Granulosa cell proliferation and differentiation are essential for follicle development. Breast cancer amplified sequence 2 (BCAS2) is necessary for spermatogenesis, oocyte development, and maintaining the genome integrity of early embryos in mice. However, the function of BCAS2 in granulosa cells is still unknown.Bcas2 in granulosa cells caused follicle development failure; the ratio of the positive cells of the cell proliferation markers PCNA and Ki67 were unchanged in granulosa cells. Specific deletion of Bcas2 caused a decrease in the BrdU-positive cell ratio, cell cycle arrest, DNA damage, and an increase in apoptosis in granulosa cells, and RPA1 was abnormally stained in granulosa cells. RNA-seq results revealed that knockout of Bcas2 results in unusual expression of cellular senescence genes. BCAS2 participated in the PRP19 complex to mediate alternative splicing (AS) of E2f3 and Flt3l mRNA to inhibit the cell cycle. Knockout of Bcas2 resulted in a significant decrease in the ratio of BrdU-positive cells in the human granulosa-like tumour (KGN) cell line.We show that conditional disruption of E2f3 and Flt3l by forming the splicing complex with CDC5L and PRP19.Our results suggest that BCAS2 may influence the proliferation and survival of granulosa cells through regulating pre-mRNA splicing of The online version contains supplementary material available at 10.1186/s13048-023-01187-1. The ovary is the female reproductive organ, consisting of oocytes and somatic cells  . PrimaryAbnormal apoptosis and degeneration of granulosa cells lead to follicular atresia , 9. SincTnp2 pre-mRNA splicing and arrested spermatogenesis [Previous studies have reported that AS plays an essential role in reproduction. In granulosa cells, the androgen receptor insertion and deletion isoforms of AS variants disrupted follicle growth and androgen metabolism, resulting in polycystic ovary syndrome (PCOS), which is a common disease that results in infertility . In maleogenesis . In oocyogenesis .Bcas2 in male germ cells affected mouse spermatogenesis and male fertility by regulating AS, which decreases the full-length formation of deleted azoospermia-like (Dazl) [Vasa-Cre mediated deletion of Bcas2 caused poor oocyte quality, abnormal oogenesis, and follicular development, possibly by regulating the AS of functional genes through the PRP19 complex [Bcas2 by Zp3-Cre mice destroyed the genomic integrity of early embryos and resulted in female infertility [BCAS2 was first found to be a highly expressed gene in human breast cancers  and invoe (Dazl) . Vasa-Cr complex . Maternaertility . HoweverFoxl2-CreERT2 mouse model to knock out Bcas2 in granulosa cells [E2f3 and Flt3l mRNA splicing through the PRP19 complex, and knocking down BCAS2 in the KGN cell line significantly reduced proliferation. Thus, BCAS2 in granulosa cells is essential for female fertility.In this study, we used a sa cells . The resBcas2F/F; Foxl2-CreERT2 lines were initially derived on a mixed background (129/C57BL/6) but maintained on C57BL/6 mice. Pups were injected with 20 mg/kg tamoxifen  at 1 day postpartum (dpp), 3 dpp, and 5 dpp to activate Cre recombinase. The cultivation, breeding, and experimental operation of the mice followed the guidelines of animal research at China Agricultural University.All mice were housed in the animal facilities of China Agricultural University with free access to food and clear water. The temperature was between 22 and 26 °C, and the light conditions were 12 h (h) in light and 12 h in darkness. The DNA was extracted from the mouse tail by the HotSHOT method, and PCR was used to distinguish different genotypes with specific primers. Taq Master Mix (2×) (Dye Plus)  was used for PCR. The common PCR conditions were as follows: 95 °C for 5 min; 36 cycles of 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 40 s; 72 °C for 5 min; and holding at 4 °C. PCR products were seperated with 2% agarose. The size of the PCR product bands was used to distinguish wild-type (WT) from floxed (Flox). The WT size was 402 bp, and the Flox size was 522 bp. PCR primer sequences are shown in Supplementary Table Ovaries were collected on different days, fixed in 4% paraformaldehyde  overnight at 4 °C. Then, the ovaries were embedded in paraffin blocks, sectioned at a thickness of 5 μm, and stained with haematoxylin and eosin (H&E). The follicles were counted and classified in every fifth section at 7 dpp, 14 dpp, and 21 dpp as follows: one layer of squamous granulosa cells we called primordial follicles, one layer of cubic granulosa cells we called primary follicles, two or more layers of granulosa cells we called secondary follicles, follicles with a large cavity multilayer and multiple layers of granulosa cells we called antral follicles, granulosa cell abnormalities and apoptosis we called atretic follicle .Sections of the ovary were dewaxed and microwaved with 0.01 M sodium citrate buffer for antigen retrieval (pH = 6.0). Nonspecific antigen was blocked with 10% normal goat serum  at room temperature (RT) for 1 h. The primary antibodies Table S were incCell immunofluorescence: The cells were fixed with precooled methanol for 20 min, washed with PBS 3 times, permeated with 0.2% Triton X-100 for 15 min, and blocked with 10% normal goat serum. Subsequent steps for immunofluorescence were performed as described above.Mice were injected with 100 mg/kg 5-bromo-2’-deoxyuridine (BrdU)  at 7 dpp, 10 dpp, or 14 dpp. After 2 h, ovarian tissue was collected, fixed, embedded, sliced, microwaved with EDTA antigen retrieval solution (pH = 9.0), and washed with PBS. BrdU antibody was added to the tissue overnight at 4 °C, and then, the tissue was washed with PBS. Secondary antibody was added at RT for 2 h, the sample was washed with PBS, and DAPI was incubated at RT for 10 min. Then, the sample was washed with PBS. Mounting and imaging were performed as above.Apoptosis of the ovarian tissue was detected by One Step TUNEL Apoptosis Assay Kit  according to a standard protocol. We chose the largest section of the ovary and counted total granulosa cell number and apoptotic cell number in the section.2 incubator. pMKO.1 GFP was a lentivirus expression vector and as a negative control. The sequences were as follows: sh-163, AAGAACTACCTGAGCTACCTG; sh-316, AATGACATTACTGCATGGCAA; sh-391, AATCTGGAACTAATGTCACAG. A total of 2.5 µg of shRNA was transfected into the KGN cell line using Lipo8000™  according to the manufacturer’s instructions. After 72 h of cell culture, 20 ng/mL BrdU  was added for 2 h.The KGN cell line  was cultured in DMEM  containing 10% fetal bovine serum  and 1% penicillin and streptomycin at 37 ℃ in a 5% COThe ovary was added to 50 µl of RIPA buffer  and mashed with a grinding rod. Then, the samples were frozen and thawed five times with liquid nitrogen and centrifuged at 12,000 rpm at 4 °C for 30 min (min). After that, the supernatant was aspirated, and the protein concentration was measured with a BCA Protein Assay Kit . Total proteins were separated by 12% sodium dodecyl sulfate-polyacrylamide and transferred to polyvinylidene difluoride membranes  by electricity . The membrane was blocked with 5% skim milk  at RT for 1 h. First, the membrane was incubated with BCAS2 antibody and GAPDH antibody overnight at 4 °C and then washed with 0.05 M Tris-buffered saline and 0.1% Tween-20  three times, incubated with secondary antibodies  and then centrifuged until the supernatant was retained. Rabbit IgG and BCAS2 antibodies were adsorbed by BeyoMag™ Protein A , and then, cell lysate was adsorbed by beads overnight at 4 °C. The beads were washed three times with TBST and boiled in 1× SDS loading buffer, and Western blot was performed as described above. Immunoblotting was performed with specific antibodies against BCAS2, PRP19, and CDC5L.The total RNA of the ovary was extracted by TRIzol Reagent , and the RNA concentration was measured by a NanoDrop . One microgram of mRNA was transcribed into cDNA using M-MLV . Quantitative PCR (qPCR) analysis was performed with Hieff UNICON® qPCR SYBR Green Master Mix  by an LightCycler® 96 . GAPDH was used as an internal reference. qPCR primer sequences are shown in Supplementary Table E2f3 and Flt3l were designed for RT‒PCR. Information on all primers is listed in Supplementary Table cDNA was used for common PCR with 2 × Taq Master Mix (Dye Plus). Primers used to determine mature mRNAs of Ovarian samples were collected at 8 dpp and sent to Novogene for RNA-seq analysis. Total RNA was isolated. The concentration was assessed with a Nanodrop  and an Agilent 2100 . The quality was determined with an Agilent 2100, and purity was determined with an Agilent 2100 and agarose gel electrophoresis. The cDNA library was generated by mRNA using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina®. The RNA-seq library was sequenced with an Illumina following sequencing. After quality control of the date, adapter reads, unknown reads, and low-quality reads were removed.The clean reads were used for analysis. rMATS software (version 3.2.5) was used for quantitative and differential analysis of AS events. The threshold for AS events with significant screening differences was a false discovery rate (FDR) of less than 0.05, while inclusion level differences greater than 5% (0.05) were considered statistically significant. Integrated Genomics Viewer  for visualization and confirmation of AS events based on RNA-seq data. Alternative 3’ splice site (A3SS) ,the 3’ end splice site is consistent but the 5’ end splice site is different, and the 5’ end exon of the second transcript is prolonged. Alternative 5’ splice site (A5SS), the 5’ end splice site is the same but the 3’ end splice site is different, and the 3’ end exon of the second transcript is prolonged. Skipped exons (SE) is when an exon is cut from the original transcript. Retained intron (RI), the second transcript is retained by retained intron to form new exons with the exons on both sides. Mutually exclusive exon (MXE), variable splicing of genes results in two different transcripts, constitutive exon which is identical and inclusive exon which is different.P < 0.05 was considered statistically significant. * P < 0.05, ** P < 0. 