quality and quantity of the isolated DNA and RNA were checked by agarose gel electrophoresis, NanoDrop 2500 , and Agilent 2100. The total RNAs of the seven samples were used to construct the RNA-Seq libraries. We used a P5 adaptor sequence P5-AATGATACGGCGACCACCGAGA (5′-3′), P5′-TTACTATGCCGCTGGTGGCTCT (3′-5′), and P7 adaptor sequence CGTATGCCGTCTTCTGCTTG-P7′ (5′-3′), GCATACGGCAGAAGACGAAC-P7 (3′-5′).High-quality transcriptomic RNA was extracted from the leaves using Plant Total RNA Mini Kit with DNase GeneaidThe transcriptome was sequenced by Novogene Technology using the Illumina HiSeq 2500 platform. After sequencing, the raw reads underwent quality control. Quality control for transcriptomic reads consists of error-rate distribution along with reads and GC content distribution. Raw transcriptomic sequencing data were preprocessed using Filter Tools from Galaxy Platform to remove reads with adaptor contamination, when uncertain nucleotides constitute more than ten percent of either read (N > 10%) and when low-quality nucleotides  constitute more than 50% of the reads. High-quality sequencing data were first assembled into contigs and assembled using Geneious Prime 2021.1.1 Align/Assemble tools. Clean reads were de novo assembled by Trinity 2.6.6 software [https://github.com/Oshlack/Corset/wiki accessed on 9 September 2021) [Trinity 2.6.6 software  was useder 2021)  to develiTAK was used to perform the transcription factor analysis of tea. De novo transcriptome filtered by Corset was used as a reference. To map reads back to the transcriptome and quantify the expression level, we used RSEM 1.2.28 . To calc2(fold change)| > 1 for the differential expression analysis. Volcano plots were used to infer the overall distribution of differentially expressed genes. The Venn diagram presents the number of genes that are uniquely expressed differentially within each group, with the overlapping regions showing the number of genes that are expressed in two or more groups. A major bioinformatics classification system, Gene Ontology (http://www.geneontology.org/ accessed on 9 September 2021), was used to unify the presentation of gene properties across all species. We used GOSeq 1.32.0 software by Young & Davidson, California, U.S. state with a corrected p-value of < 0.05 for significant enrichment. For KEGG enrichment, we used KOBAS v3.0 with a corrected p-value of <0.05 Pathway enrichment analysis identified significantly enriched metabolic pathways or signal transduction pathways associated with differentially expressed genes compared with the whole genome background (www.genome.jp) accessed on 9 September 2021.We used edgeR 3.28.0 padj < 0.005 |logTranscriptomic analysis identified a large number of potential key regulatory factors that can control the catechin content of shoot tea leaves. KEGG enrichment analyses revealed that the phenylpropanoid, flavonoid, sesquiterpenoid and triterpenoid, alpha-linolenic acid, and drug metabolism-cytochrome P450 metabolic pathways were highly enriched with DEGs. Significant DEGs mainly associated with phenylpropanoid, flavonoid, drug metabolism-cytochrome P450 pathways, and TF  appeared to be responsible for the high accumulation of secondary metabolites in ‘Kiara-8’ × ‘Sukoi’, ‘Tambi-2’ × ‘Suka Ati’, and ‘Tambi-2’ × ‘TRI-2025’ as was detected in EGCG and catechin content. The content of EGCG and catechin were higher in ‘Kiara-8’ × ‘Sukoi’, ‘Tambi-2’ × ‘Suka Ati’, and ‘Tambi-2’ × ‘TRI-2025’ than in the other hybrids and clone-cultivars. Catechin heterosis, especially in EGCG content in ‘Tambi-2’ × ‘TRI-2025’ compared with female (‘Tambi-2’) and male parents (‘TRI-2025’), and ‘Tambi-2’ × ‘Suka Ati-40’ compared with female parent (‘Tambi-2’). The accumulation pattern of tea quality-related metabolites varied depending on the accessions. These results will also provide candidate genes for the selection of breeding materials in tea plants based on tea quality-related metabolites.


Bacillus lehensis G1, led to the optimization of its purification, post-purification and crystallization processes for X-ray diffraction purpose. The workflow, conditions used and dataset obtained from each stage of the processes are presented herein. The optimization workflow has enabled the obtainment of purified, active BLEG-1 in high yield for its activity assays, crystallization and structure determination via X-ray diffraction. This is the first step to gain a better insight into its dual activity and evolutionary divergence from a structural perspective. The complete research article, including BLEG-1 dual activity analysis, is published in the International Journal of Molecular Sciences .Metallo-β-lactamases (MBLs) are class B β-lactamases from the metallo-hydrolase-like MBL-fold superfamily which act on a broad range of β-lactam antibiotics, thus conferring antibiotics resistance to bacterial pathogens. The attempt to structurally characterize BLEG-1, an evolutionary divergent B3 metallo-β-lactamase (MBL) with dual activity from • The method was optimized to increase the stability of BLEG-1 in purification, post-purification and crystallization processes.• Protein crystallization using the optimized conditions presented herein is able to produce and regenerate BLEG-1 protein crystals of medium-size, which is an advantage in X-ray diffraction.• The method can be used for relevant homologs and variants of BLEG-1 for structure-function and mechanistic studies of such proteins.  Specifications tablein silico structure-based drug design approach coupled with their in vitro analyses. In addition to this, discoveries of B3 MBLs with bifunctional properties have set forth scientific discourse and investigations on the evolution of β-lactamases. B3 MBLs have been reported to be functionally and phylogenetically diverse The production of β-lactamases is one of the common mechanisms that bacteria employ to inactivate the action of β-lactam antibiotics by cleavage amide bond of the β-lactam ring of the antibiotics before they reach the target site in bacteria. There are two types of β-lactamases, which are serine-β-lactamases (SBLs) and metallo-β-lactamases (MBLs). MBLs or class B β-lactamases belong to the metallo-hydrolase-like MBL-fold superfamily, which consists a large and ancient group of proteins with diverse catalytic functions. MBLs are characterized by the αβ/βα protein fold and the His-Xaa-His-Xaa-Asp-His motif that coordinates zinc ion(s) for their catalytic reaction https://www.rcsb.org/stats/all-released-structures, accessed on 7 Dec 2021). This is a well-established method which is able to yield high resolution data and is not limited by protein size There are several methods that can be used to determine the three-dimensional (3D) atomic structure of protein, which are the