01, *** P < 0.001, **** P < 0.0001.We used clusterProfile software for Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the differentially expressed genes. padj < 0.05 indicated significant enrichment. Mouse genome data (GRCm39) were used as the reference. We used GraphPad Prism software (version 7.0) to analyse the statistics. Student’s t test was used for this study. Data are expressed as the means ± SEMs in at least three independent experiments. Bcas2 did not have a significant change at the mRNA level at 1 dpp, 7 dpp, 14 dpp, or 21 dpp  at these stages [Bcas2F/F called PfGC-Bcas2+/+, Bcas2F/F; Foxl2-CreERT2 called PfGC-Bcas2−/−. PCR was used to detect genotypes  cell ratio or Ki67- immunoreactive (Ki67+) cell ratio  cell rate significantly decreased at 7 dpp, 10 dpp, and 14 dpp in the PfGC-Bcas2−/− granulosa cells (Fig. The absence of secondary follicles in the PfGCdpp Fig. A, C. Celtio Fig. B, D, sugBcas2+/+ mouse ovaries. Whereas the γ-H2AX-positive signal appeared in granulosa cells in PfGC-Bcas2−/− at 10 dpp, γ-H2AX-positive cells increased significantly at 14 dpp (Fig. -Bcas2−/− and PfGC-Bcas2+/+ ovaries. Intriguingly, we found that the RPA1 foci in granulosa cells were significantly increased with the deletion of Bcas2 (Fig. SBcas2+/+ granulosa cells at 7 dpp, 10 dpp, and 14 dpp. However, cell apoptosis increased in granulosa cells from 10 dpp to 14 dpp in PfGC-Bcas2−/− ovaries (Fig. To investigate whether DNA damage occurs in granulosa cells, we detected the DNA damage marker γ-H2AX using immunofluorescence. The results showed that no positive cells were observed in granulosa cells of the PfGC-dpp Fig. A, B. Givies Fig. C, D. TheBcas2−/− ovaries (Fig. Bcas2 deletion (Fig. To further explore the molecular consequences of BCAS2 depletion in granulosa cells, we performed a transcriptome analysis of ovaries at 8 dpp. RNA-seq results showed that 504 genes were differentially expressed, with 253 upregulated and 251 downregulated in the PfGC-ies Fig. A, B. Kyoion Fig. C. The acion Fig. D.Bcas2−/− ovaries, 30 splicing events were categorized as A3SS, 29 splicing events were categorized as A5SS, 79 splicing events were categorized as SE, 18 splicing events were categorized as RI, 7 splicing events were categorized as MXE (Fig. E2f3 and Flt3l genes were analyzed using IGV visualization software. The results showed that E2f3 has an A3SS AS form, and Flt3l has an SE AS form (Fig. E2f3 and Flt3l pre-mRNA in ovary (Fig. Bcas2−/− ovaries compared to that in the PfGC-Bcas2+/+ ovaries (Fig. E2f3 and Flt3l in the 8 dpp ovary (Fig. SWe analysed the AS data and found that 163 genes had undergone abnormal AS in the PfGC-Bcas2−/− ovaries decreased significantly (Fig. SBcas2−/− ovaries (Fig. SAccording to previous reports, BCAS2 is involved in mRNA splicing through the PRP19 complex , 26. To ly Fig. SC. Immunoes Fig. SD. The reBCAS2 in the KGN cell line using sh-RNA interference, and the sh-163 knockdown efficiency was the best (Fig. S+ cells of sh-163 group was significantly decreased (Fig. SBcas2 knockout mice, which provides us with a new way to explore related human diseases.To investigate the function of BCAS2 in human granulosa cells, we first detected the expression of BCAS2 in the KGN cell line, and immunofluorescence results showed that BCAS2 was expressed in the nucleus of the KGN cell line Fig. SA. We sucst Fig. SB. Prolifed Fig. SC, indicaBcas2 in granulosa cells from primary follicles by Foxl2-CreERT2 mice. The ovaries of PfGC-Bcas2−/− mice exhibited a lack of secondary follicles at 14 dpp and 21 dpp, whereas the follicles of PfGC-Bcas2+/+ ovaries developed to the antral follicle stage. Additionally, the numbers of secondary and antral follicles were significantly reduced in the ovaries of the PfGC-Bcas2−/−mice. Our study suggested that BCAS2-defficient granulosa cells undergo proliferation arrest and fail to differentiate into functional cells which leads to the phenotype of POI oocyte retardation. Therefore, the lack of BCAS2 in granulosa cells results in arrested follicle development.BCAS2 is a pre-mRNA processing factor and has been reported as a component of the spliceosome , 17, 18,+ and Ki67+ were not significantly changed at 10 dpp, by which we cannot judge the proliferative arrest state of granulosa cells after Bcas2 deletion. Hence, by incorporating BrdU, we demonstrated that the loss of BCAS2 in granulosa cells significantly reduced the number of S-phase cells in granulosa cells, suggesting that BCAS2 plays an important role in regulating the G1/S transition of the cell cycle. These results are similar to previous results showing that BCAS2 is involved in controlling Drosophila cell growth [PCNA and Ki67 are markers of cell proliferation that are expressed in the G1, S, G2, and M phases. Here, we show that PCNAl growth  and regul growth , 32.Bcas2−/− mouse ovaries. This finding indicates that DNA damage cannot be repaired in BCAS2-null granulosa cells. Moreover, RPA1 is abnormally stained in PfGC-Bcas2−/− granulosa cells. Furthermore, the TUNEL signal increased from 10 dpp to 14 dpp in granulosa cells.Cell cycle arrest is constantly accompanied by DNA damage and apoptosis . DNA damE2f3 and Flt3l. E2F3 is a family of transcription factors that regulate cellular proliferation and differentiation [Bcas2−/− mouse ovaries, which may contribute to cell cycle arrest, abnormal DNA repair, and apoptosis in granulosa cells. The probable reason why E2F3 does not change at the protein level is that there is variable splicing of E2F3 in BCAS2-null granulosa cells, and it is not significant enough to cause protein level changes. Thus, BCAS2-null in granulosa cells causes disordered E2f3 and Flt3l mRNA splicing and leads to cell cycle arrest.RNA-seq results showed that deficiency of BCAS2 in granulosa cells leads to abnormal pre-mRNA splicing of some genes associated with the cell cycle, such as ntiation –39. FLT3ntiation . Our datE2f3 and Flt3l mRNA, indicating that BCAS2 regulates AS of E2f3 and Flt3l pre-mRNA through an indirect effect. Given that BCAS2 is an important component of the spliceosome, CDC5L, PRP19 and PLRG1 were examined. PRP19 is a multifunctional protein complex that mediates pre-mRNA splicing and DNA damage responses [E2f3 and Flt3l by forming the splicing complex with CDC5L and PRP19, thus controlling the proliferation and DNA damage of granulosa cells (Fig. RIP results showed that BCAS2 antibody could not enrich esponses , 42. Theesponses . Co-IP rlls Fig. .E2f3 and Flt3l as a component of the spliceosome. These findings provide new ideas for exploring granulosa cell abnormalities and potential therapeutic targets in patients with POI.In summary, our collective findings revealed that BCAS2 is essential for the proliferation and survival of granulosa cells that may be involved in regulating pre-mRNA splicing of Additional file 1. Additional file 2.


A systematic analysis was conducted to explore the optimum raw material addition ratio and optimum sintering temperature; then, the intrinsic structure of thermal insulation bricks and their sintering formation mechanisms were revealed. The results showed that the optimal ratios of coal gangue, K-feldspar, CaCO3 and fly ash were 65 wt%, 15 wt%, 10 wt% and 10 wt%, respectively; the compressive strength of the thermal insulation brick produced under this ratio was 22.5 MPa; thermal conductivity was 0.39 W m−1 k−1. During sintering processes, mineral powders sufficiently fused to form a skeleton, and the CO2 derived from CaCO3 formed pores. The optimum sintering temperature was 1150 °C, because at this temperature, K-feldspar had the best effect in promoting the conversion of CaCO3 to Ca-feldspar. The high level of the relative crystallinity of Ca-feldspar (about 76.0%) helped raise the Si–O network’s polymerization degree (NBO/T = 1.24), finally raising the compressive strength of thermal insulation bricks. The innovative method of using coal gangue to make thermal insulation bricks not only solved the environmental pollution caused by coal gangue but also provided excellent construction materials with high practical application value.Coal gangue is a solid waste with low carbon content discharged during the course of the coal mining process. The resource utilization of coal gangue could solve environmental problems caused by its excessive production, such as soil contamination and land occupation. This study proposed to produce high-strength thermal insulation bricks using coal gangue as the primary material and three other mineral powders as auxiliary materials, including K-feldspar, CaCO Coal gangue is an industrial waste from coal mining . In Chin3 is an expansion agent. Under high-temperature conditions, CaCO3 is decomposed and produces CO2 gas, which promotes the development of pores inside the bricks. Martínez-Martinez 2−, chain-structured [Si2O6]4−, dimer-like [Si2O7]6− and monomeric [SiO4]4− [n (n represented the amount of bridging oxygen of [SiO4]) [0, Q1, Q2 and Q3 represented the structural units of SiO44−, Si2O76−, Si2O64− and Si2O52−, respectively, and their corresponding wave numbers were 840~890 cm−1, 900~950 cm−1, 960~1030 cm−1, 1050~1100 cm−1 and 1160~1190 cm−1, respectively [0 + 3 × Q1 + 2 × Q2 + 1 × Q3  . Q0, Q1,ectively . The yelectively , and the+ 1 × Q3 . NBO/T iXRD results showed that with an increase in sintering temperatures, the relative crystallinity of Ca-feldspar increased and reached a maximum of 78.4% at 1200 °C b, indica−1 to 0.04 mL g−1 (As sintering temperatures increase from 1000 °C to 1200 °C, the porosity of thermal insulation bricks gradually decreased from 19.8% to 6.6% a, and th4 mL g−1 b. High s4 mL g−1 a due to 4 mL g−1 a because4 mL g−1 a,b, whic4 mL g−1 c,d, the 4 mL g−1 e,f.3 and fly ash. The results showed that the optimum addition ratios of coal gangue, K-feldspar, CaCO3 and fly ash were 65%, 15%, 10% and 10%, respectively, and the optimum sintering temperature was 1150 °C. Thermal insulation bricks produced under this condition had a compressive strength of 22.5 MPa and thermal conductivity of 0.39 W m−1 k−1. During the sintering process, CaCO3 was decomposed into CO2 and CaO; then, CO2 promoted the formation of a porous structure and reduced the thermal conductivity. Meanwhile, K-feldspar facilitated the conversion of CaO into Ca-feldspar, increasing its mechanical strength. This study proposed an innovative method for the resource utilization of coal gangue, i.e., the production of high-strength thermal insulation bricks with the assistance of K-feldspar, CaCOIn previous studies, gangue has generally been backfilled into the ground or ground into a powder and mixed with cement as an active agent. However, the former method still has the risk of contaminating soil and groundwater, and the latter method cannot utilize a large amount of gangue because the mixing ratio of gangue in cement is too low. In summary, the use of coal gangue to produce high-strength insulation bricks not only solved the problem of coal gangue’s occupation of land and pollution of the environment but also produced excellent construction materials with high practical application value.


We delved into the design and optimization of the spinning slurry and examined how the Si3N4/BN fiber diameter affects the ceramics’ microstructure and mechanical properties. The spinning slurry exhibited exceptional stability and spinnability. Decreasing the fiber diameter contributed to material densification and improved mechanical properties. Notably, when the fiber diameter is 0.9 mm, the fabricated Si3N4/BN fibrous monolithic ceramics demonstrate a carbon content of 0.82%, a three-point bending strength of 357 ± 24 MPa, and a fracture toughness of 8.8 ± 0.36 MPa·m1/2. This investigation offers valuable insights into producing high-performance Si3N4/BN composite ceramics utilizing hot-pressing technology.Silicon nitride ceramics are regarded as a promising material for high-temperature structural applications due to their remarkable characteristics, including high strength, hardness, thermal conductivity, low dielectric properties, and resistance to creep at elevated temperatures. However, their susceptibility to catastrophic fracture at high temperatures remains a concern. Herein, Si Silicon nitride  and an interfacial phase (BN unit interfaces), and are known for their strength, wave transparency, chemical stability, and temperature resilience [3N4/BN fibrous monolithic ceramics.Sisilience ,15,16,17β-Si3N4, effectively controlling the size and distribution of fibrous large grains. Consequently, the fracture toughness of silicon nitride ceramics prepared using this method is significantly improved, reaching 11.1 MPa·m1/2. Previous studies primarily focused on incorporating β-Si3N4 rod-like seeds [3N4 fibers. However, the debinding process in these studies was conducted in an inert atmosphere, which, while enhancing the mechanical properties of Si3N4/BN fibrous monolithic ceramics, adversely affects their transparency to waves.Kiyoshi  proposedke seeds ,10 or Sike seeds ,15,16 in3N4/BN fibrous monolithic ceramics. We produced Si3N4/BN fibrous monolithic ceramics utilizing the wet-spinning–hot-pressing sintering technique, investigating their microstructure and mechanical properties. Our focus was on stabilizing the wet-spinning slurry by adjusting the solid content and sodium alginate content, without adding β-Si3N4 rod-shaped seeds or SiC whiskers. The debinding process was conducted in an ambient air environment to minimize residual carbon. We also assessed how fiber diameter (ranging from 0.42 to 1.05 mm) influences the ceramics’ microstructure and mechanical properties. These findings offer a valuable strategy for designing and optimizing the structure of fibrous monolithic ceramics to enhance their overall performance.This research aims to delve into the relationship between the wet-spinning–hot-pressing sintering process and the microstructure and mechanical properties of SiTo promote fiber formation and avert crack formation during drying, sodium alginate  was incorporated as a binder in the spinning solution. The sodium alginate binder was dissolved in deionized water, and the initial powder was combined with the sodium alginate aqueous solution through 12 h of planetary ball milling, ensuring adequate particle penetration and obtaining the spinning solution. The proportions of the initial powders are presented in The rheological behavior of the spinning precursor solution at ambient temperature was characterized using an MCR301 rotational rheometer , with a plate–plate geometry of 8 mm diameter and a gap of 2 mm. A constant frequency of 1 Hz was applied during stress scans, and oscillatory shear experiments were performed in the range of 1 to 10 with a 5 Pa amplitude. The apparent viscosity of the spinning precursor solution was recorded during strain scans from 0.1 to 10 L/s. The thermal stability of the spinning precursor solution was investigated in air, from room temperature to 1000 °C, using thermogravimetric analysis  with a heating rate of 15 °C/min.3N4/BN fibrous monolithic ceramics is depicted in 2 hydrogel bath, where the pressure of the injection pump induces rapid solidification of the Si3N4 slurry into ceramic fibers. The solidified fibers are then coated with the BN interfacial slurry by being drawn into the BN slurry pool through roller rotation. After drying at room temperature, the fibers are cut into short segments of a specific size and arranged parallel in a chromium steel mold. The assembly is pre-pressed at 20 MPa for 5 min and then heated at a rate of 1 °C/min to 600 °C in a muffle furnace to remove sodium alginate. Pure Si3N4/BN fibrous monolithic ceramics are obtained through hot pressing. This is performed in a graphite mold containing a powder bed of 50 wt.% BN and 50 wt.% Si3N4. The samples are heated to 1800 °C at a rate of 15 °C/min and held at 28 MPa for 2 h.The process flowchart of Si3N4/BN fibrous monolithic ceramics was scrutinized using a HITACHI S4800 scanning electron microscope (SEM). To supplement this, an energy dispersive spectrometer (EDS) was utilized for an in-depth analysis. The linear contraction rate was ascertained by measuring the dimensional changes between the gel and the bulk post-casting. For a comprehensive chemical composition analysis, the Si3N4/BN fibrous monolithic ceramic samples were ground and assessed for carbon elements using the Leco CS-200 carbon-sulfur analyzer.The microstructure of Si3N4/BN fibrous monolithic ceramics, were evaluated at room temperature using the INSTRON-3367 universal testing machine . To prepare for this, ceramic blocks underwent a series of processes: they were cut using an internal circular cutting machine, ground, polished to achieve a mirror finish, chamfered, and cleaned. This resulted in test samples of dimensions 3 × 4 × 36 mm and 2 × 4 × 22 mm.Mechanical properties, specifically the three-point flexural strength and fracture toughness of Siσ represents bending strength (MPa); P is the maximum applied load (N); L is the span during testing (30 mm); B is the width (approximately 4 mm); and W is the height (approximately 3 mm).For the flexural strength assessment, 3 × 4 × 36 mm specimens were selected. The tests were conducted with a 30 mm span and a loading speed of 0.5 mm/min. Results from 4–6 samples were averaged. The flexural strength was calculated using Formula (1):ICK is the fracture toughness (MPa·m1/2); P is the maximum applied load (N); L is the span (16 mm); B is the width (approximately 2 mm); and W is the height (approximately 4 mm).Fracture toughness was determined following the GB/T 23806-2009 standards , using 25 Pa) and yield stress (102–103 Pa) under high shear conditions [The rheological properties of the spinning slurry are vital for determining the quality of wet spinning. These properties are typically assessed using three parameters: modulus, yield stress, and apparent viscosity. For optimal wet spinning, the slurry should exhibit shear-thinning behavior, with an ideal storage modulus (~10nditions . The stoG′/G″-γ curve (G′) surpasses the loss modulus (G′) under the same shear strain, indicating viscoelastic behavior. However, with increasing shear stress, the two curves intersect, leading the loss modulus (G″) to exceed the storage modulus (G′). This indicates that the slurry samples can be extruded into shape with the right extrusion pressure. Nevertheless, an increase in the solid mass fraction significantly boosts both the storage (G′) and loss modulus (G″) of the slurry. For instance, at a solid mass fraction of 60 wt.% (P3), the storage modulus (G′) reaches approximately 388 Pa. This higher modulus necessitates greater extrusion pressure, increasing the risk of nozzle clogging and printing interruption. On the other hand, slurries with lower solid mass fractions (P1) have a reduced modulus, increasing the chances of splatter during spinning and affecting their self-supporting ability.The impact of different solid mass fractions on the rheological properties is demonstrated in -γ curve a shows t3 Pa·s in P1 to 1.0 × 104 Pa·s in P3. According to Woodcock’s Formula (3), when the particle diameter (d) remains constant, the distance (h) between particles is inversely related to the solid mass fraction [G) and apparent viscosities (η).The steady-state shear test results for slurries with different solid mass fractions are illustrated in fraction . This imG′0) and yield stress (τ0) are presented in G′0) of the P1 (40 wt.%) is significantly lower, along with a relatively small yield stress (τ0). The P2 (50 wt.%) has a storage modulus of approximately 205 Pa and a corresponding yield stress (τ0) of 455 Pa, both of which are within a suitable range for extrusion and indicate excellent self-supporting capability. However, the P3 slurry (60 wt.%) demonstrates a storage modulus close to 388 Pa, while the yield stress reaches 3366 Pa, making it unsuitable for wet-spinning applications. Based on the analysis mentioned above, neither the P1 slurry nor the P3 slurry is suitable for wet spinning.The statistical results for the initial storage modulus (G′) values than the loss modulus (G″) at minimal shear strains, indicating viscoelastic behavior. τ0) where the storage modulus (G′) intersects with the loss modulus (G″) under increasing shear stress, implying that the slurry containing sodium alginate can transition into a liquid sol under appropriate pressure.Fiber-reinforced cordierite ceramics are susceptible to cracks due to shrinkage stress during drying. To counteract this, hydrophilic polymers with abundant hydroxyl groups (–OH) can crosslink and reinforce the green body via hydrogen bonding. Consequently, they are employed in gel casting to prevent cracking and skinning of the green body . Sodium 2), silanol groups (≡Si–OH), and other sodium alginate molecules on the surfaces of ceramic particles through hydrogen bonding [G′0), yield stress (τ0), and initial apparent viscosity (η0) among samples P4, P2, and P5  and the cell boundaries (h-BN). Mechanical tests conducted at room temperature reveal that h-BN adopts a stratified flake structure, as demonstrated in 3N4/BN fibrous monolithic ceramics reinforced with ceramic fibers of varying diameters. Si3N4/BN-4 ceramics, fabricated with ceramic fibers of 1.05 mm diameter, exhibited a bending strength of only 240 ± 12 MPa. This lower strength is due to the larger pores formed between the coarser fibers, which are challenging to fill during the hot-pressing processes [3N4/BN-3 ceramics, using 0.9 mm diameter fibers, demonstrated an improved bending strength of 357 ± 24 MPa. However, when the fiber diameter was further reduced to 0.7 mm and 0.42 mm for Si3N4/BN-2 ceramics, a slight strength reduction was observed. This is because the smaller fiber diameter increases the density of weak interfaces in the composite, weakening the overall strength. The fracture toughness of Si3N4/BN-4, Si3N4/BN-3, Si3N4/BN-2, and Si3N4/BN-1 first increased and then decreased. Si3N4/BN-4 ceramics had a fracture toughness of only 4.4 ± 0.23 MPa·m1/2, whereas Si3N4/BN-3 ceramics exhibited a significant increase to 8.8 ± 0.36 MPa·m1/2, attributed to the higher number of weak interfaces due to the smaller fiber diameter. A subsequent reduction in the fiber diameter resulted in the fracture toughness of Si3N4/BN-2 and Si3N4/BN-1 ceramics decreasing to 3.4 ± 0.33 MPa·m1/2 and 4.6 ± 0.43 MPa·m1/2, respectively.rocesses . Consequ3N4/BN materials prepared with various fiber diameters in this study to the findings reported in the literature. The mechanical properties of Si3N4/BN materials are consistently subpar regardless of the sintering method employed. However, introducing a fibrous monolithic structure to Si3N4/BN materials has partially enhanced their mechanical properties. On the downside, the debinding process, executed in an inert atmosphere, negatively affects wave transmission performance. Additionally, the incorporation of β-Si3N4 and SiC whiskers to enhance the mechanical properties of Si3N4/BN fibrous monolithic ceramics significantly affects their wave transmission performance. In our research, the debinding process for Si3N4/BN fibrous monolithic ceramics takes place in ambient air. The carbon element content in Si3N4/BN fibrous monolithic ceramics was measured to be 0.82% using an elemental analyzer. This composition does not notably degrade their mechanical properties. The best mechanical performance was achieved with a fiber diameter of 0.90 mm, markedly outperforming other sintering techniques.3N4/BN fibrous monolithic ceramics. By incorporating a high concentration of sodium alginate, crosslinking is enhanced, leading to a boost in the slurry’s modulus and viscosity. On the other hand, a rise in solid content intensifies van der Waals interactions, which could cause potential needle blockage. To understand this better, we adjusted the mass fractions of sodium alginate and solid content, examining the rheological properties of the spinning slurry. We also explored how varying the Si3N4/BN fiber diameter impacts the ceramics’ microstructure and mechanical properties. Notably, the storage modulus of the P2 sample was approximately 205 Pa, with a yield stress (τ0) of 455 Pa. These values are ideal for extrusion. exhibiting favorable self-supporting capability, making it apt for wet spinning. The resulting Si3N4/BN fibrous monolithic ceramics exhibited a carbon content of 0.82%, a three-point bending strength of 357 ± 24 MPa, and a fracture toughness of 8.8 ± 0.36 MPa·m1/2. This investigation offers valuable insights on producing high-performance Si3N4/BN composite ceramics utilizing hot-pressing technology.This study introduces a wet-spinning–hot-pressing strategy for crafting Si