X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and cryo-electron microscopy (cryo-EM). According to the statistics in the Protein Data Bank (PDB), X-ray crystallography is still the predominant method in protein structure determination to date Luria-Bertani (LB) broth •41 M ZnSO•40 mL Bijou bottle•5 L Erlenmeyer flask•50 mL centrifuge tube•Ecotron incubation shaker •Varian Cary® 50 UV-Vis spectrophotometer •Refrigerated centrifuge 5804R Procedure1.Escherichia coli BL21(DE3) cells harbouring pET-28b(+)::Bleg-1 recombinant plasmid (from glycerol stock) were cultivated in 120 mL sterile LB broth containing 50 μg/mL of kanamycin at 37˚C for 18 h, in an incubation shaker with agitation speed of 200 rpm.Based on the method by Selvaraju et al. 2.4. The culture was cultivated at 37˚C with agitation at 200 rpm.120 mL of the starter culture was used to inoculate into 3 L sterile LB broth containing 50 μg/mL of kanamycin and 0.1 mM ZnSO3.600) of the bacterial culture reached 0.6, 0.1 mM of IPTG was added, followed by further cultivation at 20˚C for 20 h, with agitation at 200 rpm. The cells were collected by centrifugation at 9,000 x g, at 4˚C for 20 min.When the optical density at 600 nm Buffer 1 Buffer 2 Buffer 3 •Bovine serum albumin (BSA)•Syringe•Syringe filter with 0.45 µm cellulose acetate membrane •TM FF column 5 mL pre-packed HisTrap•SnakeSkin Dialysis Tubing, 10 kDa MWCO •15 mL centrifuge tube•5 L Erlenmeyer flask•Plastic cuvette•Refrigerated centrifuge 5804R •Q55 Sonicator with 1/4’’ (6mm) probe •ÄKTAPrime plus system •Mini-PROTEAN tetra cell vertical gel electrophoresis system •Varian Cary® 50 UV-Vis spectrophotometer Procedure1.Cell pellet collected from 3 L bacterial culture in step 1 was resuspended in buffer 1, and lyzed using a sonicator with 1/4’’ (6mm) probe, at 40% amplitude, 15 s ON and 15 s OFF pulses, for 12 min, on ice.2.The cell lysate was then centrifuged at 9,000 x g, 4˚C for 20 min, in which the supernatant was subsequently passed through a 0.45 µm cellulose acetate membrane by syringe filtration.3.TM FF column at flowrate of 1.0 mL/min, which was connected to ÄKTAPrime plus system and pre-equilibrated with buffer 1.The filtered supernatant was loaded into a 5 mL pre-packed HisTrap4.His-tagged protein bound on the resin was washed with 20 column volume (CV) of buffer 1 and eluted in a linear gradient of imidazole (50–500 mM imidazole) using 20 CV of buffer 2.5.The elution fractions were collected, analyzed by SDS-PAGE (12%), pooled in a dialysis tubing and dialyzed against 4 L of buffer 3 (i.e. protein-to-buffer ratio of 1:160), at 4°C for 18 h.6.Concentrations of protein were determined by Bradford assay Step 3: Optimization of His-tag cleavageMaterials and apparatus•2HPO4–NaH2PO4 buffer, 0.1 mM ZnSO4, 5% (v/v) glycerol; pH 7.4)Buffer A Buffer B •1 U/µL thrombin•5 L Erlenmeyer flask•1.5 mL microcentrifuge tube•Waterbath WNB 14 •Mini-PROTEAN tetra cell vertical gel electrophoresis system Procedure1.Purified His-tagged fusion BLEG-1 obtained via Ni-NTA affinity chromatography was dialyzed using similar method mentioned in step 2 with buffer A and buffer B, respectively.2.One mg/mL of the dialyzed protein sample was then added with 1 U/µL of thrombin enzyme, and incubated in waterbath at 4°C and 25°C, for 24 h.3.The mixtures were aliquoted at time intervals of 0, 2, 4, 8, 16 and 24 h, and subjected to SDS-PAGE (12%) analysis.Step 4: His-tagged cleavage and second-step BLEG-1 purification via anion-exchange chromatographyMaterials and apparatus•1 U/µL thrombin•2HPO4–NaH2PO4 buffer, 20 mM NaCl; pH 7.4)Buffer 3 Buffer 4  glycerol; pH 7.4)Buffer 5 5 mL Q-Sepharose•Bradford reagent •SnakeSkin Dialysis Tubing, 10 kDa MWCO •15 mL centrifuge tube•5 L Erlenmeyer flask•Waterbath WNB 14 •ÄKTAPrime plus system •Mini-PROTEAN tetra cell vertical gel electrophoresis system •Varian Cary® 50 UV-Vis spectrophotometer Procedure1.1 U/µL of thrombin enzyme was added to per mg/mL of dialyzed His-tagged fusion BLEG-1 obtained in step 2, and incubated at 25°C for 16 h.2.BLEG-1 with and without the addition of thrombin were analyzed by SDS-PAGE (12%).3.TM FF column at flowrate of 1.0 mL/min, which was connected to ÄKTAPrime plus system and pre-equilibrated with buffer 3.Thrombin-treated BLEG-1 was loaded into a 5 mL Q-Sepharose4.The bound protein was washed with 12 CV of buffer 3 and eluted in a linear gradient of NaCl (20–500 mM NaCl) using 20 CV of buffer 4.5.The eluates were collected and analyzed via SDS-PAGE (12%), pooled and dialyzed against 4 L of buffer 5 at 4°C for 18 h using the same dialysis tubing and protein-to-buffer ratio as mentioned in Step 2.6.Concentrations of protein were determined using the same method in Step 2.Step 5: BLEG-1 crystallization and optimizationMaterials and apparatus•Vivaspin 6, 10 kDa MWCO Polyethersulfone centrifugal concentrator •TM and Crystal Screen 2TM Crystal Screen 1•TM and PEG/Ion Screen 2TM PEG/Ion Screen 1•TM Index Screen•TM JCSG plus•NaI•50% (w/v) Polyethylene glycol (PEG) 3,350 Monodisperse•INTELLI-PLATE® 96 Well plates •Crystal clear sealing tape •Refrigerated centrifuge 5804R •Oryx8 protein crystallization robot .•TM chilling incubator EchoThermProcedure1.For pre-screening of BLEG-1 concentration for crystallization, purified BLEG-1 was concentrated to 3 mg/mL and 17 mg/mL via centrifugation with speed of 8,000 x g at 4°C, using Vivaspin 6 centrifugal concentrator with MWCO of 10 kDa.2.Pre-screening of the crystallization formulations and conditions for BLEG-1 were conducted using the commercial crystallization screening kits as mentioned in the “materials and apparatus” section.3.Crystallization was carried out on INTELLI-PLATE® 96 Well crystallization plates that were pre-filled with 240 µL of crystallization formulations in the reagent reservoirs, using 3 and 17 mg/mL of purified BLEG-1, through sitting-drop vapour diffusion method by Oryx8 protein crystallization robot.4.By mixing 1 µL of protein with 1 µL and 0.5 µL of formulation respectively, droplets of 1:1 and 2:1 (protein-to-formulation) ratio were made in the drop wells of crystallization plates.5.The crystallization plates were sealed and incubated at 4°C.6.Subsequent optimization of BLEG-1 crystallization conditions was performed by varying the concentrations of NaI  and PEG3350 ), using 17 mg/mL of purified BLEG-1, at 1:1 protein-to-formulation ratio, and incubated at 4°C and 20°C.E. coli BL21(DE3) after cultivation in LB broth with 0.1 mM IPTG induction at 20˚C for 20 h. BLEG-1 recombinant protein was purified to near homogeneity using Ni-NTA affinity chromatography ), in 1:1 protein-to-formulation ratio, after 14 days of incubation at 4°C  polyethylene glycol (PEG) 3350 (i.e. Tube 10 of PEG/Ion Screen 1n at 4°C . To furtn at 4°C  and 3. Tn at 4°C . ProteinThe 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.


As total joint replacement is widely applied for severe arthropathy, peri-prosthetic aseptic loosening as one of the main causes of implant failure has drawn wide attention. Wear particles such as titanium particles (TiPs) derived from prosthesis can initiate macrophages inflammation and sequentially activate osteoclasts, which results in bone resorption and osteolysis for long-term. Therefore, inhibiting wear particles induced macrophages inflammation is considered as a promising therapy for AL. In this research, we found that the inhibition of p110δ, a member of class IA PI3Ks family, could significantly dampen the TiPs-induced secretion of TNFα and IL-6. By the transfection of siRNA targeting p110δ, we confirmed that p110δ was responsible for TNFα and IL-6 trafficking out of Golgi complex without affecting their expression in TiPs-treated macrophages. As the upstream transcription-repressor of p110δ, Krüppel-like factor 4 (KLF4), targeted by miR-92a, could also attenuate TiPs-induced inflammation by mediating NF-κB pathway and M1/M2 polarization. To further ascertain the roles of KLF4/p110δ, TiPs-induced mice cranial osteolysis model was established and vivo experiments validated that KLF4-knockdown could exacerbate TiPs-induced osteolysis, which was strikingly ameliorated by knockdown of p110δ. In summary, our study suggests the key role of miR-92a/KLF4/p110δ signal in TiPs-induced macrophages inflammation and osteolysis. As an effective therapy to restore joint function, total joint replacement is an alternative for severe arthropathy and the demand for total hip or knee arthroplasty is growing rapidly . HoweverClass I PI3Ks family, which can be further divided into class IA  or class IB (p110γ), is ubiquitously reported to control cell survival, migration, differentiation and it involves in the progress of various diseases including skeletal-related inflammatory diseases –10. FormKrüppel-like factor 4 (KLF4) containing three zinc-finger motifs is a member of SP/KLF factors, which functions as a transcription factor in diverse physiological process . Apart fIn this study, we revealed the pro-inflammatory role of p110δ, which was mediated by miR-92a/KLF4 in TiPs-stimulated macrophages. Firstly, we screened p110δ as a TiPs-induced inflammation relative factor and confirmed that its expression was facilitated toward TiPs stimulation. With the application of IC87114 or si-p110δ, we found that p110δ involved in the transport of TNFα and IL-6 from Golgi complex to plasma membrane. KLF4, targeted by miR-92a, was predicted as a transcriptional repressor of p110δ and it could also attenuate the TiPs activated NF-κB pathway and M1/M2 macrophages polarization ratio. In vivo experiments, the role of KLF4 and p110δ were further confirmed in TiPs-induced mice cranial osteolysis, suggesting p110δ as a pro-osteolysis factor while KLF4 was an anti-osteolysis factor.p < 0.05; 1.61 ± 0.09 vs 0.38 ± 0.03 for IL-6, p < 0.05) in AL group upon TiPs infiltration  staining to detected the productions of TNFα and IL-6. We found stronger TNFα and IL-6 staining with higher IS score  and STRING database (https://string-db.org/), we discovered that the production of p110δ was upregulated upon various inflammatory stimulation including TNFα, IL-1β, interferon γ, and LPS, and there was a strong connection between p110δ and pro-inflammatory factors.   Figs.  and S1C.5) Figs. .Fig. 2InNext, the activity of p110δ was inhibited by IC87114, a p110δ selective inhibitor, to explore the role of p110δ. Co-treated with TiPs for 8 h, IC87114 led to attenuated TNFα and IL-6 secretions and the concentration of 10 μM exhibited the most promising inhibitory effect without affecting cell viability Fig. E–G. But Since that TNFα needed to be cleaved by TNFα-converting enzyme (TACE) into mature form for secretion, we could detect the surface or intracellular distribution of TNFα by the application of TAPI-1, an inhibitor of TACE. Flow cytometry (FCM) assay revealed that TiPs-induced surface level of TNFα declined from 52.82 ± 2.28 to 27.57 ± 1.37 while intracellular TNFα was significantly cemented due to the presence of IC87114, indicating that the inhibition of p110δ increased residual TNFα and reduced the secreted TNFα Figs.  and S1D.Given the effect of IC87114, we then designed p110δ-knockdown macrophages for further study. Three different siRNAs targeting mouse p110δ were transfected and macrophages with the minimal level of p110δ were screened Fig. S. Next, wNext, Brefeldin A (BFA), an inhibitor to hinder the protein trafficking from Endoplasmic Reticulum (ER) to Golgi complex, was applied for WB assay. After macrophages were co-treated with BFA and TiPs, we found that protein levels of TNFα and IL-6 were unaffected upon p110δ-knockdown, suggesting that p110δ was responsible for post-trafficking process of TNFα and IL-6 Fig. . For furNext, we wondered whether extra p110δ could facilitate TNFα and IL-6 secretions. Intriguingly, we generated p110δ-overexpressed macrophages and found that the overexpression of p110δ merely affected the serum levels of TNFα and IL-6 Fig. SC–E. SimiNext, downstream factors and signaling pathways of p110δ were also further explored. We performed WB assay and found comparable activation of NF-κB and MAPK signaling pathways upon different levels of p110δ Fig. H, I. It https://www.ncbi.nlm.nih.gov/), the sequence of Pik3cd (gene ID of p110δ) was acquired. In all, 2000 bp upstream and 100 bp downstream of the transcriptional initiated site was considered as the binding realm for transcription regulators. Next, UCSC (http://www.genome.ucsc.edu/) and JASPAR (http://jaspar.genereg.net/) were utilized to sift the underlying transcription factors, among which KLF4 with the highest score was selected.   macrophages  or KLF4-overexpressed   Figs.  and S4C,5) Figs. . Sequent5) Figs. . This ouThus, we performed WB assay and we discovered that upon 1 h TiPs stimulation, knockdown KLF4 facilitated the phosphorylation of p65 and IκBα while total IκBα was reduced, but overexpressed KLF4 led to contrary outcome Fig. . FurtherReportedly, KLF4 could be targeted by various microRNAs –30. Ther+, implying miR-92a could facilitate p110δ expression by targeting KLF4  or inhibitor-miR-92a (Anti-92a) transfected macrophages for gain- or loss- function experiments. As demonstrated in WB and qPCR assays, increased miR-92a obviously reduced the expression of KLF4 in both protein and mRNA levels, while impaired miR-92a exhibited reverse effect Fig. C, D. To LF4 Fig. G, H. ConLF4 Fig. . Meanwhited Fig. . Taken tMethods section. At day 7 after surgery, we could detect strong luciferase signal, which meant that macrophages remained on the calvaria.  