To date, there is no cure for Parkinson’s disease (PD). There is a pressing need for anti-neurodegenerative therapeutics that can slow or halt PD progression by targeting underlying disease mechanisms. Specifically, preventing the build-up of alpha-synuclein (αSyn) and its aggregated and mutated forms is a key therapeutic target. In this study, an adeno-associated viral vector loaded with the A53T gene mutation was used to induce rapid αSyn-associated PD pathogenesis in C57BL/6 mice. We tested the ability of a novel therapeutic, a single chain fragment variable (scFv) antibody with specificity only for pathologic forms of αSyn, to protect against αSyn-induced neurodegeneration, after unilateral viral vector injection in the substantia nigra. Additionally, polyanhydride nanoparticles, which provide sustained release of therapeutics with dose-sparing properties, were used as a delivery platform for the scFv. Through bi-weekly behavioral assessments and across multiple post-mortem immunochemical analyses, we found that the scFv-based therapies allowed the mice to recover motor activity and reduce overall αSyn expression in the substantia nigra. In summary, these novel scFv-based therapies, which are specific exclusively for pathological aggregates of αSyn, show early promise in blocking PD progression in a surrogate mouse PD model. The variable regions of heavy chain (HC) and light chain (LC) genes were isolated using standard molecular cloning methods as described in E. coli was grown at 37 °C in 5 mL terrific broth (TB) media shaking at 250 rpm overnight. Stocks were stored in 15% glycerol at −80 °C until further use. E. coli was then incubated in 250 mL of a 3 L Fernbach flask  at a starting OD560 of 0.05 at 37 °C, 180 rpm for ~2–3 h, until reaching an OD560 of 0.6. Then, the temperature was reduced to 20 °C, and 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to induce over-expression and cells were grown overnight for 24 h. Cells were centrifuged at 6000× g for 30 min, the supernatant was decanted, and cell pellets were frozen at −80 °C.scFv for biodistribution experiments was recombinantly produced in a non-pathogenic 2 was added to the lysate and mixed thoroughly, followed by end-over-end incubation at room temperature for 20 min. The lysates were centrifuged at 15,000× g for 30 min at 4 °C and the soluble fraction protein extract supernatant was collected for purification.To collect recombinant scFv protein, the frozen cell pellets were suspended in a lysis buffer of 20 mM PBS, 100 mM NaCl, and 200 µg/mL chicken egg white lysozyme with EDTA-free protease inhibitors (pH 7.4). The cells were lysed for 3 h in 4 °C in an end-over-end rotator. Then, 20 µg/mL DNAse and 10 mM MgClg, and supernatant was discarded. The resin was resuspended in two resin-bed volumes of an equilibration buffer, containing 20 mM sodium phosphate with 300 mM sodium chloride , and 10 mM imidazole (pH 7.4), followed by centrifugation for 2 min at 700× g. Then, the protein extract was mixed 1:1 with equilibration buffer and 20 column volumes were added to the resin and mixed for 2 h in an end-over-end rotator. The tube was centrifuged for 2 min at 700× g and collected for re-incubation. The resin was washed 5–8 × in 2 column volumes in a wash buffer of PBS with 25 mM imidazole (pH 7.4) followed by centrifugation until the OD280/260 reached baseline levels. Then, the scFv was eluted by incubating the resin for 5 min in 2 column volumes in an elution buffer of PBS in increasing imidazole concentrations, including 2 × each of 250 mM, 350 mM, and 500 mM imidazole, centrifuging between each elution step. Once protein concentrations by OD280/260 again reached baseline, wash buffer was added to rinse the resin 2×, followed by centrifugation. scFv buffer was exchanged for 1× PBS using 10 kDa molecular weight cut-off spin-concentration columns  and stored at 4 °C until further use.The soluble fraction supernatant was further purified by a batch method using a polyhistidine-tag resin. Specifically, 5 mL of a nickel–nitrilotriacetic acid resin slurry (50% slurry in 20% ethanol) was added to a 50 mL conical tube. The tube was centrifuged for 2 min at 700× The presence of purified protein was confirmed by SDS-PAGE and anti-Myc tag dot blot. Specifically, SDS-PAGE and dot blot studies were performed to evaluate the molecular weight and presence of purified scFv, respectively. Briefly, 7 µL scFv sample was added to 63 µL of a solvent containing 4× Laemmli sample buffer  diluted 1:4 into 1× PBS with 10 mM dithiothreitol , which was then heated for 7 min at 100 °C. Approximately 40 µL was loaded into polyacrylamide gels  for SDS-PAGE studies at 200 V constant voltage at 4 °C for 30 min in 10× Tris/Glycine/SDS buffer  diluted 10× in dH2O, and visualized via Coomassie blue staining. To evaluate 1-day biodistribution in mice, scFv was first conjugated to VivoTag 800  using the manufacturer’s protocol. To use scFv for treatment over a 10-week period following AAV injection into mice, endotoxins were first purified from scFv using endotoxin removal resin  using the manufacturer’s protocol.αSyn RT-QuIC assay was performed based on previously established protocols with minor modifications to test the activity of antibody on seeding kinetics . In brie2: pentane ratio. NP morphology and size were determined using field-emission scanning electron microscopy . ImageJ 1.43u software  was utilized to quantify NP size and size distribution. NP release kinetics and encapsulation efficiency were performed in vitro as described previously [scFv at 1% w/w was encapsulated into 10:90 CPTEG:SA NPs by an anti-solvent nano-encapsulation method at room temperature, as described previously . Briefly® Spectrum live animal imaging system (Perkin Elmer) at an excitation wavelength of 745 nm and an emission wavelength of 800 nm. To estimate the concentration of scFv in organs, a standard curve of scFv from 0.3–10 µg/mL was obtained on a 96-well plate in triplicate and imaged using the same settings as that for tissues.To evaluate biodistribution of scFv in C57BL/6 mice, 100 µL soluble scFv or NPs was administered IV via lateral tail vein injection at 20 µg scFv /kg mouse body weight using a 26-gauge needle. To assist with IV injection, a mouse restraint device was used, together with a heating lamp to dilate the tail vein. Control mice were injected with PBS only. Mice were euthanized and the brain was dissected for further analysis at 2 h and 24 h post-injection. The experiments were performed in quadruplicate. To assess fluorescence levels in mice administered soluble scFv or NPs, brains were dissected and imaged using the IVISAAV1/2-A53T-αSyn viral vector was purchased from Vigene . Deeply anesthetized mice were stereotactically injected unilaterally into the left SN with a microinjector at a rate of 0.5 µL/min with either 1.5 µL of PBS (control) or 1.5 µL of AAV1/2 expressing human mutated A53T-αSyn at a concentration of 5 × 1012 GC/mL. According to the mouse brain atlas of Paxinos and Franklin, the coordinates from Bregma: AP −3.1 mm; ML −1.4 mm; DV −4.4 mm were applied for incision . Six micOpen-field and rotarod experiments were performed every two weeks as described previously . Briefly2O with 20% methanol.Brain SN designated for Western blot analysis were micro-dissected and stored at −80 °C until further use. Western blot lysates from the SN were prepared in RIPA buffer with protease and phosphatase inhibitors. Specifically, RIPA buffer was added, and samples were homogenized and then sonicated for 5 min on ice (30 s on/off cycle). Protein estimation was performed using a Bradford assay. Samples of appropriate dilution were added to protein assay dye reagent concentrate  diluted 1:5 in nanopure water and absorbance was measured at 595 nm. A standard curve of BSA between 2–14 µg/mL was compared for quantification. Then, 25 µg per sample was added 1:1 to a 5:1 mixture of 2× Laemmli sample buffer  to beta-mercaptoethanol, or approximately 20 µL, into polyacrylamide gels  for an SDS-PAGE run at 100 V constant voltage for 1 h 30 min at 4 °C in 10× Tris/Glycine/SDS buffer diluted 10× in dH2O. Following SDS-PAGE, proteins were transferred to a nitrocellulose membrane at 80 V constant voltage at 4 °C for 3 h using a transfer buffer of 10× Tris/Glycine  diluted 10× in dThe membranes were blocked for 1 h in intercept blocking buffer  at room temperature. Membranes were cut into sections according to molecular weight and incubated with either anti-tyrosine hydroxylase-mouse mAb at 1:2000  or anti-αSyn mouse mAb at 1:1000  in blocking buffer with 0.05% T-20 for 16 h at room temperature. Membranes were washed 5× with 1× PBS + 0.05% T-20 (wash buffer), then incubated with 800CW Donkey anti-Mouse IgG secondary  1:15000 in blocking buffer + 0.05% T-20 for 1 h at room temperature. Membranes were washed 5× in wash buffer, then imaged using a Li-Cor Odyssey infrared scanner . Membranes were incubated with anti-beta actin-rabbit mAb  1:1000 + 680RD Donkey anti-Rabbit IgG secondary  1:15,000 in blocking buffer + 0.05% T-20 for 1 h at room temperature. Membranes were washed 5× in wash buffer then imaged once more.agg, αSyn, BSA (non-specific control), or SN brain homogenate used for Western blotting . After letting the blots dry, 5 mL of 5 wt % skim milk in 1× PBS was added to the blots inside microcentrifuge tubes, and the blots were blocked on a Labquake tube shaker/rotator (Fisher Scientific) for 60 min at room temperature (RT). The blots were rinsed three times with 1× PBS; then, 5 mL of 10 μg/mL purified singularly specific monoclonal antibody IgM 3C5 or 5H6 developed and purified in-house according to previous work in a dilution buffer of 0.1 wt % T-20 in 1× PBS, and the blots were incubated on a tube rotator for 90 min at RT [Nitrocellulose membranes were spotted twice with 2.5 μLof 1 μg/μL αSynin at RT . The bloBrain STR were micro-dissected, preserved in antioxidant mixture containing 0.2 M perchloric acid, 0.1% NA2S2O5, 0.05% NaEDTA, and isoproterenol as an internal standard as described previously and immediately stored in dry ice . The samAnimals were transcranially perfused with PBS and 4% PFA and stored in 4% PFA at 4 °C until all blood vessels were visibly transparent. SN and STR sections were dissected and placed in cassettes which were stored in 4% PFA for 2 d at 4 °C. Tissues were embedded in paraffin, and 5 µm slide sections were mounted onto positively charged slides by the Iowa State University clinical pathology laboratory. To process tissues for immunohistochemistry, slide sections of SN and STR were de-paraffinized by heating at 60 °C for 30 min and then using a gradient solvent method with xylene (2×), 50/50 xylene/ethanol, 100% ethanol, 90%, ethanol in water, 70% ethanol, and 50% ethanol. Following rinses in water and then 3× in PBS, antigen retrieval was achieved by keeping sections at 80 °C for 30 min in 10 mM sodium citrate buffer, pH 6.For preparation of slides for fluorescent imaging, slides were pre-incubated for 1 h in a blocking buffer containing 10% donkey serum (DS), 2% bovine serum