while more osteoclasts were detected in TiPs+sh-KLF4 group    Figs.  and S5B.5) Figs. , D. In cIn this study, we revealed the pro-inflammatory role of p110δ, which was mediated by miR-92a/KLF4 in TiPs-stimulated macrophages. Firstly, we screened p110δ as a TiPs-induced inflammation relative factor and confirmed that its expression was facilitated toward TiPs stimulation. With the application of IC87114 or si-p110δ, we found that p110δ involved in the transport of TNFα and IL-6 from Golgi complex to plasma membrane. KLF4, targeted by miR-92a, was predicted as a transcriptional repressor of p110δ and it could also attenuate the TiPs activated NF-κB pathway and M1/M2 macrophages polarization ratio. In vivo experiments, the role of KLF4 and p110δ were further confirmed in TiPs-induced mice cranial osteolysis, suggesting p110δ as a pro-osteolysis factor while KLF4 was an anti-osteolysis factor.Ubiquitous reports indicated that wear particles induced prosthetic aseptic loosening was initiated by a series of pro-inflammatory factors from macrophages –39. Our After we discovered that the inhibition or knockdown of p110δ could not impaired the mRNA expression of TNFα and IL-6, we assumed that p110δ functioned in the post-transcriptional modification process or protein trafficking of TNFα and IL-6. Next, p110δ-knockdown macrophages were constructed and BFA was applied to block protein trafficking from Endoplasmic Reticulum (ER) to Golgi complex for WB assay. If p110δ mediated the post-transcriptional modification process, the knockdown level of p110δ would lead to reduced TNFα and IL-6 protein levels in BFA-treated macrophages. Else, the knockdown level of p110δ would not affect the protein levels of TNFα and IL-6 since the treatment of BFA had already abrogated their transport from ER to Golgi complex. As a result, comparable protein levels of TNFα and IL-6 were detected between p110δ-knockdown group and control group after 8 h BFA and TiPs treatment, validating that p110δ was responsible for TNFα and IL-6 transport, and this point of view was further confirmed by IF staining and FCM assay.Intriguingly, we also found that p110δ overexpression could not promoted TNFα and IL-6 secretions in TiPs-stimulated macrophages. This reminded us that the trafficking was a multi factors-controlled process and the overexpressed p110δ might not be enough to facilitated the protein transport. On the other hand, whether the trafficking process was the bottleneck of inflammatory cytokines secretion was under discussion, and some argued that the accumulation of trafficking protein was temporary. Rather than transport, synthesis was the rate-limit step . And theAfter we found that KLF4 was the transcription repressor of p110δ, further role of KLF4 was confirmed, and our findings were aligned with the previous reports that KLF4 was an anti-inflammatory factor –47. UndeApproved by the ethics committee, synovial membranes of 15 patients, including 8 femoral head necrosis (FHN) patients  and 7 hip prosthetic aseptic loosening (AL) patients , from Department of Orthopaedic Surgery, Sun Yat-sen Memorial Hospital, Sun Yat-sen University were collected following the guidelines for human care. After 1 day fixation in 4% paraformaldehyde, specimens were decalcified in EDTA decalcifying solution . Sealed in paraffin, synovium was made into section for IHC assays. Specific information of subjects was mentioned in our previous study . All patRabbit antibody against phospho-SAPK/JNK , phospho-p44/42 MAPK , phospho-p38 MAPK , GAPDH , phospho-NF-κB p65 , NF-κB p65 , phospho-IKKα/β , IKKβ , phospho-IκBα , and IκBα  were purchased from Cell Signaling Technology . Rabbit antibody against PI3Kinase p110 delta  was obtained from Abcam . PE-Cyanine7-conjugated TNF alpha Monoclonal Antibody , PE-Cyanine7 conjugated iNOS monoclonal antibody , APC conjugated CD206 monoclonal antibody , mouse antibody against TGN38 monoclonal antibody  was purchased from ThermoFisher Scientific . Rabbit antibody against β-tubulin , KLF4 , P230 , TNF-α , IL-6, and IL-6  was purchased from ABclonal . Mouse antibody against Dyn2  was purchased from Santa Cruz Biotechnology . p110δ inhibitor , Dyn2 inhibitor , AKT inhibitor , and TAPI-1 (S7434) was purchased from Selleck . Rabbit antibody against IL-6  was purchased from Proteintech . LPS was purchase from Sigma-Aldrich .+ and KLF4+ respectively. Plasmids named Vector were taken as negative control. Recombinant lentiviruses were constructed to stably knockdown p110δ/KLF4 or to stably overexpress p110δ/KLF4, denoting as sh-p110δ/sh-KLF4 and OE-p110δ/OE-KLF4 respectively, with lentiviruses named sh-NC or OE-vector as negative control. To generated miR-92a-altered macrophages, mimics-miR-92a (Pre-92a) and relative negative-control microRNAs (Pre-NC), inhibitor-miR-92a (Anti-92a) and relative negative-control microRNAs (Anti-NC) were obtained from RiboBio. The lentivirus expressing firefly luciferase (fluc-lentivirus) from OBiO Technology  was applied for the construction of bioluminescent reporter cells to detect the bioluminescence signal.To generated gene-altered macrophages, siRNA, plasmids or recombinant lentiviruses were obtained from GenePharma . SiRNAs targeting p110δ (GeneID: 18707)/KLF4 (GeneID: 16600) were constructed and denoted as si-p110δ and si-KLF4 respectively. Si-NC was taken as negative control. P110δ/KLF4 overexpressed plasmids were constructed and denoted as p110δ−1 . Processed TiPs were then diluted with PBS into the concentration of 1.8 × 10−3g/mL for experiments.The procedure to generate sterilized TiPs was described in our previous study . Briefly2 circumstance, RAW264.7  were commonly cultivated in the growth medium-high glucose Dulbecco’s modified Eagle’s medium (DMEM)  containing 10% fetal bovine serum (FBS)  and 1% penicillin-streptomycin solution . Before stimulation, cells were seeded in plates for 24 h until 70–80% confluence.Under 37 °C and 5% CO7 transducing unit (TU) per mL lentiviruses contained in cultured medium was applied for 24–48 h transfection.As for transient transfection, Lipofectamine RNAiMAX Reagent  and plasmids were diluted and mixed as a system by Opti-MEM Medium  for 24–48 h transfection according to the specification. Similarly, miR-92a mimics or miR-92a inhibitor were transfected with the assistance of Lipofectamine 3000 . As for stable transfection, 1 × 103 for 24 h, TiPs with or without IC87114 were applied for stimulation for 16 h. Sequentially, Cell Counting Kit-8  was utilized according to the specification. After 37°C incubation for 2 h, cell viability was detected by gauging the absorbance at 450 nm.After seeded in 96-well plate in the concentration of 5 × 10TM Reagent  and was sequentially purified by chloroform, isopropyl alcohol, ethanol, 75% ethanol and RNase-Free Water. RNA concentration was measured by NanoDrop 2000 . Then, RNA solution was blended with PrimeScript RT Master Mix  and according to the specification, reverse transcription was conducted. After obtaining cDNA solution, UNICONTM qPCR SYBR Green Master Mix , specific primers and RNase-Free Water were applied for qualitive real-time PCR by Roche LightCycler 96 Real-Time PCR system . The primers of the target genes were listed in Table Total RNA was extracted by TRIzolLysate solution