albumin, and 0.05% Tween-20 (T-20) in PBS. Then, slides were incubated in 2% DS and 0.05% T-20 with 1:2000 rabbit anti-tyrosine hydroxylase polyclonal antibody  and 1:500 mouse anti-s129 αSyn  for 16 h at room temperature. After washing the tissues 6 × 10 min in 1× PBS, donkey anti-mouse 555  or 647  and donkey anti-rabbit 488  was incubated in 2% DS with 0.05% T-20 for 1 h. Sections were washed again 6 × 5 min in 1× PBS, then Hoechst (1:5000) was incubated in slides for 5 min. Slides were washed 2 × 5 min and then mounted using Fluoromount™ aqueous mounting medium  and allowed to dry for at least 18–24 h.For preparation of slides for bright-field imaging, tissues were additionally incubated in methanol + 3% hydrogen peroxide , 3× in 1× PBS, then pre-incubated for 1 h in a blocking buffer containing 10% normalized goat serum , 2% bovine serum albumin , and 0.5% Triton-X in 1× PBS. Blocking buffer was removed and slides were then incubated in 2% NGS and 0.05% T-20 with 1:1600 rabbit anti-tyrosine hydroxylase polyclonal antibody for 16 h at 4 °C. After washing the tissues 6 × 5 min in 1× pbs, goat anti-rabbit  was incubated in 2% NGS with 0.05% T-20 for 1 h. Tissues were washed 6 × 5 min in 1× PBS, then incubated with ABC solution , which was pre-equilibrated for at least 30 min prior to incubation for 1 h at RT. Tissues were washed 3 × 5 min in PBS, then incubated with 2 mM 3,3′-diaminobenzidine + 0.005% hydrogen peroxide (DAB) in 1× PBS for 1 min until color development, then immediately exchanged for PBS and washed 4 × 5 min. Tissues were dried overnight, then dehydrated by a gradient solvent method in reverse order from that presented above, then DPX mounting medium  was added to the slides and samples were covered with coverslips and allowed to dry for at least 18–24 h. Both fluorescent and brightfield images were taken using a BZ-X810 All-in-One Fluorescence Microscope . SN and STR regions for each set of slides were imaged under the same conditions. Densitometric analysis of specified regions from fluorescence microscopic images were further analyzed by ImageJ to evaluate TH and αSyn expression.Two methods of normalization were used in motor and immunohistochemical microscopic analysis. The first normalization method involved calculating the percent of ipsilateral/contralateral control, i.e., the level of expression of proteins and neurotransmitters of mice unilaterally injected with AAV were compared to levels in the non-injected, or contralateral, site. The normalized ipsilateral/contralateral control is expressed as:X is the relative protein or catecholamine expression from the same mouse for both values. The second normalization method was used only for the cumulative score behavioral data in motor and immunohistochemical microscopic analysis. Specifically, data were normalized based on basal activity for each individual assessment prior to cumulating data into a behavior score and analysis, due to differences in units across individual behavior assessments. All Western, dot blot, and immunohistochemical images were analyzed by the ImageJ densitometric analysis–gel analysis tool. Individual and cumulative score behavioral data were log-transformed prior to analysis due to high variability within sample groups. Statistical analysis was performed using GraphPad® Prism software. Specifically, a one-way ANOVA using Dunnett’s multiple comparisons (two-tailed) was used for all percent ipsilateral/contralateral control comparisons. All statistical tests were set at multiple levels to denote varying degrees of significance including: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001. Additionally, linear mixed models (LMMs) were used for analyzing the repeated measures behavioral data using R coding software. Further, the Benjamini–Hoch method was used to correct for multiple comparisons in the behavioral data. All comparisons denoting statistical significance was defined as p < 0.05.Here, Sample sizes for each experiment were as follows: SEM—n = 4 images counting 100 particles per image; zeta potential and release—n = 3; biodistribution—n = 4 per group, per time point; animal behavior—n = 8 for scFv and NP, n = 7 for AAV, n = 6 for control; catecholamine and dot and Western blot analysis—n = 4 for AAV, scFv and NP, n = 3 for control; immunohistochemistry (brightfield and fluorescence) analysis—n= 3 for control and AAV, n = 4 for scFv and NP. No outliers were excluded from the datasets. All release, dot, and Western blot individual behavior assessment data are presented in motor and immunohistochemical microscopic analysis; catecholamine and immunohistochemistry data are presented as mean ± SEM. Cumulative behavior score data in motor and immunohistochemical microscopic analysis and individual behavioral assessment data in The purified scFv had a molecular weight of approximately 27 kDa and displayed activity against anti-myc tag A,B. An R10:90 CPTEG:SA copolymer in molecular weight range of 15,000–20,000 g/mol was used for synthesizing NPs loaded with scFv, as demonstrated previously . AdditioAfter quantifying fluorescence intensity, it was found that the mass of scFv in the brain remained constant for one day after IV administration C. Howevep < 0.05 are indicated. After normalizing and compiling all individual assessments, a trend indicating an overall decrease in motor function over time in mice injected with AAV without treatment compared to PBS control was observed . The forepaw use studies did not show statistically significant differences between the groups  displayed a trend of recovered motor performance to basal levels, particularly at later times I. Specife groups J.agg in AAV-treated mice in a dot immunoblot , a 25% reduction in dopaminergic neurons was seen only 6 months after transduction ,13. HencDue to the various individual assessments and variability that is commonly observed between mice in each group, behavioral data can become convoluted. Therefore, we used a cumulative behavior score that compiled normalized data from four separate behavioral assessments , all of which are expected to be affected by AAV injection, into a single value. The rationale for this inclusion is to allow for a comprehensive evaluation of the total motor impairment at any given time throughout the course of the study.In our studies, a slightly longer endpoint of 12 weeks post-injection was chosen to allow time for a more pronounced decrease in motor activity. It was found from VersaMax and rotarod behavioral assessments that there was a trend of consistent decrease in motor activity over time among AAV-injected mice, suggesting that this model can produce rapid behavioral deficits. Basal activity was assessed based on the average of 0–2 weeks data before initiation of treatment. Both time points were used for basal data as no significant differences were observed in behavioral activity between 0–2 weeks after AAV injection. Additionally, using two time points for measuring activity allowed for more robust statistical analysis of trends in behavior using the LMM discussed above. It was found that after an initial depression in activity across all groups at week 4, the scFv-treated groups (as well as control) recovered back to basal activity. In contrast, animals that received only AAV  did not recover . ForepawTo support this finding, immunochemical analyses were performed on the SN and STR. Some previous work has shown that this AAV1/2-A53T model can successfully produce rapid nigrostriatal degeneration in C57BL/6 mice . Other sagg, because each form provides unique insights into the mechanisms at play and the extent of disease.In addition to TH loss, because this is an αSyn over-expression model, the levels of αSyn were expected to be higher in the ipsilateral SN of injected mice. The exact mechanisms of αSyn pathology in PD are still not fully known, because protein aggregation plays a role in many neurodegenerative diseases . What isagg in early-stage disease, this may indicate that the mice were at an early stage of the degenerative process [In our studies, a pronounced increase in αSyns129 levels was observed after AAV injection B. Becaus process . Both so process  and coul process .The scFv described in this preliminary study, which is specific exclusively for pathological aggregates of αSyn, shows early promise in blocking PD progression in a surrogate mouse PD model, although further studies will be needed to definitively determine its use as a therapeutic agent in PD patients. Especially encouraging is the recent accelerated approval by the United States Federal and Drug Administration (FDA) of Aduhelm (Aducanumab) developed by Biogen for management of Alzheimer’s disease (AD) , a progrThere is an urgent unmet need for novel and efficacious preventive and therapeutic management modalities for degenerative neurological disorders, including PD. It is imperative that such a therapeutic management blocks PD progression without eliciting toxic systemic deleterious effects. Among disease-slowing treatment options, anti-αSyn-targeting antibody therapeutics are becoming a well-studied area of research due to the prevalence of αSyn pathology in PD. As a prelude to in vivo determination of therapeutic potential, we first validated the robustness of the AAV1/2-A53T model in mice, which showed similar pathology to that shown in previous studies. Then, by assessing motor activity and protein, protection against SN AAV injection was observed in animals receiving both soluble scFV and NP-encapsulated scFv treatments. These scFv-based formulations and potential cocktail treatment regimens combining soluble and nanoparticle-encapsulated scFv should be further optimized and evaluated in additional proxy animal models to assess their use as a therapeutic modality to slow down or halt PD progression.


These results were determined with single-crystal X-ray diffraction analysis technology, ESI-MS NMR spectroscopy, etc. Importantly, the photoquenching effect of Cp* groups and the discrepancy of intermolecular π–π stacking interactions between building block and half-sandwich fragments promote markedly different photothermal conversion results. These results will further push the synthesis of topological structures and the development of photothermal conversion materials.Due to their fascinating topological structures and application prospects, coordination supramolecular complexes have continuously been studied by scientists. However, the controlled construction and property study of organometallic handcuffs remains a significant and challenging research subject in the area of supramolecular chemistry. Hence, a series of tetranuclear organometallic and heterometallic handcuffs bearing different size and metal types were rationally designed and successfully synthesized by utilizing a quadridentate pyridyl ligand (tetra-(3-pyridylphenyl)ethylene) based on three Cp*Rh (Cp* =  Therefore, building blocks B2 and B3 were individually introduced into the system to carry out a reaction with ligand L1. Once the two reaction processes were completed, the NMR exploration experiments of complexes 2 and 3 were performed. The 1H NMR of complex 1 exhibited a clear set of proton signals  further demonstrated the presence of complex 2 ]2− [opba = o-phenylenebis(oxamato)] (B5) were deliberately selected to synthesize a bigger organometallic assembly, in which the Rh–Rh nonbonding distances were 11.80 and 10.67 Å. Through the self-assembly of ligand L1 and building blocks B4 and B5, two new gray and green centrosymmetric complexes 4 and 5 were obtained in the yields of 85.8% and 83.6% , further determining the stable presence of 4  between Cp* and the phenyl group could be observed, reflecting strong intermolecular interactions. These interactions caused the formation of two-dimensional layered structures.  