consisted of RIPA buffer , PMSF , and phosphatase inhibitor cocktail  was applied for protein extraction. After centrifugation, the protein concentration was gauged and normalized by Bicinchoninic acid (BCA) protein assay . Separated and transferred to the polyvinylidene fluoride (PVDF) membranes , protein was blocked with 5% Bovine Serum Albumin (BSA) . Overnight incubation of primary antibodies at 4°C and 1 h incubation of HRP-linked secondary antibody at room temperature were sequentially conducted. With the assistance of Super ECL Detection Reagent , immunoblotting results were obtained with digital imaging system (Syngen G:BOX Chmi XT4).To estimate serum levels of TNFα and IL-6, supernatant was collected and purified by centrifugation. Referring to specification, instant enzyme-linked immunosorbent assay (ELISA) was operated with specific kit .4 macrophages were cultivated in a confocal dish for 24 h . After stimulation, 4% paraformaldehyde were applied for fixation. For intracellular TNFα and IL-6 detection, 0.1% Triton X-100 was implemented for permeabilization. As for surface TNFα detection, 10 μM TAPI-1 was applied to inhibit TNFα secretion and cells were carried out IF staining without permeabilization. Blocked with 1% BSA for 30 min, cells were sequentially incubated with primary and secondary antibodies, followed by DAPI  for antifade fluorescence disposal. Confocal microscopy  was operated for imaging and further analysis was carried out by ZEN 2011 (blue edition) software (version 1.0).Before stimulation, 5 × 10Cells were harvested by 0.25% Trypsin-EDTA  and were washed with PBS. Then, fixation was performed with Fixation Medium (Medium A)  and Permeabilization Medium (Medium B)  was applied for permeabilization to detect intracellular TNFα, iNOS or CD206. Blocked with 1% BSA for 30 min, cells were incubated with PE-Cyanine7-conjugated TNFα antibody, or PE-Cyanine7-conjugated iNOS antibody, or APC conjugated CD206 antibody for 20 min. Further analysis was conducted with Flow Cytometry Instruments .+ plasmids together with plasmids loading wild-type Pik3cd promoter (Pik3cd-WT) or mutant-type (Pik3cd-MT) Pik3cd promoter were transfected. After 48 h, luciferase activity was detected with Firefly/Renilla Luciferase Assay Reagent by SYNERGY H1 (Bio-Tek). pRL-TK plasmids expressing Renilla luciferase were applied for collation. Similarly, Pre-NC/Pre-92a and plasmids containing wild type (KLF4 3′UTR-WT) or mutant type 3′‐UTR (KLF4 3′UTR-MT) of KLF4 were transfected into HEK-293T for exploring the relationship between miR-92a and KLF4.In order to confirm the relation between p110δ and KLF4, HEK-293T cells  were seeded in 24-well plates and after 24 h, cells reached a confluence of 70%-80%. According to the manufacturer’s specifications of Dual-Glo Luciferase Assay System, Vector/KLF43 gelatin sponges were employed as retainers for injection. Divided into six groups with six mice for each, mice were treated with different injections as followed: (1) 100 μl PBS was applied in Control group; (2)100 μl PBS with 3 mg TiPs was applied in TiPs group; (3) 100 μl PBS with 0.5 × 106 NC-stably knockdown RAW264.7 and 3 mg TiPs were applied in TiPs+sh-NC group; (4) 100 μl PBS with 0.5 × 106 p110δ stably-knockdown RAW264.7 and 3 mg TiPs were applied in TiPs+sh-p110δ group; (5) 100 μl PBS with 0.5 × 106 KLF4 stably-knockdown RAW264.7 and 3 mg TiPs were applied in TiPs+sh-KLF4 group; (6) 100 μl PBS with 0.5 × 106 KLF4 and p110δ stably-knockdown RAW264.7 and 3 mg TiPs were applied in TiPs+sh-KLF4&sh-p110δ group. Apart from that, a bioluminescence group was applied: 100 μl PBS with 0.5 × 106 bioluminescent reporter cells were injected and 7 days after surgery, bioluminescence signal were detected. A week after the procedure, mice calvarias were isolated for micro-CT imaging or IHC assay. No blinding was applied in the procedure and mice were randomly divided into six groups. All the animal procedures were performed under the guidelines for the care and use of laboratory animals of Sun Yat-sen University and were approved by the Animal Ethical and Welfare Committee of Sun Yat-sen University Cancer Center (L102042020120Q).In order to construct the mice cranial osteolysis model, male BALB/c nude mice aging 10 weeks  were acquired for vivo experiments. After anesthesia, a 10-mm midline sagittal incision was operated to uncover calvarias and 0.5 × 0.5 × 0.5 cm2O2 incubation was utilized to quench endogenous peroxidaseactivity. After incubated in 3% bovine serum albumin (BSA)  for 30 min, sections were next incubated in diluted primary antibody followed by incubation of HRP conjugated Goat Anti-Rabbit IgG H&L antibody . DAB Horseradish Peroxidase Color Development Kit  was applied for visualization. Nuclear staining was processed with hematoxylin and sections were eventually sealed off by neutral balsam . Biomicroscope  was applied for observation with Bresalier’s analysis for quantitative analysis. As for Bresalier’s analysis, 10 fields of each section were randomly chosen to estimate the staining score  and the scale of each staining level (Fx). Eventually, average intensity score (IS) was calculated as follows: ∑(0 × F0 + 1 × F1 + 2 × F2 + 3 × F3).Calvarias or human synovial membranes loaded by sections were embedded in paraffin. Heated at 60 °C for 2 h, the sections were sequentially treated by xylene, ethanol, 90% ethanol, 80% ethanol, 70% ethanol, 60% ethanol and PBS for deparaffinization and rehydration. Then pepsin  was applied to restore antigenicity for 20 min and 25 min 3% H3 region was set as region of interest (ROI) for gauging bone mineral density (BMD), bone volume (BV)/total volume (TV) and osteolysis area.After fixed in the formalin, calvarias were scanned using a high-resolution in vivo micro-CT imaging system  and further analysed by the affiliated software (ZZKS-Micro-CT4.1). The radiographic projection was performed under the condition of 60 kV and 667 μA within 240 ms. All the projection frames were recorded five times for average. After the reconstruction 3D images were obtained by the bundled manufacturer’s reconstruction software, a 1 × 3 × 3 mmp value < 0.05 was considered statistically significant compared with the negative group. *p < 0.05, **p < 0.01, ***p < 0.001. For synovium staining, seven patients with prosthetic aseptic loosening and eight patients with femoral head necrosis were included to ensure the reliability. For vivo study, six mice were included to ensure the reliability. For vitro experiments, assays repeated three time to ensure the reliability.Data were analysed by GraphPad Prism (version 8.0.2) and displayed in the form of mean ± SEM. All values were assessed by the Kolmogorov–Smirnov test to verify data normality. T-test was performed to compare the statistical difference between two groups while One-way ANOVA or two-way ANOVA was performed for more than two groups. The statistical data were compared and Supplementary materials- merged western blotsFig. 2C-GAPDH for p110δFig. 2C-p110δFig. 3C-IL-6Fig. 3C-TNFαFig. 3C-β tubulin for IL-6Fig. 3C-β tubulin for TNFαFig.3H-GAPDH for MAPK signaling pathwayFig.3H-p-ErkFig.3H-p-JNKFig.3I-p-IκBαFig.3H-p-p38Fig.3I-p-p65Fig.3I-β Tubulin for NF-κB signaling pathwayFig.4C-GAPDH for p110δFig.4C-p110δFig.5D-IκBαFig.5D-p-IκBαFig.5D-p-p65Fig.5D-p65Fig.5D-β Tubulin for NF-κB signaling pathwayFig.6D-GAPDH for KLF4Fig.6D-KLF4Fig.6H-GAPDH for p110δFig.6H-p110δFig.6I-IκBαFig.6I-p-IκBαFig.6I-p-p65Fig.6I-p65Fig.6I-β Tubulin for NF-κB signaling pathwaySupplemental material of figures and figures caption that are not shown in the manuscript