and 3 (from 27.4 to 35.8 °C) are similar to that recorded for complex 1. Importantly, an obvious stronger photothermal conversion could be observed for molecular handcuffs 4. Thus, a temperature increase of 26.7 °C (from 27.7 to 54.4 °C) in 230 s under a power irradiation  absorption (1.0) at 730 nm and intermolecular π–π stacking interactions, leading to a good NIR photothermal conversion effect. Nevertheless, just as we expected, the weak near infrared absorption (0.22) at 730 nm and poor intermolecular π–π stacking interactions in complex 5 resulted in a very small temperature change of about 6.4 °C (from 26.2 °C to 32.4 °C), which was consistent with our initial speculation.Subsequently, the NIR photothermal conversion studies for complexes 4 at the laser power per unit area of 0.15 W/cm2 at 730 nm, we continued to study the photothermal conversion ability under different laser power per unit areas: 0.15, 0.3, 0.45 W/cm2 at 730 nm , suggesting that in a special near infrared light region, the increase in laser power per unit area could result in the enhancing of the photothermal conversion performance.Due to the excellent photothermal conversion performance of complex t 730 nm a. Resultordingly b. SpecifB1, B2, B3, B4 and [Cp*2Rh2(LCu)]Cl2 (B5) were synthesized based on a previously reported method in the literature. NMR spectra were taken at room temperature using Bruker AVANCE I 500 spectrometers. Elemental analyses were collected on an Elementar Vario EL III analyzer. All reagents and solvents were commercially purchased and used without further purification. The starting materials L1 and the five Cp*Rh based building blocks B1, B2, B3, B4 and B5. The change of the width and length of building blocks did not cause the change of the type of molecular handcuffs. And, these complexes were characterized by single crystal X-ray diffraction analysis, NMR spectra and UV–Vis spectra. Interestingly, the photothermal conversion studies for these complexes are carefully performed, reflecting different photothermal conversion capacities. Discrepancy in photothermal conversion comes from the different conjugative effects of building blocks B1–B5. A strong conjugation effect and π–π stacking interactions induced good photothermal conversion results. We believe that our results may be useful for constructing various Cp*-based photothermal conversion materials and promoting the rapid development of fine materials and smart materials. In summary, a series of organometallic molecular handcuffs bearing different sizes and species of metal atoms were successfully acquired through the self-assembly of quadridentate pyridyl ligand


Pseudomonas fluorescens antibacterial natural product that inhibits threonyl-tRNA synthetase (ThrRS). It acts as a broad-spectrum antibiotic against a range of clinically relevant pathogens and comprises a strained β-lactone ring decorated with catechol and 4-nitro-benzyl moieties. The catechol moiety is widespread in nature and its role in the coordination of ferric iron has been well-characterised in siderophores and Trojan horse antibiotics. Here we use a combination of mutasynthesis, bioassays, enzyme assays and metal binding studies to delineate the role of the catechol moiety in the bioactivity of obafluorin. We use P. fluorescens biosynthetic mutants to generate obafluorin analogues with modified catechol moieties. We demonstrate that an intact catechol is required for both antibacterial activity and inhibition of the ThrRS molecular target. Although recent work showed that the obafluorin catechol coordinates Zn2+ in the ThrRS active site, we find that obafluorin is a weak Zn2+ binder in vitro, contrasting with a strong, specific 1 : 1 interaction with Fe3+. We use bioassays with siderophore transporter mutants to probe the role of the obafluorin catechol in Fe3+-mediated uptake. Surprisingly, obafluorin does not behave as a Trojan horse antibiotic but instead exhibits increased antibacterial activity in the presence of Fe3+. We further demonstrate that Fe3+ binding prevents the hydrolytic breakdown of the β-lactone ring, revealing a hitherto unreported function for the catechol moiety in natural product bioactivity.Obafluorin is a  Complex formation with ferric iron protects the β-lactone moiety of obafluorin from hydrolysis and potentiates antibiotic activity. This worrying development has been highlighted by several high-profile reports, emphasising the need to increase the supply of antimicrobials effective against drug-resistant microorganisms.Pseudomonas fluorescens ATCC 39502 (hereafter P. fluorescens WT).9,10 Although 1 undergoes facile ring-opening in the presence of nucleophiles, and under mildly basic conditions, it exhibits broad-spectrum antibacterial activity. It inhibits both Gram-positive and Gram-negative clinically relevant pathogens, including methicillin-resistant Staphylococcus aureus (MRSA), E. coli and Pseudomonas aeruginosa,9,11,12 and when dosed systemically can protect mice infected with Streptococcus pyogenes with no observable toxicity.10One such example is obafluorin (1), a structurally unique β-lactone antibiotic produced by 12 ThrRS is one of a suite of essential housekeeping aminoacyl tRNA synthetase (aaRS) enzymes, each one responsible for loading a specific amino acid on to its cognate tRNA.13 In ThrRS, an essential Zn2+ ion in the active site enables the discrimination between threonine and serine for effective translation.14 Despite relatively high conservation of aaRSs across the bacterial kingdom, divergencies have been exploited by natural product antibiotics that selectively target those of competing organisms.15,16 The LeuRS inhibitor Agrocin 84 is used to treat the plant pathogen crown gall disease17 and the IleRS inhibitor mupirocin is used clinically as a topical treatment for skin infections.18 In addition to 1, ThrRS is also targeted by borrelidin, a structurally distinct polyketide natural product, which acts by simultaneously occupying the threonine, ATP and tRNA catalytic subsites.19,201-Producing strains carry obaO, an additional copy of ThrRS encoded by the 1 biosynthetic gene cluster (BGC), which can confer transferable resistance to 1-sensitive bacterial strains12 This immunity strategy has been observed for other aaRS-targeting natural products.21–23We previously showed that the target of 1 in sensitive organisms is threonyl-tRNA synthetase (ThrRS).P. fluorescens has been delineated in full and proceeds by the condensation of 2,3-dihydroxybenzoic acid  and -2-amino-3-hydroxy-4-(4-nitrophenyl)butanoate (AHNB) catalysed by the non-ribosomal peptide synthetase (NRPS) ObaI , with product release occurring via the formation of the β-lactone ring.24–26 We showed that the genes obaJLN are responsible for the biosynthesis of 2,3-DHBA, and that deletion of obaL leads to the abolition of 1 production. This phenotype can be rescued via the exogenous addition of 2,3-DHBA to P. fluorescens ΔobaL cultures.24 However, when the immunity determinant obaO is also absent, the addition of 2,3-DHBA instead abolishes growth.12The biosynthesis of 1 in 27 that is observed in a number of natural products, including enterobactin,28 bacillibactin,29 vibriobactin,30 and myxochelin.31 Therefore, we hypothesised that metal binding may be involved with the mechanism of action of 1. This could be via interactions with the ThrRS target directly and/or facilitating uptake via a Trojan horse antibiotic (THA) strategy, whereby the catechol enables the compound to hijack the active siderophore uptake system to cross the otherwise impenetrable Gram-negative membrane.32 The aaRS inhibitors albomycin, microcin C-like compounds, and agrocin 84 have been shown to act as THAs.33 Once inside the cell, the THA is then able to act on its target, in the case of 1via the highly reactive β-lactone group that is essential for antibacterial activity, as shown previously10 and confirmed in this work.The 2,3-DHBA catechol moiety is a common siderophore motifvia chemical hydrolysis. Using a selection of bioindicator strains we demonstrate that the modified analogues lack bioactivity in cellular assays. We show that this is mirrored in vitro using aminoacylation assays with both the E. coli ThrRS (EcThrRS) and the P. fluorescens housekeeping ThrRS (PfThrRS) in the producer. Taken together, these data demonstrate that the catechol moiety is essential for 1 bioactivity, most likely via a direct interaction with the ThrRS target.Here, we used a mutasynthesis strategy to produce analogues of 1 with modified catechol moieties. We demonstrate that these analogues are produced in the absence of the immunity determinant ObaO, with no significant detriment to growth, in stark contrast to 1 itself. We go on to purify these congeners, alongside ring-open 1 analogues which are accessed E. coli ThrRS, in which the phenol groups of the catechol coordinate the essential Zn2+ ion present in the active site.34 We further demonstrate that despite this interaction in the molecular target, 1 shows weak binding of Zn2+ in solution, but instead shows a strong, specific 1 : 1 interaction with Fe3+. Surprisingly, this interaction did not appear to facilitate active cellular uptake, as experiments with E. coli mutants deficient in Fe3+ uptake transporters demonstrated 1 did not function as a THA. Instead, iron binding appears to prevent the hydrolytic breakdown of 1, which is associated with increased levels of the active lactone versus the ring-open form during production by P. fluorescens and with increased potency against sensitive organisms.During the preparation of this manuscript, the interaction of the catechol with the target was verified by the publication of the crystal structure of 1 covalently bound to the P. fluorescens ΔobaL that is deficient in 1 production, a phenotype that is rescued by the exogenous addition of 2,3-DHBA.24 We fed this strain with 2-hydroxybenzoic acid (2-HBA), 3-hydroxybenzoic acid (3-HBA) and benzoic acid (BA). Although none of the resultant cultures displayed the characteristic purple colour associated with 1 production, HPLC of extracts revealed several new peaks with retention times shifted relative to 1  and 3 (182 mg). The low solubility of 4 meant it could be readily precipitated from its crude extract by suspension in water/acetonitrile (1 : 1) to yield pure 4 (397 mg) without the need for further chromatography. The structures were confirmed by full 1D and 2D NMR characterisation  assay3+ and/or a range of other biologically relevant metal ions and analysed using high resolution electrospray ionisation mass spectrometry (ESI-HRMS) (3+ a mass shift from 359 Da ([M + H]+) to 412 Da was observed, which corresponds to the [M − 2H + Fe3+]+ species. This indicates that ferric iron binds to 1via the deprotonated catechol, a motif often observed for catecholate siderophores.36 No mass shift was observed for Fe2+, Zn2+, Mg2+, or Ga3+. Addition of Mn2+ resulted in a peak consistent with a [M − H + Mn2+]+ species,37 but in contrast to incubation with Fe3+, the [M + H]+ peak was not fully depleted. Furthermore, in the presence of a mixture of all metal ions tested, only the [M − 2H + Fe3+]+ ion was observed, suggesting that the binding affinity of 1 for Fe3+ is greater than that of Mn2+  . Upon ad3+ ions, and to confirm the 1 : 1 stoichiometry observed in the MS experiments, we utilised Job's method of continuous variation.38 An absorbance maximum was observed at a mole fraction of 0.5 indicative of 1 : 1 complex with Fe3+, consistent with the salicylic acid (2-HBA) positive control -resorcinol (PAR) assay. Zn2+ binding is observed as a colour change from orange to yellow, resulting from a decrease in Zn(PAR)2 at 495 nm and increase in free PAR at 410 nm.39 Only at the highest molar ratio of 25 : 1 compound : Zn2+ was a modest decrease in the Zn(PAR)2 peak and slight increase in the free PAR peak observed for 1, but not 2–4, suggestive of some weak Zn2+ binding via the catechol. In contrast, the N,N,N′,N′-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN) Zn2+ binding positive control completely displaced PAR at molar ratios of >1  of 2 μg mL−1 and 4 μg mL−1 against MRSA and B. subtilis, respectively  was 4 μg mL−1; this suggests that uptake is the main barrier to activity against Gram-negative organisms. Saccharomyces cerevisiae was included as a representative eukaryotic strain, against which 1 showed activity only at the greatest concentration tested of 1000 μg mL−1.With compounds 2–7 in hand, we went on to test their activity against a range of bioindicator strains. We previously established a spot-on-lawn method for 1 bioassays, given that 1 reacts with both filter disks and media components in microbroth dilution assays.ectively . Whilst In contrast, the modified catechol analogues 2, 3 and 4, showed little or no activity against all the strains tested. This