This article has been corrected: The authors found two mistakes in the legend to Figure 8:increases production of NFkB, TNF-α and IL-6 by suppressing miR-let-7g-5p expression”;in the title, “ET-1 suppresses production of NFkB, TNF-α and IL-6 by increasing miR-let-7g-5p expression,” the authors accidentally transposed the words "suppresses" and "increasing." The correct text should be “ET-1 8B, the word “melatonin” should be replaced with “ET-1."in the text of the legend for panel Figure 8 is presented below.The corrected legend to Figure 8. ET-1 increases production of NFkB, TNF‐α and IL‐6 by suppressing miR‐let‐7g-5p expression. (A) Open-source software  sought to identify miRNAs that could possibly interfere with NFkB, IL-6 and TNF- α transcription. (B) Cells were incubated with ET-1 (0-50 nM) for 24 h and miR‐let‐7g-5p expression was examined by qPCR. (C) Cells were pretreated with BQ123+BQ788, Ly294002, Akt inhibitor for 30 min, then stimulated with ET-1 for 24 h. miR‐let‐7g-5p expression was examined by qPCR.  Cells were transfected with the miR‐let‐7g-5p mimic and then treated with ET-1 (50 nM). TNF‐α and IL‐6 expression was evaluated by qPCR. The wild-type and mutant Ikbkb 3′-UTRs contained the miR-let‐7g-5p binding site. (F) Cells were transfected with the miR‐let‐7g-5p mimic and then treated with ET-1 (50 nM). (G) Cells were transfected with 3′-UTR plasmids as indicated then stimulated with ET-1 dose concentration. Then, cells were transfected with indicated luciferase plasmids for 24 h then stimulated with ET-1 for 24 h. Relative luciferase activity was measured. Results are expressed as the mean ± SEM. *P < 0.05 compared with controls; #P < 0.05 compared with the melatonin‐treated group.


H,6H-bispyrazine with CuI led to the formation of a two-dimensional copper(I) coordination polymer with CuI in a trigonal–pyramidal coordination environment defined by two S and two I atoms.The reaction of the pyrazinethiophane ligand 3,4,8,10,11,13-hexahydro-1 H,6H-bispyrazine (L) with CuI led to the formation of a two-dimensional coordination polymer, incorporating a [Cu2I2] motif. These units are linked via the four S atoms of the ligand to form the title two-dimensional coordination polymer, polypyrazine]di-μ-iodido-dicopper(I)], [Cu2I2(C12H16N2S4)]n, (I). The asymmetric unit is composed of a ligand molecule, two copper(I) atoms and two I− ions. Both copper(I) atoms are fourfold S2I2 coordinate with almost regular trigonal-pyramidal environments. In the crystal, the layers, lying parallel to (102), are linked by C—H⋯I hydrogen bonds, forming a supramolecular framework.The reaction of ligand 3,4,8,10,11,13-hexahydro-1 H,6H-bispyrazine (L), with silver(I) nitrate leads to the formation of a two-dimensional coordination polymer, with the silver(I) atom coordinated by three S atoms of the ligand and an O atom of the nitrate anion pyrazine]di-μ-iodido-dicopper(I)], incorporating a [Cu2I2] motif , and there are C—H⋯S and C—H⋯I intralayer hydrogen bonds present  atoms are considered to be fourfold S2I2 coordinate. The fourfold index parameter τ4 is 0.89 for atom Cu1 and 0.84 for atom Cu2  atoms.Selected bond lengths and bond angles involving the copper(I) atoms in pseudo-twofold rotation axis bisecting bonds C1—C2 and C6—C7 in one ring and bonds C3—C4 and C10—C11 in the second ring.In the complex, the ligand is step-shaped, as in the solid-state structure of the ligand itself . The Cu⋯Cu distances of the majority of these compounds vary from ca 2.580 to 3.087 Å (largest observed distance is 3.706 Å). For the majority of the compounds, the Cu—S bond lengths vary from 2.246 to 2.374 Å (largest observed distance is 2.531 Å), while the Cu—I bond lengths vary from 2.498 to 2.762 Å (largest observed bond length is 3.086 Å). It is evident from Table 2I fall within these limits.A search of the Cambridge Structural Database   Fig. 2 are link2) Fig. 2. There aH,6H-bispyrazine (L), have been reported  in CHCl3 (10 ml) was introduced into a 16 mm diameter glass tube and layered with MeCN (2 ml) as a buffer zone. Then a solution of CuI  in MeCN (5 ml) was added very gently to avoid possible mixing. The glass tube was sealed under an atmosphere of nitrogen and left in the dark at room temperature for at least 3 weeks, whereupon pale-yellow block-like crystals of complex I were isolated at the interface between the two solutions. Analysis for C12H16N2S4Cu2I2 (Mr = 697.46); calculated (%): C 20.66, H 2.32, N 4.02; found (%): C 20.90, H 2.31, N 3.93. The IR spectrum for I is shown in Fig. S1 of the supporting information.Rint = 0.0. No suitable ψ scans could be found so the crystal was equated to a sphere and the ABSSphere absorption correction was applied  I, Global. DOI: 10.1107/S2414314620004678/wm4128Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2414314620004678/wm4128sup3.tifScheme for CSD search. DOI: 10.1107/S2414314620004678/wm4128sup4.pdfCSD Search. DOI: Click here for additional data file.10.1107/S2414314620004678/wm4128sup5.tifIR spectrum for complex I. DOI: 1994655CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


By analyzing the expression signatures of this SSC-enriched population along with other germinal progenitors, we established an exhaustive transcriptome of human spermatogenesis. Transcriptome profiling of the human β-2M−SPα-6+THY1+ population and comparison with the profile of mouse undifferentiated spermatogonia provide insights into the molecular networks and key transcriptional regulators regulating human SSCs, including the basic-helix-loop-helix (bHLH) transcriptional repressor HES1, which we show to be implicated in maintenance of SSCs in vitro.Male infertility is responsible for approximately half of all cases of reproductive issues. Spermatogenesis originates in a small pool of spermatogonial stem cells (SSCs), which are of interest for therapy of infertility but remain not well defined in humans. Using multiparametric analysis of the side population (SP) phenotype and the α-6 integrin, THY1, and β-2 microglobulin cell markers, we identified a population of human primitive undifferentiated spermatogonia with the phenotype β-2 microglobulin (β-2M) •−SPα-6+THY1+ undifferentiated spermatogonia are enriched in stem cellsHuman β-2M•Comparative transcriptomics analysis of human and murine spermatogonia•in vitroHES1 is involved in the physiology of SSCs  −SPα-6+THY1+. They established