clearly demonstrates that the intact catechol is essential for bioactivity. The ring-open analogues 5, 6 and 7 were also inactive as anticipated  to the growth agar. For THAs a decrease in MIC is anticipated upon iron depletion as the target strain is expected to actively transport the THA-Fe3+ complex into the cell.40,41 However, no change in the MIC was observed when compared to normal assay conditions, consistent with the results of the assay under iron replete conditions above and the conclusion that 1 does not act as a THA  FhuA, FecA, CirA, FepA, FhuE and Fiu have been sequentially deleted.42 It has been shown that the TBDTs CirA and Fiu are responsible for the uptake of catecholate containing compounds,43 including THAs.40 Therefore, if 1 acts as a THA we would expect mutants E. coli BW25113 ΔfhuAΔfecAΔcirA (Δ3) and/or ΔfhuAΔfecAΔcirAΔfepAΔfhuEΔfiu (Δ6) to have increased 1 MICs when grown under iron depleted conditions. When grown in the presence of bipy (150 μM) Δ6 did not grow at all, consistent with a complete lack of iron uptake, whereas mutant Δ3 grew normally and the 1 MIC was unchanged in the presence of bipy. Furthermore, as for E. coli 25922, the MIC of 1 against E. coli BW25113 WT, Δ3 and Δ6 decreased ≥256-fold upon addition of Fe3+  on the antibacterial activity against E. coli 25922 and S. aureus of several antibiotics which operate through a range of mechanisms. Whilst we observed some impact on MIC, overall, the addition of Fe3+ tended to increase rather than decrease MIC values, and effects were much less marked than for 1  and found that this protected 1 against hydrolysis at all pH values  and MS after incubation for 30 min at half unit intervals between pH 6.0 and 8.0. We observed pH-dependent 1 hydrolysis, with little change observed at pH 6.0, but with the majority of 1 in the hydrolysed form at pH 8.0 . To testH values . To dete9,10 We recently showed that 1 inhibits bacterial growth through the inhibition of ThrRS in sensitive organisms, a target that is distinct from any clinically utilised antibiotic.5 The results here shed further light on the mechanism by which 1 inhibits ThrRS by identifying the catechol moiety as an essential molecular feature required for antibiosis and the inhibition of ThrRS. This data is consistent with work published during the preparation of this manuscript which described a crystal structure of 1 bound to ThrRS from E. coli, and which showed that the phenol groups of the catechol form key interactions with the essential Zn2+ ion present in the active site.34 Our results further show that 1 is a weak binder of Zn2+ in solution, suggesting that the Zn2+ interaction is specific to the ThrRS active site and that the 1 mode of action does not involve the modulation of zinc homeostasis as do dithiolopyrrolone antibiotics.44,45In this study we investigated 1, an antibiotic discovered almost 40 years ago.3+ ions and that this association protects 1 from hydrolysis in solution. This is important as an intact β-lactone moiety is required for the antibacterial activity. Moreover, the presence of ferric iron increases the antibacterial activity of 1 against E. coli and P. aeruginosa, with the MIC decreasing by over two orders of magnitude in spot-on-lawn bioassays. This decrease is dramatic, and these MICs are in line with those observed without the addition of ferric iron for Gram-positive organisms and for E. coli NR698 which has a compromised outer membrane. Taken together these observations suggest that, in addition to protecting 1 from hydrolysis, and thereby maintaining effective concentrations of the active species, the association with Fe3+ ions may increase transport across the Gram-negative cell outer membrane. Our work ruled out the likelihood that 1 operates as a THA, suggesting that increased transport is due to a passive mechanism. While the presence of iron is important for the activity of some antibiotics such as streptonigrin,46 or the ability of siderophores to scavenge iron leading to antimicrobial activity,27 the multiple roles of the 1 catechol moiety in direct target interaction, compound stability and potentially uptake is without precedent. However, how these effects play out under iron limited physiological conditions remains to be determined.Additionally, our results show that 1 readily forms a 1 : 1 complex with Fe3+. This is consistent with loss of the protective effect for analogues 2 and 3, whereas the benzoic acid analogue 4 is stable to hydrolysis at pH 8 ] was purchased from Moravek Inc. ‘Microtitre plate’ refers to Greiner bio-one, flat bottom 96 well suspension culture plates.Solvents used for extractions and HPLC analysis were bought from Fisher Scientific. Reagents and chemicals were purchased from Alfa-Aesar and Sigma-Aldrich (Merck). P. fluorescens, 30 °C for S. cerevisiae and 37 °C for E. coli, B. subtilis and S. aureus. All strains were maintained on solid LB medium with appropriate selection. Growth temperatures were 28 °C for P. fluorescens ATCC 39502 strains were maintained on LB agar plates supplemented with nitrofurantoin (100 μg mL−1). Strains were grown in 1 production medium (OPM), comprising: yeast extract 0.5%, d-glucose 0.5%, MgSO4·7H2O 0.01%, and FeSO4 0.01%, dissolved in Milli-Q (Merck Millipore) filtered water. A toothpick was used to inoculate 100 mL of OPM starter culture in a 250 mL Erlenmeyer flask from a single colony, with subsequent growth for 24 h at 25 °C, 300 rpm. 1 mL of this culture was used to inoculate 100 mL OPM production cultures in a 500 mL Erlenmeyer flask. Where required, production cultures were supplemented with: 2,3-DHBA , 2-HBA , 3-HBA  or BA  in DMSO. For growth curves, production cultures were grown at 25 °C, 300 rpm and OD600 measurements relative to an OPM blank were recorded every hour for 12 h, in biological triplicate. For metabolite analysis, after 14 h of growth, 1 mL of culture broth was extracted with an equal volume of ethyl acetate by mixing at 1400 rpm for 15 min. Samples were then centrifuged (15 682 × g for 15 min), and the organic phase was collected and evaporated. The resulting organic extract was dissolved in acetonitrile (ACN) (250 μL) and centrifuged (15 682 × g for 20 min) to remove any remaining cell debris, before HPLC analyses. For photographs, a 20 mL aliquot of the culture at 14 h was transferred to a screw-cap vial.Wild-type and mutant P. fluorescens cultures were analysed on an Agilent 1100 system using a Gemini 3 μm NX-C18 110 Å, 150 × 4.6 mm column (Phenomenex) with a gradient elution: ACN/0.1% (v/v) TFA (H2O) gradient from 10/90 to 100/0 0–15 min, 100/0 for 15–16 min, gradient to 10/90 16–16.50 min and 10/90 for 16.50–23 min. The flow rate was 1 mL min−1 and the injection volume of each sample 10 μL. DAD signals were analysed at 270 nm, with a bandwidth of 4 nm.Extracts from 18, 100 × 4.6 mm, 5 μM column (Phenomenex) was used with a gradient elution: ACN/0.1% (v/v) FA (H2O) gradient from 5/95 for 0–0.5 min, 5/95 to 98/2 0.5–12 min, 98/2 for 12–13 min, gradient to 5/95 13–13.5 min and 5/95 for 13.5–15 min. The flow rate was 0.6 mL min−1 and the injection volume of each sample 5 μL. DAD signals were analysed at 270 nm, with a bandwidth of 4 nm. MS were recorded in positive mode using an atmospheric pressure ionisation electrospray (API-ES) ion source with a capillary voltage of 3000 V. The drying gas temperature was 350 °C with a flow rate of 12 L min−1. The scan parameters were: mass range = 200–1500; fragmentor = 70; gain EMV = 1.0; threshold = 150; step-size = 0.10. Data were analysed with Agilent ChemStation software.Hydrolysis of 1–4 was analysed on an Agilent 1290 system fitted with an Agilent LC/MSD MS spectrometer. A Kinetex XB-C18, 100 × 4.6 mm, 5 μM column (Phenomenex) was used with a gradient elution: ACN/0.1% (v/v) FA (H2O) gradient from 5/95 for 0–0.5 min, 5/95 to 98/2 0.5–12 min, 98/2 for 12–13 min, gradient to 5/95 13–13.5 min and 5/95 for 13.5–15 min. The flow rate was 0.6 mL min−1 and the injection volume of each sample 5 μL. DAD signals were acquired at 270, with a bandwidth of 4 nm. HRMS were recorded in positive mode using a dual Agilent Jet Stream (AJS) ESI ion source with a capillary voltage of 3500 V and a nozzle voltage of 1000 V. The drying gas temperature was 320 °C with a flow of 8 L min−1, and the sheath gas temperature was 350 °C with a sheath gas flow of 11 L min−1. Reference mass correction was enabled with monitoring of reference masses with m/z 121.050873 and 922.009798. Data were analysed with Agilent MassHunter software.LC-HRMS of side products of hydrolysis I–VI .NMR spectra were recorded on a Bruker AVANCE III 400 MHz spectrometer at 298 K. Chemical shifts are reported in parts per million (ppm) relative to the solvent residual peak of acetone (−1 using a Harvard Apparatus syringe pump. The mass spectrometer was controlled by Masslynx 4.1 software (Waters). It was operated in positive ion mode and calibrated using sodium iodide. The sample was analysed for 1 min with a 1 s MS scan time over the range of 50–1200 m/z with 3000 V capillary voltage, 40 V cone voltage, 115 °C cone temperature. Leu-enkephalin peptide  was infused at 10 μL min−1 as a lock mass (m/z = 556.2766) and measured every 10 s. Spectra were generated in Masslynx 4.1 and peaks were centred using automatic peak detection with lock mass correction.For high-resolution electrospray mass spectrometry (HR-ESI-MS) samples were diluted into 50% ACN/0.1% formic acid and infused into a Synapt G2-Si mass spectrometer  at 10 μL minThe specific optical rotation of compounds was measured with a Model 341 Polarimeter .P. fluorescens ATCC 39502  and incubated at 25 °C with shaking at 300 rpm. After 24 h, 8 L of OPM (16 × 500 mL in 2 L flasks) were each inoculated with 5 mL from the starter culture; for analogue production 2 mL of a 100 mM stock solution of either 2-HBA, 3-HBA or BA in DMSO was added to give a final concentration of 0.4 mM. After incubation for a further 14 h at 25 °C with orbital shaking at 250 rpm, ethyl acetate (500 mL) was added to each flask and the cultures were shaken vigorously and then left to stand for ∼2 h. The organic phase was then separated, and the solvent removed under reduced pressure.OPM was inoculated with a single colony of 18 30 g cartridge using a Biotage Isolera™ system with UV absorbance monitoring at 276 nm. Mobile phase A: water; mobile phase B: ACN; flow rate: 25 mL min−1. Elution started from 3% B for 1.5 column volumes (CV), then gradient to 30% B over 1 CV, then gradient to 80% B over 7.5 CV, then gradient to 100% over 1 CV, and holding at 100% B for 1.5 CV. The fractions containing the desired compound were combined and the solvent was removed under reduced pressure. Biotage fractionation of the extract containing either 1 or 2 gave sufficiently pure compound that no further purification was required. Biotage fractionation of the extract containing 3 gave material which required further purification by the method described for preparative HPLC. The fraction containing 3 was collected and dried under reduced pressure.Extracts containing 1, 2 or 3 were dissolved in ACN/water  and fractionated over a Biotage CThe extract containing 4 was dissolved in ACN/water . Drawing this solution up through a long needle (Sterican 0.80 × 120 mm 21G) caused immediate precipitation in the syringe. The precipitant was carefully filtered, dried under a stream of nitrogen, and washed with ice cold acetone (3 mL) to give 4. Additional 4 was recovered by addition of water (1 mL) to the acetone, causing it to precipitate.The process was repeated nine times at 8 L scale to give 72 L of producing culture for 1, and three times at 8 L scale giving 24 L of producing culture for 2, 3, and 4.−1) as a light purple powder; [α]D +69°  ); HRMS (ESI) m/z: calculated for C17H15N2O7 ([M + H]+) = 359.0874, observed = 359.0872 ([M + H]+), Δ = −0.6 ppm. For tabulated NMR see Table S2 (ESI1: 1468 mg (20.4 mg mLle S2 ESI.