transcriptome profiling of this primitive undifferentiated spermatogonium along with other germinal progenitors, providing an exhaustive transcriptome of human spermatogenesis.Givelet et al. identified a population of human primitive undifferentiated spermatogonia, highly enriched in stem cells, with the phenotype β-2M Throughout a male’s reproductive life, the pool of spermatogonial stem cells (SSCs) can self-renew or differentiate before undergoing meiosis and then spermiogenesis to produce sperm. SSCs constitute a potential source of cells that can be used to develop regenerative medicines for infertility, especially after cancer treatments during childhood. Testicular transplantation of SSCs has been found to lead to efficient production of functional sperm and restoration of fertility in several animal models , includisingle (As) spermatogonia in the adult testis (p) and aligned  spermatogonia, which are collectively called undifferentiated spermatogonia. However, this hierarchical model has been questioned because of recent evidence showing some equipotency in terms of regenerative potential among GFRA1+ undifferentiated spermatogonia, with stemness apparently not restricted to only As spermatogonia -based method that enabled us to purify a population of primitive undifferentiated spermatogonia, highly enriched in SSCs, and five premeiotic, meiotic, and postmeiotic populations of human germ cells. We established an exhaustive transcriptome of human spermatogenesis and examined the dynamics of gene expression throughout the developmental process. This study provides insights into the molecular networks potentially implicated in regulation of primitive spermatogonia and SSCs in humans.Bcrp1/Abcg2 , 3 (2N DNA content), and 4 (N DNA content) were assumed to correspond to spermatocyte I, spermatocyte II, and haploid (N) spermatids cells, respectively. To further this characterization, the SP population was divided based on α-6 integrin and THY1 expression. β-2M−α-6+THY-1+ cells and β-2M−α-6medTHY1− cells represented, respectively, 0.77% ± 0.2% and 0.95% ± 0.2% of the β-2M− cell population (n = 5). Using the opposite gating scheme, we confirmed that β-2M−α-6+THY1+ cells were found mainly in the SP population and represented 69.7% ± 2.7% (n = 11) of the SP cells  marker and FGFR3 and UTF1 undifferentiated spermatogonial markers were detected at very low levels, the β-2M+ population being composed of somatic cells. The lower expression level of α-6 integrin observed in the β-2M−SP α-6medTHY1− fraction, in addition to the lack of expression of markers of primitive spermatogonia and meiotic cells  suggested that subpopulations 2 (4N), 3 (2N), and 4 (N) corresponded to the spermatocyte I, spermatocyte II, and spermatid populations, respectively. These data show that the β-2M−SPα-6+THY1+ population contains primitive spermatogonia and lacks somatic cells.We and others have shown previously that, in mice, SSCs and spermatogonial progenitors express α-6 integrin and Thy-1 and harbor the side population (SP) phenotype according to an analysis of vital DNA dye efflux by the (ATP Binding Cassette) ABC transporter p1/Abcg2 . Assuminp1/Abcg2 A to charp1/Abcg2 A. ViableSP cells B–S1H. β-rker CD9 B and S1Ind GFRA1 C, we foupulation A. The viic cells B. On thepulation B and S2Bic cells C and S2ABCRP1/ABCG2, an ABC transporter, has been shown to play a major role in development of the SP phenotype in stem and progenitor cells during murine hematopoiesis and spermatogenesis and in human cancer stem-like cells  into busulfan-depleted testes of NOD−Scid/IL2Rγc−/− (NSG) humanized mice. As a control, a population including all germinal cells excluding SPα-6+ THY1+cells  was sorted and transplanted . The rate of colonization by human SSCs was estimated via immunofluorescence analysis using an antibody against human DNA nuclear protein in serial sections of recipient testes  are devoid of SSCs  and fibroblast growth factor 2 (FGF2) up to 2 weeks of culture . A principal-component analysis (PCA) of these genes revealed a clear trend for the transcriptomes to diverge along the germinal differentiation process . The contribution of sexual chromosomes to the transcriptional program decreased, respectively, from 15.4%  to 0.92% of the transcriptome  and 0.65%  to 0% of the transcriptome  for X and Y chromosomes  . We obseomosomes .−SPα-6+THY1+ transcriptome compared with β-2M−SPα-6medTHY1− at the transition of immature spermatogonia to differentiated spermatogonia: genes in Xq21, Xq22, Xp22, Xq23, Xq26, Yp11, and Yq11 chromosomal regions (UTY and USP9Y genes), AZFb , and AZFc, in which microdeletions are associated with fertility issues  were distributed in clusters 1, 2, and 3, corresponding to states 0 and 1 in SSC development  of the transcriptome of PLPPR3+ cells was found in the β-2M-SPα-6medTHY1− population  and in transcriptional regulation of gene expression /bone morphogenetic protein (BMP)/SMAD pathways  because they play crucial roles in differentiation. Among the 492 TRs that varied across the stages of spermatogenesis . The genes involved in the inflammatory transcriptional program were enriched in β-2M− SPα-6+THY1+ cells  transcriptional repressor HES1, which is involved in development of hematopoietic, neural, and intestinal stem cells , and SSC functionality was assessed 7 days later, as described previously  indicates that the combination of the 4 markers used here improved the definition of this stem cell subpopulation compared with previous strategies . Gene Ontology annotations highlighted cell migration and motility as top biological processes in human β-2Mntiation . TGF/BMPntiation . EMT is ntiation . Inflammntiation , has alsin vitro when Hes1 expression was reduced. Constitutive downregulation of Hes1 can affect stem cells and can lead to neuronal differentiation .Testicular single-cell suspensions were prepared from human biopsies. Vybrant staining (1 μg/mL) and immunolabeling of α-6 integrin, THY1, and β2M of the cell suspensions were performed as described previously . This prRNA samples were analyzed using an Affymetrix Human Gene 2.1 ST Array and an Affymetrix GeneChip Mouse Gene 2.0 ST Array (Thermo Fisher Scientific).∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.All values are shown as mean ± SEM. Statistical analyses were performed by Student’s t test (GraphPad Prism software): ns, not significant, p > 0.05; GSE155509.The accession number for the microarray data reported in this paper is GEO: M.G., V.F., B.L., V.B.-L., and P.F. provided project design. M.G., V.F., B.L., and P.F. performed experiments and analyzed and interpreted data. A.S.G., C.L., I.H., A.J., F.H., I.A., and L.R. participated in experimentation and provided comments. F.D., S.J., and F.L. participated in microarray and bioinformatics analyses. F.P. provided NSG mice. N.T., C.P., J.P.W., and V.B.-L. supervised sample acquisition. M.G., V.F., and P.F. prepared the manuscript.The authors declare no competing interests.


The remarkable rise of the current COVID-19 pandemic to every part of the globe has raised key concerns for the current public healthcare system. The spike (S) protein of SARS-CoV-2 shows an important part in the cell membrane fusion and receptor recognition. It is a key target for vaccine production. Several researchers studied the nature of this protein under various environmental conditions. In this work, we applied molecular modeling and extensive molecular dynamics simulation approaches at 0°C (273.15 K), 20°C (293.15 K), 40°C (313.15 K), and 60°C (333.15 K) to study the detailed conformational alterations in the SARS-CoV-2 S protein. Our aim is to understand the influence of temperatures on the structure, function, and dynamics of the S protein of SARS-CoV-2. The structural deviations, and atomic and residual fluctuations were least at low (0°C) and high (60°C) temperature. Even the internal residues of the SARS-CoV-2 S protein are not accessible to solvent at high temperature. Furthermore, there was no unfolding of SARS-CoV-2 spike S reported at higher temperature. The most stable conformations of the SARS-CoV-2 S protein were reported at 20°C, but the free energy minimum region of the SARS-CoV-2 S protein was sharper at 40°C than other temperatures. Our findings revealed that higher temperatures have little or no influence on the stability and folding of the SARS-CoV-2 S protein. The outbreaks of Severe Acute Respiratory Syndrome CoV 1 (SARS-CoV-1), Middle-East Respiratory Syndrome CoV (MERS-CoV), and Severe Acute Respiratory Syndrome CoV 2 (SARS-CoV-2) were caused by zoonotic viruses in 2003, 2012, and 2019–2020 with a fatality ratio of 10%, 35%, and 5%, respectively . The IntThe Spike (S) protein of SARS-CoV-2 performs a vital part in the cell membrane fusion and receptor recognition. It has two subunits such as S1 and S2. A receptor-binding domain (RBD) is present on the S1 subunit. The RBD recognizes and attaches to the host receptor angiotensin-converting enzyme 2 (ACE-2). The membrane fusion (MF) is facilitated by the S2 subunit by making 6 helical bundles through two heptad repeat (HR) domains . The S pThe S proteins are covered with polysaccharide for camouflage and escaping the host immune system in the course of entry . The S pThe SARS-CoV-2 S protein is an important target for vaccine production. In this work, we applied molecular modeling and molecular dynamics simulations approaches at 0°C (273.15 K), 20°C (293.15 K), 40°C (313.15 K), and 60°C (333.15 K) to study the detailed conformational variations in the SARS-CoV-2 S protein. It is worth noting that, experimentally, in terms of information on inactivation of viruses, data in the range 40–60°C are essential . Our finThe structures of S protein of SARS-CoV-2 (PDB: 6vsb) were taken from PDB . The misvia GROMACS 2018.2 , 20°C (293.15 K), 40°C (313.15 K), and 60°C (333.15 K). The complete MD simulation procedure is cited in prior publications (MD simulations were achieved on SARS-CoV-2 spike protein at 0°C (273.15 K), 20°C (293.15 K), 40°C (313.15 K), and 60°C (333.15 K) S 2018.2  using a S 2018.2 . Na+ andications .ED was obtained for the SARS-CoV-2 S protein at 0°C (273.15 K), 20°C (293.15 K), 40°C (313.15 K), and 60°C (333.15 K). It is estimated as:ri denotes the coordinate, ith Cα atom, N signifies the Cα atoms, and <ri > indicates time average over all configurations , 20°C (293.15 K), 40°C (313.15 K), and 60°C (333.15 K) . The GFEkB, T, and PPC1, PC2), 20°C (293.15 K), 40°C (313.15 K), and 60°C (333.15 K) respectively.The SARS-CoV-2 spike protein comprises N-terminal, TM, and C-terminal segments . It consRg were considered throughout 100-ns MD simulations at 0°C (273.15 K), 20°C (293.15 K), 40°C (313.15 K), and 60°C (333.15 K), respectively (Rg) values for the SARS-CoV-2 S protein at 0, 20, 40, and 60°C was found to be 4.09, 4.37, 4.32, and 3.48 nm, respectively. The Rg is described as the allotment of atoms of a molecule around its axis. The calculation of Rg is a significant indicator that is broadly used in calculating the structural activity. At different temperatures, there is a conformational change in the SARS-CoV-2 S protein that changes the radius of gyration. It was estimated that the SARS-CoV-2 S protein is tightly packed at 60°C. At 20°C–40°C, it shows high fluctuations throughout the time scale.To investigate the structural dynamics of the SARS-CoV-2 S protein, the RMSD, RMSF, and the ectively . The meaectively . It has 2, respectively  of the SARS-CoV-2 S protein at 0, 20, 40, and 60°C, respectively. The mean H-bonds between M-S chains of the SARS-CoV-2 S protein were found to be 413.43, 407.89, 417.99, and 417.51 at 0, 20, 40, and 60°C, respectively . The str2 for the SARS-CoV-2 S protein at 0, 20, 40, and 60°C, respectively. It was higher at 20–40°C (It shows global expansion of the SARS-CoV-2 S protein at 0, 20, 40, and 60°C. It estimates mean atomic motions of the SARS-CoV-2 S protein at 0, 20, 40, and 60°C. The eigenvalues were 8,360.61, 33,665.60, 53,083.50, and 8,911.24 nm 20–40°C . The aveThe GFE landscape exhibited diverse forms for the SARS-CoV-2 S protein at 0, 20, 40, and 60°C . Every aRecently, several inhibitors and their mode of action have been demonstrated . EdwardsPreviously, we published several articles based on finding potential inhibitors of SARS-CoV-2. In the present work, we focused on the SARS-CoV-2 S protein as it performs a vital part in the cell membrane fusion and receptor recognition. Researchers have demonstrated the nature of the S protein of SARS-CoV-2 on diverse environmental conditions. We applied molecular modeling and extensive molecular dynamics simulations approaches at different temperatures to investigate the structural conformational of SARS-CoV-2 spike protein. There are several hypotheses proposed regarding the temperature dependence of the COVID-19 transmission. We concluded that temperature has no effect or has the least effect on the structure conformations of S protein of SARS-CoV-2. Minor changes were reported in the structure and thermodynamic properties that are mentioned in this paper.


P21. The crystal packing is characterized by C—H⋯N and C—H⋯O contacts.The title compound crystallizes in the monoclinic space group  15H14ClN5O2, the terminal phenyl ring and the adjacent furan ring subtend a dihedral angle of 6.77 (17)°. The 4-chloro-5-(dimethylamino)-pyridazin-3(2H)-one group is linked to the oxadiazole ring by a methylene bridge, and the dihedral angle between the pyridazine and oxadiazole rings is 88.66 (14)°. In the crystal, C—H⋯O and C—H⋯N hydrogen bonds extend the structure into a three-dimensional network.In the structure of the title compound, C Afterwards, the reaction mixture was cooled and poured into 60 ml of ice–water. The precipitate formed was collected by filtration and then dried to obtain the pure title compound . It was recrystallized from mixed solvents of ethyl acetate and petroleum (3:5) to give crystals suitable for X-ray diffraction (m.p. 427–429 K).To a 100 ml round-bottom flask, 4,5-dichloro-2-[ methyl]pyridazin-3: 3.18 , 5.62 , 7.54 , 7.67 , 8.06 . IR : 2973, 2937, 1632, 1600, 1520, 1482, 1449, 1216, 1146, 752, 710.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S241431462200342X/vm4052sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S241431462200342X/vm4052Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S241431462200342X/vm4052Isup3.cmlSupporting information file. DOI: 2162169CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Scientific Reports 10.1038/s41598-022-23575-1, published online 29 November 2022Correction to: In the original version of the Article, Figure 2 [(emu/g)2] × 103’ was incorrectly given as ‘Magnetization [(emu/g)2] × 103’.In addition, in Figure 1, panel (c), the label of the Y-axis ‘MagnetizationThe original Article has been corrected.