−1) as an off-white powder; [α]D +115° ; HRMS (ESI) m/z: calculated for C17H15N2O6 ([M + H]+) 343.0925, observed 343.0930 [M + H]+, Δ = 1.5 ppm. For tabulated NMR see Table S3 , as an off-white powder; [α]D +66° ; HRMS (ESI) m/z: calculated for C17H15N2O6+ ([M + H]+) = 343.0925, observed [M + H]+ = 343.0933, Δ = 2.3 ppm. For tabulated NMR see Table S4  as a bright white powder; [α]D +45° ; HRMS (ESI) m/z: calculated for C17H15N2O5 ([M + H]+) = 327.0975, observed [M + H]+ = 327.0963, Δ = −3.7 ppm. For tabulated NMR see Table S5 ; HRMS (ESI) m/z: calculated for C17H17N2O8+ ([M + H]+) = 377.0979, observed [M + H]+ = 377.0976, Δ = −0.8 ppm. For tabulated NMR see Table S6  was dissolved in aqueous NaOH  and stirred for 3 h at room temperature. Hydrochloric acid  was then added, and the reaction mixture dried under reduced pressure. The resulting dry mixture was dissolved in water/acetonitrile  and fractionated using the preparative HPLC method. The major fraction was dried under reduced pressure to give 5  as a brown oil; [le S6 ESI.α]D +51° ; HRMS (ESI) m/z: calculated for C18H19N2O8+ ([M + H]+) = 391.1136, observed [M + H]+ = 391.1131, Δ = −1.3 ppm. For tabulated NMR see Table S7  was added to a solution of 1  in dry methanol (2 mL) under argon. The reaction mixture was stirred for 3 h at room temperature, after which time it was dried under vacuum. The residue was dissolved in water/acetonitrile  and fractionated using the preparative HPLC method. The single major fraction was dried under reduced pressure to give 6  as a brown oil; D +41° ; HRMS (ESI) m/z: calculated for C17H17N2O6+ ([M + H]+) = 345.1081, observed [M + H]+ = 345.1071, Δ = −2.9 ppm. For tabulated NMR see Table S8 (ESILiOH·Hle S8 ESI.3·6H2O (1.5 mL of 1 mM dissolved in 10 mM HCl). The resulting solution was gradually added to an aqueous solution of hexadecyltrimethyl-ammonium bromide  with stirring. To this, MES buffer  was added and the final volume was adjusted to 100 mL with H2O. To prepare the CAS assay solution, CAS (7.5 mL of 2 mM in water) was mixed with FeClA dilution series (50 mM to 50 μM) of 1–4 were prepared in DMSO. In a microtitre plate, 90 μL of CAS solution was added to 10 μL of compound each dilution, to give final analyte concentrations of 5000 to 5 μM. The negative control was 10% v/v DMSO in CAS solution. The microtitre plates were imaged with a Perfection V600 scanner (Epson) and UV spectra were recorded from 220 to 800 nm on a microplate reader (Clariostar).2 solution, aqueous ZnSO4  was added to PAR solution in PBS . To prepare the Zn(PAR)N-acetyl-threonine (N-Ac-Thr) and N,N,N′,N′-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN) were prepared in DMSO. In a microtitre plate, 10 μL of each compound dilution was added to 90 μL of Zn(PAR)2 solution, to give final analyte concentrations of 250–5 μM. The negative control was 10% v/v DMSO in Zn(PAR)2 solution, and the positive controls were 10% v/v DMSO in PAR solution, and the dilution series of TPEN in Zn(PAR)2 solution. The microtitre plates were imaged with a Perfection V600 scanner (Epson) and UV spectra were recorded from 220–800 nm on a microplate reader (Clariostar).A dilution series (2500 to 50 μM) of 1–7, 2,3-DHBA, 2-HBA, 3-HBA, 4·7H2O, FeCl3·6H2O, ZnSO4·7H2O, MgCl2·6H2O, MnCl2·4H2O or Ga(NO3)3⋅92OH, or an equimolar mixture of all salts to give final concentrations of 100 μg mL−11 (279 μM) and 1 mM for each metal ion. Compounds 2–4 (100 μg mL−1) were mixed with FeCl3·6H2O (1 mM). Samples were analysed on a Synapt G2-Si mass spectrometer as described above.To assess the metal binding 1 was mixed with aqueous solutions of FeSO3+ complex, the change in absorbance was measured by UV-visible spectroscopy across a series of molar ratios. Samples with a mole fraction range of 1:Fe3+ from 0.1 to 0.9 were prepared as follows: aliquots of 1 (from 10 to 90 μL of a 1 mM stock solution in DMSO) and Fe(NO3)3·9H2O (from 90 to 10 μL of a 1 mM stock solution in water) were mixed in a 96-well microtitre plate. The solutions were left at room temperature for 15 min to equilibrate, then the UV-visible spectrum was measured using a microplate reader (Clariostar). The λmax of the 1-Fe3+ complex was determined as 690 nm, and the absorbance for each sample was plotted against the mole fraction to give a Job plot. Salicylic acid  was used as a positive control, as it forms a 1 : 1 complex with Fe3+ with a λmax at 535 nm. [Reid 2008]51 This analysis produced a maximum at 0.5 molar ratio indicating a 1 : 1 1 : Fe3+ complex.To determine the stoichiometry of the 1-Fe12 using Gibson assembly (Plasmids for protein expression were generated as previously describedassembly .E. coli NiCo21(DE3) (NEB) carrying pET28a(+)-EcThrRS, pET28a(+)-PfThrRS or pET28a(+)-obaO was used for protein expression and purification. Immobilised metal affinity chromatography (IMAC) and size exclusion chromatography (SEC) was used as previously described.24 Proteins were purified in a buffer containing 20 mM Tris–HCl at pH 8.0 with 500 mM NaCl and 10 mM MgCl2. The IMAC elution buffer also contained 250 mM imidazole.Thr for aminoacylation assays, tRNAThr was overexpressed in E. coli and purified by phenol extraction, as described in Avcilar-Kucukgoze et al.52 with some modifications as follows. A LB starter culture (10 mL) was inoculated from a single colony of E. coli MRE600:pWFW1015 and incubated for 16 h at 37 °C with 250 rpm shaking. This was used to inoculate LB (1 L containing 100 μg mL−1 carbenicillin in a 2 L Erlenmeyer flask) and incubated at 30 °C until the OD600 reached 0.4–0.6, and then induced with IPTG  and grown for a further 16 h at 30 °C. The cells were collected by centrifugation at 5000 × g for 25 min at 4 °C, and total RNA extracted by resuspension of the pellet in solution 1 (8 mL) followed by the addition of acid phenol ; . The resulting emulsion was shaken on an orbital incubator at 215 rpm for 30 min at 37 °C, then centrifuged at 5000 × g for 15 min at 4 °C and the resulting aqueous layer was collected and transferred to a clean tube. The phenol/interface layer was extracted a second time by addition of Solution 1 (14 mL) followed by orbital shaking at 215 rpm for 15 min at 37 °C. This was followed by centrifugation at 5000 × g for 15 min at 4 °C and the aqueous phases were combined. Subsequent precipitation of total nucleic acids was achieved by addition of NaCl solution (5 M) to give a final concentration of 0.2 M, followed by 1 volume of isopropanol. The mixture was allowed to sit at room temperature briefly before centrifugation at 14 500 × g for 15 min at room temperature. The resulting nucleic acid pellet was washed with ice cold 70% ethanol, then air dried for 5–10 min.To obtain purified tRNAg for 20 min at 4 °C. The supernatant was collected, and the remaining pellet was again vortexed in cold NaCl solution  and centrifuged again. The supernatants were combined, and the soluble nucleic acids were precipitated by addition of 2 volumes of cold ethanol and standing for 30 min at −20 °C, followed by centrifugation at 14 500 × g for 5 min at 4 °C. The resulting pellet was washed with 70% ethanol and air dried for 5–10 min. Contaminating DNA was removed by heating the pellet in NaOAc solution  at 60 °C until it dissolved, followed by addition of isopropanol (3.4 mL) and standing for 10 min at room temperature. This was then centrifuged at 14 500 × g for 5 min at room temperature and the supernatant collected. The precipitation of any remaining tRNAs was achieved by addition of isopropanol (2.3 mL) and standing for 30 min at −20 °C. The suspension was then centrifuged at 14 500 × g for 15 min at 4 °C, the pellet washed with 70% ethanol and air dried for 5–10 min and the resulting dry pellet dissolved in DEPC-treated water (500 μL). The tRNA was deacylated by addition of Tris buffer  and incubated for 45 min at 37 °C. This was followed by addition of NaOAc solution  and ethanol (1.6 mL), followed by standing for 30 min at −80 °C. The resulting suspension was then centrifuged at 16 100 × g for 25 min at 4 °C, and the pellet washed with 70% ethanol and air dried. The pellet was finally dissolved in DEPC-treated water (250–300 μL). The concentration and quality of tRNA was assessed by Nanodrop using a A260/A280 ratio .The contaminating rRNA was then removed by dissolution of tRNAs by vortex mixing in ice cold NaCl solution , followed by centrifugation at 9500 × et al.53 was used, where active enzyme was incubated with its necessary substrates and its activity measured using 14C labeled Thr and a liquid scintillation counter. Purified ThrRS protein (10 nM) was pre-incubated with varying concentrations of compounds 1–4 or ACN for 10 min. After pre-incubation, ThrRS samples were added to a master reaction mixture with the final component concentrations of HEPES , ATP (4 mM), MgCl2 (10 mM), l-[U-14C]threonine , and tRNAThr (5 μM). This mixture was incubated at room temperature for 10 min, with samples taken at 1, 2.5, 5, and 10 min. At each time point, a 7 μL aliquot was spotted onto a 5% trichloroacetic acid (TCA) presoaked 3 MM Whatman paper (Cytiva) squares (repeated in triplicate). After allowing the spots to dry, the Whatman paper was washed three times with 5% TCA, and once with 95% EtOH. The Whatman paper was dried and the counts on each square of paper analyzed using a Tri-Carb 2910 TR liquid scintillation counter using Hydrofluor Liquid Scintillation Fluid . To calculate IC50 values, fractional initial velocities (velocity of inhibited reaction/velocity in the absence of inhibitor) were plotted against the log of 1 concentration and then fitted to To measure the effects of 1–4 on ThrRS canonical tRNA charging activity, an assay modified from that reported by Ruan 600 of 0.3–0.4. These cultures were then diluted 1 : 10 with molten soft nutrient agar (SNA), before pouring into appropriately sized Petri dishes to set. Serial dilutions of the test compounds  were prepared in acetonitrile (ACN), and 4 μL of each sample was applied directly onto the SNA surface. Kanamycin (50 μg mL−1), apramycin (50 μg mL−1) or aureobasidin A (0.2 μg mL−1) were used as positive controls, as appropriate, and ACN or water (4 μL) were used as negative controls. Plates were incubated for 16–18 h and the MIC was defined as the lowest concentration of compound that resulted in a zone of inhibition. Experiments were carried out in at least triplicate for each strain.Indicator strains were grown for 16–18 h in LB (5 mL) by inoculation from a single colony. An aliquot of the resulting culture (500 μL) was then used to inoculate LB (50 mL in 250 mL Erlenmeyer flasks), followed by orbital shaking at 250 rpm until they reached an OD3)3·9H2O stock (400 mM) was added to give a final concentration of 1.5 or 2.0 mM; for iron depletion, bipy stock (1 M) was added to give a final concentration of 150 μM. For MRSA, 1 was further diluted to 0.5 0.25 and 0.125 μg mL−1 due to a decrease in MIC under excess iron conditions. The general effect of excess iron on the activity of alternative antibiotics was determined by performing a similar serial dilution of carbenicillin, kanamycin, streptomycin, nitrofurantoin and chloramphenicol in the presence of Fe3+.The method was the same as for the antibacterial MIC assays described above, with the following modifications: for excess iron, Fe(NO3)3.9H2O (11.1 mM) were also prepared at pH 6.0, 6.5, 7.0, 7.5, 8.0 respectively. Stock solutions of 1–4 (10 mM) were prepared in DMSO. For each hydrolysis experiment, stock solution of 1–4 (10 μL) was added the HEPES buffer (90 μL) or the HEPES + Fe(NO3)3.9H2O buffer (90 μL), to give final concentrations of HEPES (100 mM), 1–4 (1 mM) and Fe3+ (0 or 1 mM). The reaction mixtures were shaken using a bench top vortex and left to stand for 30 min, after which the samples were analysed by HPLC using the Analytical HPLC method. All stock solutions of 1–4 were tested at every pH, with and without Fe3+ and all experiments were repeated in triplicate.Five solutions of HEPES buffer (111 mM) were prepared at pH 6.0, 6.5, 7.0, 7.5 and 8.0 respectively. A further five solution of HEPES buffer (111 mM) with Fe(NOS. F. D.: conceptualization; methodology; validation; investigation; supervision; writing – original draft; writing – reviewing & editing; visualization. M. J. D.: conceptualisation; methodology; investigation. E. S. H.: validation; investigation; supervision; writing – review & editing; visualization. J. D. L.: methodology; investigation; formal analysis; visualisation. T. A. S.: conceptualization; investigation; S. A.: investigation. C. S. F.: methodology. B. W.: conceptualization; methodology; resources; writing – reviewing & editing; supervision; project administration; funding acquisition.The authors declare no competing interests.CB-004-D3CB00127J-s001